Compositions and methods for split artr-seq

Abstract

Aspects of the present disclosure are directed to at least methods and compositions for profiling of RNA-binding protein binding sites by in-situ reverse transcription-based sequencing using split reverse transcriptase enzymes. Provided compositions and associated profiling methods can capture both stable and transient interactions between RBPs and their RNA substrates, especially when the interaction is dynamic or materials are limited. Also disclosed herein are compositions, methods, and kits suitable for profiling of RNA-binding protein binding sites by in-situ reverse transcription-based sequencing.

Description

COMPOSITIONS AND METHODS FOR SPLIT ARTR-SEQCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No.63 / 743,914 filed January 10, 2025, which is incorporated by reference herein in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under HG008935 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on January 8, 2026, is named ARCD_P0858WO_Sequence_Listing.xml and is 256,144 bytes in size.BACKGROUNDI. Field of the Invention

[0004] Aspects of this invention relate to at least the field of molecular biology. More particularly, aspects concern at least methods and compositions for characterizing RNA-binding protein binding and RNA modification sites by in-situ reverse transcription-based sequencing.II. Background

[0005] RNA-binding proteins (RBPs) dynamically interact with their RNA targets to regulate RNA fate in all aspects, including transcription, splicing, modification, localization, translation and degradation. The dysfunction of RBPs or their binding to RNA substrates can lead to various defects or even diseases. Effective methods to capture RBP-RNA interactions, in particular dynamic or even transient interactions, are critical for obtaining better understandings of RBPs and their functional effects on target RNAs.

[0006] The widely used approaches to identify RNA substrates of RBPs are based on immunoprecipitation (IP) of the specific RBP along with their bound RNAs, either through direct RNA immunoprecipitation (RIP) or through cross-linking immunoprecipitation (CLIP) assisted by covalent capture. Substrate RNAs bound by specific RBP can be enriched through either RIP or CLIP using the antibody against the RBP, followed by high-throughput301320428.1 - 1 -sequencing (seq) to profile RBP targets at the whole transcriptome level. CLIP-seq captures RBP binding sites on substrate RNAs via covalent crosslinking. RNase treatment digests the RBP-free regions of RNAs, which can provide a higher resolution of the binding sites. Modulations of CLIP-seq such as PAR-CLIP or eCLIP approaches further improve the efficiency of crosslinking or specificity and resolution of the binding site assignment. While these methods have been very effective and widely used, they also have limitations. All these methods are IP based and often require large amounts of starting materials due to the low efficiency of IP; the UV crosslinking in CLIP -based methods is also a low-efficiency chemical reaction. Recently reported targeted RNA immunoprecipitation sequencing (tRIP-seq) and linear amplification of complementary DNA ends sequencing (LACE-seq) can be applied in low-input samples but at the cost of significantly reducing the complexity of libraries.

[0007] Targets of RNA-binding Proteins Identified by Editing (TRIBE) and Surveying Targets by APOB EC -Mediated Profiling (STAMP) type approaches fuse RBPs with an RNA base editor to introduce mutations nearby RBP binding sites, which bypasses IP to identify RBP binding sites. These methods could be readily applied to study RNA binding by RBPs in live cells and with limited materials down to single-cell level. However, just like other methods, these editing-based methods also have limitations. They can require the manipulation of genomes through the insertion of base editing proteins in germlines or cell lines, hindering their application in primary cells and tissue samples. Induction of the editing protein expression typically takes ~ 24 hours or longer, which cannot be applied to monitor dynamic RNA binding by RBPs. These base editors have their own sequence preferences, and their fusion to RBPs could change the native binding profile of the target RBP. The entire procedure is also more complicated in comparison to CLIP or RIP type approaches. CUT&Tag strategy, is a recent method that was developed to study RBP-RNA interaction. This method profiles RBP-RNA interaction by oligo(dT) primer-initiated reverse transcription and Tn5 tagmentation of the resulting full-length RNA-cDNA heteroduplex. The method can identify RBP binding in polyadenylated RNAs but is ineffective to non-polyadenylated RNAs and RBP binding in the cytoplasm because CUT&Tag strategy needs to be performed in isolated nuclei. Due to the low efficiency of the Tn5 enzyme on heteroduplex, it still can require ~ 100 k cells to obtain sufficient binding signals.

[0008] As described above, there exists a need for methods and compositions for sensitively profiling RNA targets of RBPs in situ with good sequencing quality, minimal sample sizes, and / or a short time frame.301320428.1 - 2 -SUMMARY OF THE INVENTION

[0009] In some aspects, the present disclosure encompasses a system for profiling RNA-protein interactions comprising a first polypeptide construct comprising an N-terminal fragment of a reverse transcriptase, or a variant thereof, and a first dimerization domain; and a second polypeptide construct comprising a C-terminal fragment of the reverse transcriptase, or a variant thereof, and a second dimerization domain that specifically binds to the first dimerization domain; and wherein the dimerization of the first polypeptide construct and the second polypeptide at the dimerization domain reconstitutes the reverse transcriptase.

[0010] In some aspects, the first polypeptide construct comprises a first targeting moiety. In some aspects, the second polypeptide construct comprises a second targeting moiety. In some aspects, the reverse transcriptase comprises Moloney murine leukemia virus (MMLV) RTase, human immunodeficiency virus (HIV) RTase, Avian Myeloblastosis Virus (AMV) RTase or a variant thereof. In some aspects, the N-terminal fragment of the reverse transcriptase enzyme is encoded by a nucleic acid sequence as set forth is any one of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 116, 148, 168, 172, 176, or 180, or a nucleic acid sequence at least 60% identical thereto. In some aspects, the C-terminal fragment of the reverse transcriptase enzyme is encoded by a nucleic acid sequence as set forth is any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 114, 115, 150, 170, 174, 178, 182, or 184, or a nucleic acid sequence at least 60% identical thereto. In some aspects, the N-terminal fragment comprises an amino acid sequence as set for in any one of SEQ ID NOs: 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 145, 149, 169, 173, 177, or 181, an amino acid sequence at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto. In some aspects, the fragment of the reverse transcriptase is a C-terminal fragment comprising an amino acid sequence as set for in any one of SEQ ID NOs: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 143, 144, 151, 171, 175, 179, 183, or 185, or an amino acid sequence at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein).

[0011] In some aspects, the first and / or the second targeting moiety is a protein; an oligonucleotide, a nucleoside analog, a nucleotide, a receptor, a ligand, an antibody, a nanobody, a small molecule, or any combination thereof. In some aspects, the first and the301320428.1 - 3 -second targeting moiety target different targets. In some aspects, the first and / or the second targeting moiety comprises a Fc or Fab binding protein. In some aspects, the first and / or the second targeting moiety comprises an oligonucleotide or a variant thereof. In some aspects, the oligonucleotide comprises a barcode, indices, affinity tag, label, a modified nucleotide, or any combination thereof. In some aspects, the affinity tag comprises a streptavidin, or an avidin tag. In some aspects, the first and / or the second targeting moiety comprises a small molecule.

[0012] In some aspects, the Fc or Fab binding protein comprises protein A, protein G, protein A / G (pAG), protein L, anti-rabbit IgG, anti-mouse IgG, or a nanobody. In some aspects, the Fc or Fab binding protein comprises an anti-mouse or an anti-rabbit nanobody which specifically binds the Fc, or the Fab region of an antibody. In some aspects, the nanobody is encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOS: 152, 154, 156 or 158, or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein). In some aspects, the nanobody comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 153, 155, 157, or 159 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein). In some aspects, the Fc binding protein is encoded by a a nucleic acid sequence as set forth in any one of SEQ ID NOS: 53, 55, 57, or 59, or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein). In some aspects, the Fc binding protein comprises an amino acid sequence as set forth in SEQ ID NOS: 54, 56, 58, or 60, or a or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein).

[0013] In some aspects, the first and / or the second dimerization moieties comprise binding regions of a rapamycin mediated dimerization domain, a leucine zipper, an SH3 domain, a PDZ domain, an Fc domain, an Fc binding domain, a basic helix-loop-helix domain (bHLH), a zinc finger domain, a tet repressor dimerization domain, or a Halo-tag ligand, a Halo-tag, or any combination of. In some aspects, the first and the second dimerization domain comprises, consists essentially of, or consists of a rapamycin mediated dimerization domain. In some aspects, the first polypeptide construct comprises an FRB domain, and the second polypeptide comprises an FKBP domain. In some aspects, the second polypeptide construct comprises an FRB domain, and the first polypeptide comprises a FKBP domain. In some aspects, the FRB domain is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 33, or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein), and comprises an amino acid sequence as set forth in301320428.1 - 4 -SEQ ID NO: 34, or an amino acid sequence at least about 90% identical thereto; and the FKBP domain is encoded by a nucleic acid sequence as set forth in SEQ ID NOS: 31, or a sequence at least about 90% identical thereto, and comprises an amino acid sequence as set forth in SEQ ID NO: 32, or an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein).

[0014] In some aspects, the first polypeptide further comprises a split N-intein (Npu DnaE-N) and the second polypeptide further comprises a split C-intein (Npu DnaE-C). In some aspects, the split N-intein is encoded by a polynucleotide comprising a nucleic acid sequence as set forth in SEQ ID NO: 160, or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein), and comprises an amino acid sequence as set forth in SEQ ID NOS: 161, or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein); and the split C-intein is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 162, or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein), and comprises an amino acid sequence as set forth in SEQ ID NOS: 163, or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein). In some aspects, the first and / or the second polypeptide construct further comprises one or more linker sequences. In some aspects, the one or more linker sequences are 2-100 amino acids in length. In some aspects, the linker is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 41, 43, 45, 47, 49, 164 or 166 or a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein), and comprises an amino acid sequence as set forth in SEQ ID NOs: 42, 44, 46, 48, 50, 165, or 167 or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein).

[0015] In some aspects, the first and / or the second polypeptide construct further comprises a fluorophore. In some aspects, the fluorophore comprises Green Fluorescent Protein (GFP), eGFP, Red Fluorescent Protein (RFP), Teal Fluorescent Protein (TFP), Blue Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), miRFP, cerulean fluorescent protein (CFP), eCyanFP, mCherry, mVenus, mOrange, mTurquoise, tdTomato, aminocoumarin, fluorescein, texas red, Alexa Fluor dyes (e.g. Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 350, Alexa Fluor 532, and Alexa Fluor 700), Cy dyes (e.g. Cy3, Cy5), DyLight dyes, FITC, or Rhodamine, or functional variants thereof. In some aspects, the first301320428.1 - 5 -and / or the second polypeptide construct further comprises a purification and / or a solubilization tag. In some aspects, the purification and / or a solubilization tag comprises a maltose binding protein (MBP) tag, a GST-tag, a FLAG tag, an HA tag, a His-tag, a SUMO-tag, a Trx-tag, a Halo-tag ligand, a Halo-tag, or any combination of. In some aspects, the first and / or the second polypeptide construct further comprises a peptide leader sequence.

[0016] In some aspects, the first polypeptide is encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOS: 168, 172, 176, or 180, or a nucleic acid sequence at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein). In some aspects, the second polypeptide is encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOS: 170, 174, 178, 182, or 184, or a nucleic acid sequence at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein). In some aspects, the first polypeptide comprises an amino acid sequence as set for in any one of SEQ ID NOs: 145, 169, 173, 177, or 181, or an amino acid sequence at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein). In some aspects, the second polypeptide comprises an amino acid sequence as set for in any one of SEQ ID NOs: 143, 144, 171, 175, 179, 183, or 185, or an amino acid sequence at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein).

[0017] In some aspects, the present disclosure encompasses a method of determining one or more RNA interaction sites of a RNA-binding Protein (RBP) in a biological sample, comprising incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; incubating the primary complex with one or more secondary binding agents that specifically bind the RBP-targeting agent, to form a secondary complex; incubating the first or the secondary complex with the system described above to form a tertiary complex; adding a transcriptase mix to obtain cDNAs; and sequencing the cDNA to determine the one or more RNA interaction sites of the RBP.301320428.1 - 6 -

[0018] In some aspects, the present disclosure encompasses a method of determining one or more RNA interaction sites of a RNA-binding Protein (RBP) in a biological sample, comprising incubating a RBP-targeting agent with the biological sample, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; incubating the primary complex with one or more secondary binding agents that specifically bind the RBP-targeting agent, to form a secondary complex; incubating the first or the secondary complex with a system comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first or the secondary complex to form a tertiary complex; incubating the tertiary complex with a transcriptase mix and a system comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; and sequencing the cDNA to determine the one or more RNA interaction sites of the RBP. In some aspects, the first fragment of the reverse transcriptase is a N-terminal fragment, or a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof; or the C-terminal fragment, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase or any variant thereof. In some aspects, the second fragment of the reverse transcriptase is a N-terminal fragment, or a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof; or the C-terminal fragment, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase or any variant thereof. In some aspects, the method further comprises adding an inducer of dimerization, wherein the addition of the inducer of dimerization brings the first polypeptide construct and the second polypeptide construct in proximity to form a functional reverse transcriptase. In some aspects, the addition of the inducer of dimerization brings the first polypeptide construct and the second polypeptide construct in proximity allowing the splicing together of the N-terminal fragment of the reverse transcriptase to the C-terminal fragment of the reverse transcriptase, thereby forming a functional reverse transcriptase. In some aspects, the method further comprises one or more wash steps between any of the steps of the method. In some aspects, the first dimerization domain is FKBP, or a variant thereof and the second dimerization domain is FRB, or a variant thereof, or the second dimerization domain is FKBP, or a variant thereof and the first dimerization domain is FRB, or a variant thereof, and wherein the inducer of dimerization is rapamycin. In some aspects, the301320428.1 - 7 -first dimerization domain is a Halo-tag, or a variant thereof, or a Halo-tag ligand or a variant thereof; wherein the second dimerization is a Halo-tag, or a variant thereof, or a Halo-tag ligand or a variant thereof and wherein the binding of the first and the second dimerization domains can be induced by alkyl chloride.

[0019] In some aspects, the transcriptase mix comprises one or more adapter-RT primer, wherein the one or more adapter RT-primer each comprises an adapter primer sequence and an RT primer sequence. In some aspects, at least one of the one or more RT primer sequence is a random RT primer. In some aspects, the random RT primer comprises at least 7 nucleotides. In some aspects, the random RT primer is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more, nucleotides in length. In some aspects, the adapter primer sequence comprises a sequencing barcode. In some aspects, the transcriptase mix further comprises non-labeled dNTPs, labeled dNTPs, or any combination thereof. In some aspects, the labeled dNTPs are biotinylated dNTPs, wherein the biotinylated dNTPs comprises biotin- 16-dUTP, or biotin-16-dCTP, or both. In some aspects, the labeled dNTP and the non-labeled dNTP are at a ratio of at least 0.5:1, 1:1, or 2:1. In some aspects, the RT primer sequence further comprises an azide functional group. In some aspects, the adapter-RT primer comprises a nucleotide sequence as set forth in as set forth in SEQ ID NO: 51, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein).

[0020] In some aspects, the biological sample is a RNA-protein complex, a cell, or a tissue section. In some aspects, the method further comprises one or more of permeabilizing the biological sample, fixing the biological sample with a fixing agent, and quenching the fixing agent. In some aspects, the fixing agent comprises formaldehyde, paraformaldehyde, and / or glutaraldehyde, and the quenching agent comprises glycine. In some aspects, the RBP is a transcription factor, a splicing factor, RNA helicase, ribonuclease, RNA polymerase, translation initiation factor, or ribosomal protein. In some aspects, the RBP comprises YTHDF1, YTHDF2, YTHDC1, HuR, PTB, Musashi, eIF4E, FMRP, LARP1, IMP, hnRNP family proteins, Lin28, AUF1, IGF2BP, FUBP1, LIN28B, RBM5, FUS, TIA1, TTP, QKI, MBNL, CELF, NONO, DDX5, RBM10, SAFB, TDP-43, Ataxin-2, hnRNP A / B, C9orf72, hnRNP H / F, Matrin 3 (MATR3), Pur-alpha, TAF15, Huntingtin, RBFOX, SMN, ELAVL, Ro (SSA) and La (SSB) Proteins, hnRNP, Roquin, Staufenl, NF90 / NF110, ILF3, SF3B1, SRSF2, U2AF1, ZRSR2, PRPF8, PRPF31, SNRNP200, HNRNPA1, HNRNPA2B1, NELFE, CPEB1, SRSF1, NO VAI, NOVA2, G3BP1, PTBP1, RBFOX2, and / or HNRNPC. In some aspects, the RBP-targeting agent and / or the secondary binding agent comprises an antibody or functional301320428.1 - 8 -variant thereof, wherein the antibody or functional variant thereof comprises a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody variant, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, single chain antibody, single domain antibody, nanobodies, diabodies, a bi-specific antibody, a multi-specific antibody, or a DARPin. In some aspects, the RBP-targeting agent and / or the secondary binding agent is labeled, wherein the label comprises a radioisotopes, a hapten, a fluorescent label, a fluorescent polypeptide, a phosphorescent molecule, a chemiluminescent molecule, a chromophore, a luminescent molecule, a photoaffinity molecule, a colored particle, and / or a ligand. In some aspects, the RBP-targeting agent is linked to a functionalized DNA barcode via an amino spacer, optionally wherein the functionalized DNA barcode comprises an alkyne (3'-O-propargyl N 2'-5' linked) functionalized DNA barcode. In some aspects, the alkyne functionalized barcodes comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs: 65-112, or a nucleic acid sequence at least 80% identical thereto. In some aspects, the steps (a) - (c) are conducted in-situ.

[0021] In some aspects, the method further comprises imaging the biological sample after steps (a)-(c). In some aspects, the biological sample comprises less than or equal to 1000, 750, 500, 100, 50, or 20 cells, or wherein the biological sample comprises a single cell, wherein the biological sample comprises less than 5 tissue sections, or wherein the biological sample comprises a single tissue section. In some aspects, the method does not comprise any one or more of ultraviolet cross-linking, immunoprecipitation, use of base editing proteins, dissociating the one or more tissue section into single cells, oligo(dT) primer initiated reverse transcription, Tn5 tagmentation. In some aspects, the method detects transient and / or dynamic RNA-RBP interactions, wherein the transient and / or dynamic RNA-RBP interactions occur on a timescale within 10 minutes. In some aspects, the method can be used to determine one or more interaction sites of the RBP with RNA in the cytoplasm, or nucleus, or both. In some aspects, the method is used to measures relative binding strength of the RBP to the RNA in comparison to one or more other RBPs to the RNA. In some aspects, the cDNA comprises one or more labeled nucleotides, wherein the one or more labeled nucleotides are labeled with a fluorescent label, and / or are biotinylated. In some aspects, the method further comprises purifying the cDNA with a streptavidin comprising agent, wherein the streptavidin comprising agent comprises, a bead, a plate, a magnetic bead, an agarose bead, a microtiter plate, a nanoparticle, and / or a membrane. In some aspects, two or more unique RBP targeting agents that interact with one or more RBPs are used in step (a). In some aspects, each of the two or more unique RBP targeting agents comprise a unique functionalized DNA barcode linked via301320428.1 - 9 -an amino spacer. In some aspects, the functionalized DNA barcode comprises an alkyne (3'-O-propargyl N 2'-5' linked) functionalized DNA barcode. In some aspects, the alkyne functionalized barcodes comprise a nucleic acid sequence as set forth in any one of SEQ ID NO: 65-112, or a sequence at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical thereto (or any percentage derivable therein).

[0022] In some aspects, the present disclosure encompasses a method of in-situ imaging of one or more RNA interaction sites of an RNA-binding Protein (RBP) in a biological sample bound to a solid surface, comprising incubating an RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; incubating the first complex with one or more secondary binding agents that specifically binds the RBP-targeting agent, to form a secondary complex; contacting the first or the secondary complex with the system described above to form a tertiary complex; adding a transcriptase mix to obtain cDNAs; and imaging the solid surface.

[0023] In some aspects, the present disclosure encompasses a method of in-situ imaging of one or more RNA interaction sites of an RNA-binding Protein (RBP) in a biological sample bound to a solid surface, comprising incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; incubating the first complex with one or more secondary binding agents that specifically binds the RBP-targeting agent, to form a secondary complex; incubating the first or the secondary complex with a system comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first or the secondary complex to form a tertiary complex; incubating the tertiary complex with a transcriptase mix and a system comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; and imaging the solid surface. In some aspects, the imaging is done using fluorescence microscopy. In some aspects, the solid surface comprises a slide, a multi-well plate, a capillary, or a microfluidic chamber. In some aspects, the method further comprises sequencing the cDNA. In some aspects, the sequencing is performed using Next Generation Sequencing (NGS) techniques. In some aspects, the sequencing is done using a single cell genomic imaging techniques. In some aspects, the single301320428.1 - 10 -cell genomic imaging technique comprises, consists essentially of, or consists of spatial transcriptomics, MERFISH, SeqFISH, STARmap, Slide-Seq, Visium Spatial Gene Expression, or deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq). In some aspects, the single cell genomic imaging technique is a microfluidic based technique comprising ligating a first set and a second set of spatial barcodes to the cDNA of step (c), prior to step (d), wherein the first set of spatial barcodes are contacted to the cDNA horizontally using a first multi-channel microfluidic chip, and wherein the second set of spatial barcodes are contacted to the solid surface vertically using a second multi-channel microfluidic chip. In some aspects, the first set of spatial barcodes and second set of spatial barcodes form a 2D spatial barcode array.

[0024] In some aspects, the present disclosure encompasses a kit comprising the system described above. In some aspects, the kit further comprises a transcriptase mix as disclosed herein.

[0025] In some aspects, the present disclosure encompasses a method of determining the one or more RBP binding sites on an mRNA throughout an mRNA cycle, the method comprising incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; incubating the first complex with one or more secondary binding agents that specifically binds the RBP-targeting agent, to form a secondary complex; incubating the first or the secondary complex with the system comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a first targeting moiety; wherein the first targeting moiety binds the first or the secondary complex to form a tertiary complex; incubating the tertiary complex with a transcriptase mix and a system comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase, a second dimerization domain and a second targeting moiety, wherein the second dimerization domain binds the first dimerization domain, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA, and wherein the second targeting moiety binds a specific site on the mRNA; and sequencing the cDNA. In some aspects, the second targeting moiety comprises a nucleoside analog. In some aspects, the nucleoside analog is a 5-ethyluridine.

[0026] In some aspects, the present disclosure encompasses a method of determining one or more RNA interaction sites of a first RNA-binding Protein (RBP) and a second RBP, in a biological sample, comprising incubating a first RBP-targeting agent comprising a functionalized first DNA barcode, with the first RBP, wherein the first RBP-targeting agent301320428.1 - 11 -specifically binds the first RBP to form a first complex; incubating a second RBP-targeting agent comprising a functionalized second DNA barcode, with the second RBP, wherein the second RBP-targeting agent specifically binds the second RBP to form a second complex; incubating the first complex and the second complex with a system comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first complex; and a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form a first barcoded cDNA library and a second barcoded cDNA library; amplifying and sequencing the first and the second barcoded cDNA library; and obtaining one or more interaction site of the first RBP and the second RBP by deconvoluting the sequenced cDNA library based on the first and the second DNA barcode. In some aspects, the transcriptase system comprise an RT primer sequence comprising a functional group and biotinylated dNTPs. In some aspects, the functional group is an azide functional group. In some aspects, the biotinylated dNTPs, and the RT primer sequence comprising the azide functional group, are incorporated into the cDNA to form proximal azide labeled biotinylated cDNAs during reverse transcription in step c. In some aspects, the functionalized DNA barcode comprises an alkyne (3'-O-propargyl N 2'-5' linked) functionalized DNA barcode. In some aspects, the alkyne functionalized barcodes comprise a nucleic acid sequence as set forth in any one of SEQ ID NO: 65-112, or a sequence at least 80% identical thereto. In some aspects, the method further comprises incorporating the alkyne functionalized first and / or second DNA barcode into the cDNA by reacting the alkyne functionalized first DNA barcode and / or second DNA barcode with the proximal azide labeled biotinylated cDNA using in-situ copper catalyzed azide-alkyne cycloaddition (CuAAC), to obtain a first barcoded biotinylated cDNA library and / or the second biotinylated cDNA library. In some aspects, the method further comprises purifying the first and / or the second barcoded biotinylated cDNA library over a streptavidin column prior to amplifying and sequencing. In some aspects, the method further comprises processing the CuAAC using a Klenow Fragment DNA polymerase for second strand synthesis. In some aspects, the one or more interaction sites of the first RBP and the second RBP are obtained by deconvoluting the sequenced data based on the first and the second DNA barcodes incorporated into the cDNA.

[0027] In some aspects, the present disclosure encompasses a method of determining spatial distribution of a RNA modification sites on a biological sample bound to a solid surface,301320428.1 - 12 -comprising incubating a modification-targeting agent that specifically binds the modification site on the RNA to form a primary complex; incubating the primary complex with a secondary binding agent that specifically bind the primary complex to form a secondary complex; incubating the first or the secondary complex with a system comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a first targeting moiety; wherein the first targeting moiety binds the first or the secondary complex to form a tertiary complex; incubating the tertiary complex with a transcriptase mix and a system comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase, a second dimerization domain and optionally a second targeting moiety, wherein the second dimerization domain binds the first dimerization domain, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; optionally incorporating labelled barcodes into the cDNA; and sequencing and imaging the biological sample using a single cell genomic imaging technique to determine the one or more modification sites.

[0028] Also disclosed herein are the following enumerated Aspects 1-188.

[0029] Aspect 1 is a polypeptide construct comprising: a) a fragment of a reverse transcriptase enzyme, or a variant thereof; and b) a dimerization moiety.

[0030] Aspect 2 is the polypeptide construct of aspect 1, further comprising a targeting moiety.

[0031] Aspect 3 is the polypeptide construct of aspect 1 or 2, wherein the fragment of the reverse transcriptase enzyme is a N-terminal fragment, or a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof.

[0032] Aspect 4 is the polypeptide construct of aspect 1 or 2, wherein the fragment of the reverse transcriptase enzyme is a C-terminal fragment, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase, or any variant thereof.

[0033] Aspect 5 is the polypeptide construct of any one of aspects 1-4, wherein the fragment of the reverse transcriptase enzyme comprises an amino acid sequence with, with at least, or with at most 50%, 60%, 70%, 80%, 90%, 100% amino acid identity to the overlapping fragment of the reverse transcriptase.

[0034] Aspect 6 is the polypeptide construct of any one of aspects 1-5, wherein the fragment of the reverse transcriptase enzyme is, is less than, or is about 5%, 10%, 20%, 25%, 30%, 35%, 40% functional as compared to the wild-type RT enzyme, as measured using a reverse transcriptase enzyme assay.301320428.1 - 13-

[0035] Aspect 7 is the polypeptide construct of any one of aspects 1-6, wherein the reverse transcriptase enzyme comprises Moloney murine leukemia virus (MMLV) RTase, human immunodeficiency virus (HIV) RTase, Avian Myeloblastosis Virus (AMV) RTase or a variant thereof.

[0036] Aspect 8 is the polypeptide construct of aspect 7, wherein the reverse transcriptase enzyme comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 6, or 147 or an amino acid sequence at least 60% identical thereto, or a variant thereof.

[0037] Aspect 9 is the polypeptide construct of any one of aspects 7-8, wherein the fragment of the reverse transcriptase enzyme is encoded by a polynucleotide sequence comprising a nucleic acid sequence as set forth in any one of SEQ ID NOs: 7-30 or a nucleic acid sequence at least 60% identical thereto, or a variant thereof.

[0038] Aspect 10 is the polypeptide construct of any one of aspects 7-9, wherein the fragment of the reverse transcriptase enzyme comprises an amino acid sequence comprising as set forth in any one of SEQ ID NOs: 117-142 or an amino acid sequence at least 60% identical thereto, or a variant thereof.

[0039] Aspect 11 is the polypeptide construct of any one of aspects 3-10, wherein the targeting moiety is a Fc binding protein, or a functional fragment or variant thereof; an antibody, or a functional fragment or variant thereof; an oligonucleotide, a nucleoside analog, a nucleotide, a receptor, a ligand, a small molecule, or any combination thereof.

[0040] Aspect 12 is the polypeptide construct of aspect 11, wherein the targeting moiety comprises a Fc binding protein or a variant thereof.

[0041] Aspect 13 is the polypeptide construct of aspect 11, wherein the targeting moiety comprises an antibody or variant thereof.

[0042] Aspect 14 is the polypeptide construct of aspect 11, wherein the targeting moiety comprises an oligonucleotide or a variant thereof.

[0043] Aspect 15 is the polypeptide construct of aspect 14, wherein the oligonucleotide comprises a barcode, indices, affinity tag, label, a modified nucleotide, or any combination thereof.

[0044] Aspect 16 is the polypeptide construct of aspect 15, wherein the affinity tag comprises a streptavidin, or an avidin tag.

[0045] Aspect 17 is the polypeptide construct of aspect 11, wherein the targeting moiety comprises a small molecule.301320428.1 - 14-

[0046] Aspect 18 is the polypeptide construct of aspect 11, wherein the Fc binding protein comprises protein A, protein G, protein A / G (pAG), protein L, anti-rabbit IgG, anti-mouse IgG, or a variant thereof, or any combination thereof.

[0047] Aspect 19 is the polypeptide construct of aspect 16, wherein the Fc binding protein comprises pAG.

[0048] Aspect 20 is the polypeptide construct of any one of aspects 1-19, wherein the dimerization domain comprises, consists essentially of, or consists of a rapamycin mediated dimerization domain (FKBP and FRB), a leucine zipper, an SH3 domain, a PDZ domain, an Fc domain, a basic helix-loop-helix domain (bHLH), a zinc finger domain, a tet repressor dimerization domain, a Halo-tag ligand, a Halo-tag ligand, a Halo-tag, or any combination of, or any combination of.

[0049] Aspect 21 is the polypeptide construct of any one of aspects 1-20, wherein the dimerization domain comprises, consists essentially of, or consists of a rapamycin mediated dimerization domain.

[0050] Aspect 22 is the polypeptide construct of aspect 21, wherein the fragment of the reverse transcriptase enzyme comprises the N-terminal fragment of the reverse transcriptase enzyme, or a fragment starting at least within the first 20 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof, and the dimerization moiety is a FRB domain, or alternatively, a FKBP domain.

[0051] Aspect 23 is the polypeptide construct of aspect 22, wherein the fragment of the reverse transcriptase enzyme, or a variant thereof, comprises the C-terminal fragment of the reverse transcriptase enzyme, or a variant thereof, and the dimerization moiety is a FRB domain, or alternatively, a FKBP domain.

[0052] Aspect 24 is the polypeptide construct of aspect 20, wherein the dimerization domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 32 or 34, or a sequence at least 80% identical thereto.

[0053] Aspect 25 is the polypeptide construct of any one of aspects 1-24, further comprising one or more linker sequences.

[0054] Aspect 26 is the polypeptide construct of any one of aspects 1-25, wherein the one or more linker sequences are at least 2-100 amino acids in length, optionally 2-10 amino acids, or 11-20 amino acids, or 21-30 amino acids, 31-40 amino acids, or 41-50 amino acids, or 51-60 amino acids, 61-70 amino acids, or 71-80 amino acids, or 81-90 amino acids, 91-100 amino acids in length.301320428.1 - 15-

[0055] Aspect 27 is the polypeptide construct of aspect 20, wherein the linker comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 42, 44, 46, 48 and 50, or a sequence at least 80% identical thereto.

[0056] Aspect 28 is the polypeptide construct of any one of aspects 1-27, further comprising a fluorophore.

[0057] Aspect 29 is the polypeptide construct of aspect 28, wherein the fluorophore comprises Green Fluorescent Protein (GFP), eGFP, Red Fluorescent Protein (RFP), Teal Fluorescent Protein (TFP), Blue Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), miRFP, cerulean fluorescent protein (CFP), eCyanFP, mCherry, mVenus, mOrange, mTurquoise, tdTomato, aminocoumarin, fluorescein, texas red, Alexa Fluor dyes (e.g. Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 350, Alexa Fluor 532, and Alexa Fluor 700), Cy dyes (e.g. Cy3, Cy5), DyLight dyes, FITC, or Rhodamine, or functional variants thereof.

[0058] Aspect 30 is the polypeptide construct of any one of aspects 1-29, further comprising a purification and / or a solubilization tag.

[0059] Aspect 31 is the polypeptide construct of aspect 30, wherein the purification and / or a solubilization tag comprises a maltose binding protein (MBP) tag, a GST-tag, a FLAG tag, an HA tag, a His-tag, a SUMO-tag, a Trx-tag, a Halo-tag ligand, a Halo-tag, or any combination of, or any combination thereof.

[0060] Aspect 32 is the polypeptide construct of aspect 31, wherein the purification and or a solubilization tag comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 36, 38, and 40 or a sequence at least 80% identical thereto.

[0061] Aspect 33 is the polypeptide construct of any one of aspects 1-32, further comprising a peptide leader sequence.

[0062] Aspect 34 is a composition comprising: a) a first polypeptide construct, wherein the first polypeptide construct comprises a N-terminal fragment of a reverse transcriptase, or a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof, a first dimerization moiety; and / or b) a second polypeptide construct, wherein the second polypeptide construct comprises a C-terminal fragment of the reverse transcriptase, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase, or any variant thereof, a second dimerization moiety that specifically binds to the first dimerization moiety.

[0063] Aspect 35 is the composition of aspect 34, wherein the first polypeptide construct comprises a targeting moiety.301320428.1 - 16-

[0064] Aspect 36 is the composition of aspect 34 or 35, wherein the second polypeptide construct comprises a targeting moiety.

[0065] Aspect 37 is the composition of any one of aspects 35-36, wherein the first and the second targeting moiety are the same or are different targeting moieties.

[0066] Aspect 38 is the composition of any one of aspects 34-37, wherein the composition further comprises one or more stabilizing agents comprising glycerol, trehalose, sucrose, polyethylene glycol (PEG), bovine serum albumin (BSA), ammonium sulfate, dithiothreitol (DTT), reducing agents, EDTA, magnesium ions, potassium chloride, sodium azide, buffering agents and cryoprotectants.

[0067] Aspect 39 is the composition of any one of aspects 34-38, wherein the reverse transcriptase comprises Moloney murine leukemia virus (MMLV) RTase, human immunodeficiency virus (HIV) RTase, Avian Myeloblastosis Virus (AMV) RTase or a variant thereof.

[0068] Aspect 40 is the composition of aspect 39, wherein the N-terminal fragment of the reverse transcriptase enzyme is encoded by a polynucleotide sequence comprising a nucleic acid sequence as set forth is any one of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29 or an amino acid sequence at least 60% identical thereto, or a variant thereof.

[0069] Aspect 41 is the composition of aspect 40, wherein the C-terminal fragment of the reverse transcriptase enzyme is encoded by a polynucleotide sequence comprising a nucleic acid sequence as set forth is any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30, or an amino acid sequence at least 60% identical thereto, or a variant thereof.

[0070] Aspect 42 is the composition of aspect 40, wherein the fragment of the RTase is an N-terminal fragment comprising an amino acid sequence as set for in any one of SEQ ID NOs: 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139 or 141 or a sequence at least 60% identical thereto.

[0071] Aspect 43 is the composition of aspect 41, the fragment of the RTase is an C-terminal fragment comprising an amino acid sequence as set for in any one of SEQ ID NOs: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, or 142 or a sequence at least 60% identical thereto.

[0072] Aspect 44 is the composition of any one of aspects 35-41, wherein the first and / or the second targeting moiety is a Fc binding protein, or a functional fragment or variant thereof; an antibody, or a functional fragment or variant thereof; an oligonucleotide, a nucleoside analog, a nucleotide, a receptor, a ligand, a small molecule, or any combination thereof.301320428.1 - 17 -

[0073] Aspect 45 is the composition of aspect 44, wherein the first and / or the second targeting moiety comprises a Fc binding protein or a variant thereof.

[0074] Aspect 46 is the composition of aspect 44, wherein the first and / or the second targeting moiety comprises an antibody or variant thereof.

[0075] Aspect 47 is the composition of aspect 44, wherein the first and / or the second targeting moiety comprises an oligonucleotide or a variant thereof.

[0076] Aspect 48 is the composition of aspect 47, wherein the oligonucleotide comprises a barcode, indices, affinity tag, label, a modified nucleotide, or any combination thereof.

[0077] Aspect 49 is the composition of aspect 48, wherein the affinity tag comprises a streptavidin, or an avidin tag.

[0078] Aspect 50 is the composition of aspect 44, wherein the first and / or the second targeting moiety comprises a small molecule.

[0079] Aspect 51 is the composition of aspect 44, wherein the Fc binding protein comprises protein A, protein G, protein A / G (pAG), protein L, anti-rabbit IgG, anti-mouse IgG, or a variant thereof, or any combination thereof.

[0080] Aspect 52 is the composition of aspect 49, wherein the Fc binding protein comprises pAG.

[0081] Aspect 53 is the composition of any one of aspects 34-52, wherein the first and the second dimerization moieties comprise binding regions of a rapamycin mediated dimerization domain (FKBP and FRB), a leucine zipper, an SH3 domain, a PDZ domain, an Fc domain, an Fc binding domain, a basic helix-loop-helix domain (bHLH), a zinc finger domain, a tet repressor dimerization domain, or a Halo-tag ligand, a Halo-tag, or any combination of.

[0082] Aspect 54 is the composition of any one of aspects 34-53, wherein the first and the second dimerization domain comprises, consists essentially of, or consists of a rapamycin mediated dimerization domain.

[0083] Aspect 55 is the composition of any one of aspects 34-54, wherein the first polypeptide construct comprises an FRB domain, or alternatively, a FKBP domain.

[0084] Aspect 56 is the composition of aspect 34-54, wherein the second polypeptide construct comprises an FRB domain, or alternatively, a FKBP domain.

[0085] Aspect 57 is the composition of any one of aspects 34-56, wherein the first and / or the second polypeptide construct further comprises one or more linker sequences.

[0086] Aspect 58 is the composition of aspect 57, wherein the one or more linker sequences are at least 2-50 amino acids in length.301320428.1 - 18-

[0087] Aspect 59 is the composition of aspect 58, wherein the one or more linker sequences are 2-100 amino acids in length, optionally 2-10 amino acids, or 11-20 amino acids, or 21-30 amino acids, 31-40 amino acids, or 41-50 amino acids, or 51-60 amino acids, 61-70 amino acids, or 71-80 amino acids, or 81-90 amino acids, 91-100 amino acids in length.

[0088] Aspect 60 is the composition of aspect 58, wherein the linker comprises an amino acid sequence as set forth in SEQ ID NOs: 42, 44, 46, 48 and 50, or a sequence at least 80% identical thereto.

[0089] Aspect 61 is the composition of any one of aspects 34-60, wherein the first and / or the second polypeptide construct further comprises a fluorophore.

[0090] Aspect 62 is the composition of aspect 61, wherein the fluorophore comprises Green Fluorescent Protein (GFP), eGFP, Red Fluorescent Protein (RFP), Teal Fluorescent Protein (TFP), Blue Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), miRFP, cerulean fluorescent protein (CFP), eCyanFP, mCherry, mVenus, mOrange, mTurquoise, tdTomato, aminocoumarin, fluorescein, texas red, Alexa Fluor dyes (e.g. Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 350, Alexa Fluor 532, and Alexa Fluor 700), Cy dyes (e.g. Cy3, Cy5), DyLight dyes, FITC, or Rhodamine, or functional variants thereof.

[0091] Aspect 63 is the composition of any one of aspects 34-62, wherein the first and / or the second polypeptide construct further comprises a purification and / or a solubilization tag.

[0092] Aspect 64 is the composition of aspect 63, wherein the purification and / or a solubilization tag comprises a maltose binding protein (MBP) tag, a GST-tag, a FLAG tag, an HA tag, a His-tag, a SUMO-tag, a Trx-tag, a Halo-tag ligand, a Halo-tag, or any combination of.

[0093] Aspect 65 is the composition of aspect 64, wherein the purification and or a solubilization tag comprises an amino acid sequence as set forth in SEQ ID NOs: 36, 38, and 40, or a sequence at least 80% identical thereto.

[0094] Aspect 66 is the composition of any one of aspects 34-65, wherein the first and / or the second polypeptide construct further comprises a peptide leader sequence.

[0095] Aspect 67 is the composition of any one of aspects 35-66, wherein the first and the second targeting moieties are the same, or different.

[0096] Aspect 68 is a method of determining one or more RNA interaction sites of a RNA-binding Protein (RBP) in a biological sample, comprising: a) incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; b) incubating the first complex with one or more secondary binding agents that301320428.1 - 19-specifically bind the RBP-targeting agent, to form a secondary complex; c) incubating the first or the secondary complex with the composition of any one of aspects 34-67, and a transcriptase mix to obtain cDNA; and d) sequencing the cDNA to determine the one or more RNA interaction sites of the RBP.

[0097] Aspect 69 is the method of aspect 68, wherein step (c) further comprises adding an inducer of dimerization of the first polypeptide construct and the second polypeptide construct.

[0098] Aspect 70 is the method of aspect 69, wherein the inducer of dimerization is rapamycin.

[0099] Aspect 71 is the method of aspect 68, the composition comprises the first polypeptide construct of aspect 34; and wherein step (c) further comprises one or more wash steps prior to incubation with a second composition of any one of aspects 34-67, wherein the second composition comprises the second polypeptide construct of aspect 34.

[0100] Aspect 72 is the method of aspect 68, the composition comprises the second polypeptide construct of aspect 34; and wherein step (c) further comprises one or more wash steps prior to incubation with a second composition of any one of aspects 34-67, wherein the second composition comprises the first polypeptide construct of aspect 34.

[0101] Aspect 73 is a method of determining one or more RNA interaction sites of a RNA-binding Protein (RBP) in a biological sample, comprising: a) incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; b) incubating the first complex with one or more secondary binding agents that specifically bind the RBP-targeting agent, to form a secondary complex; c) incubating the first or the secondary complex with a composition comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first or the secondary complex to form a tertiary complex; d) incubating the tertiary complex with a transcriptase mix and a composition comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; and e) sequencing the cDNA to determine the one or more RNA interaction sites of the RBP.

[0102] Aspect 74 is the method of aspect 73, wherein the first fragment of the reverse transcriptase is a N-terminal fragment, or a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof; or the C-terminal301320428.1 - 20 -fragment, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase or any variant thereof.

[0103] Aspect 75 is the method of aspect 73, wherein the second fragment of the reverse transcriptase is a N-terminal fragment, or a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof; or the C-terminal fragment, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase or any variant thereof.

[0104] Aspect 76 is the method of any one of aspects 73-75, wherein the first dimerization domain is FKBP, or a variant thereof, or FRB or a variant thereof.

[0105] Aspect 77 is the method of any one of aspects 73-75, wherein the second dimerization domain is FKBP, or a variant thereof, or FRB or a variant thereof.

[0106] Aspect 78 is the method of any one of aspects 73, 76-77, wherein the inducer of dimerization is rapamycin.

[0107] Aspect 79 is the method of aspect 73, wherein the transcriptase mix comprises one or more adapter-RT primer, wherein the one or more adapter RT -primer each comprises an adapter primer sequence and an RT primer sequence.

[0108] Aspect 80 is the method of aspect 79, wherein at least one of the one or more RT primer sequence is a random RT primer.

[0109] Aspect 81 is the method of aspect 80, wherein the random RT primer comprises at least 7 nucleotides.

[0110] Aspect 82 is the method of aspect 81, wherein the random RT primer is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more, nucleotides in length.

[0111] Aspect 83 is the method of aspect 79, wherein the adapter primer sequence comprises a sequencing barcode.

[0112] Aspect 84 is the method of any one of aspects 77-83, wherein the transcriptase mix further comprises non-labeled dNTPs, labeled dNTPs, or any combination thereof.

[0113] Aspect 85 is the method of aspect 84, wherein the labeled dNTPs are biotinylated dNTPs, optionally wherein the biotinylated dNTPs comprises biotin- 16-dUTP, or biotin-16-dCTP, or both.

[0114] Aspect 86 is the method of aspect 84, wherein the labeled dNTP and the non-labeled dNTP are at a ratio of at least 0.5:1, 1:1, or 2:1.

[0115] Aspect 87 is the method of any one of aspects 77-86, wherein the RT sequence primer further comprises an azide functional group.301320428.1 - 21 -

[0116] Aspect 88 is the method of any one of aspects 77-86, wherein the adapter-RT primer comprises a nucleotide sequence as set forth in as set forth in SEQ ID NO: 51, or a sequence at least 80% identical thereto.

[0117] Aspect 89 is the method of any one of aspects 73-88, wherein the biological sample is a RNA-protein complex, a cell, or a tissue section.

[0118] Aspect 90 is the method of aspect 89, further comprising fixing the biological sample with a fixing agent.

[0119] Aspect 91 is the method of aspect 90, wherein the fixing agent comprises formaldehyde, paraformaldehyde, and / or glutaraldehyde.

[0120] Aspect 92 is the method of aspect 91, wherein the fixing agent is paraformaldehyde at a concentration of about 0.1% to about 5% by volume, or at a concentration of, of at least, of about, orofmorethan0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, I.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5% by volume.

[0121] Aspect 93 is the method of aspect 90, wherein the fixing comprises incubating the biological sample and the fixing agent together for, or for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, II, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes.

[0122] Aspect 94 is the method of aspect 90, further comprising quenching of the fixing agent with a quenching agent.

[0123] Aspect 95 is the method of aspect 94, wherein the quenching agent comprises glycine.

[0124] Aspect 96 is the method of aspect 94 or aspect 95, wherein the quenching agent is at a concentration of, of greater than, of at least, of at most, or of about 25, 50, 75, 100, 125, 150, 200, 225, or250 mM.

[0125] Aspect 97 is the method of aspect 89, wherein the biological sample comprises cell and / or tissue, the method further comprising permeabilizing the cell and / or the tissue section with a permeabilizing agent.

[0126] Aspect 98 is the method of aspect 97, wherein the permeabilizing agent comprises a detergent.

[0127] Aspect 99 is the method of aspect 97, wherein the detergent comprises Triton X-100, optionally wherein the Triton-X is at a concentration of, of greater than, of at least, of at most, or of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%.301320428.1 - 22 -

[0128] Aspect 100 is the method of any one of aspects 73-99, further comprising incubating the primary and / or the secondary complex with an RNase enzyme.

[0129] Aspect 101 is the method of any one of aspects 73-100, wherein the RBP is a transcription factor, a splicing factor, RNA helicase, ribonuclease, RNA polymerase, translation initiation factor, or ribosomal protein.

[0130] Aspect 102 is the method of aspect 101, wherein the RBP comprises YTHDF1, YTHDF2, YTHDC1, HuR, PTB, Musashi, eIF4E, FMRP, LARP1, IMP, hnRNP family proteins, Lin28, AUF1, IGF2BP, FUBP1, LIN28B, RBM5, FUS, TIA1, TTP, QKI, MBNL, CELF, NONO, DDX5, RBM10, SAFB, TDP-43, Ataxin-2, hnRNP A / B, C9orf72, hnRNP H / F, Matrin 3 (MATR3), Pur-alpha, TAF15, Huntingtin, RBFOX, SMN, ELAVL, Ro (SSA) and La (SSB) Proteins, hnRNP, Roquin, Staufenl, NF90 / NF110, ILF3, SF3B1, SRSF2, U2AF1, ZRSR2, PRPF8, PRPF31, SNRNP200, HNRNPA1, HNRNPA2B1, NELFE, CPEB1, SRSF1, NO VAI, NOVA2, G3BP1, PTBP1, RBFOX2, and / or HNRNPC.

[0131] Aspect 103 is the method of any one of aspects 73-102, wherein the RBP-targeting agent specifically binds the RBP, optionally wherein the RBP-targeting agent comprises an antibody or functional variant thereof, optionally wherein the antibody or functional variant thereof comprises a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody variant, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, single chain antibody, single domain antibody, nanobodies, diabodies, a bi-specific antibody, a multispecific antibody, a DARPin, or a variant of each thereof.

[0132] Aspect 104 is the method of any one of aspects 73-103, wherein the secondary binding agent comprises an antibody or functional variant thereof, optionally wherein the antibody or functional variant thereof comprises a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody variant, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, single chain antibody, single domain antibody, nanobodies, diabodies, a bi-specific antibody, a multi-specific antibody, a DARPin, or a variant of each thereof.

[0133] Aspect 105 is the method of any one of aspects 73-104, wherein the RBP-targeting agent is labeled, optionally wherein the label comprises a radioisotopes, a hapten, a fluorescent label, a fluorescent polypeptide, a phosphorescent molecule, a chemiluminescent molecule, a chromophore, a luminescent molecule, a photoaffinity molecule, a colored particle, and / or a ligand.301320428.1 - 23 -

[0134] Aspect 106 is the method of any one of aspects 73-104, wherein the RBP-targeting agent is linked to a functionalized DNA barcode via an amino spacer, optionally wherein the functionalized DNA barcode comprises a alkyne (3'-O-propargyl N 2'-5' linked) functionalized DNA barcode.

[0135] Aspect 107 is the method of aspect 106, wherein the alkyne functionalized barcodes comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs: 65-112, or a nucleic acid sequence at least 80% identical thereto.

[0136] Aspect 108 is the method of any one of aspects 73-107, wherein the secondary binding agent is labeled, optionally wherein the label comprises a radioisotopes, a hapten, a fluorescent label, a fluorescent polypeptide, a phosphorescent molecule, a chemiluminescent molecule, a chromophore, a luminescent molecule, a photoaffinity molecule, a colored particle, and / or a ligand.

[0137] Aspect 109 is the method of aspect 108, wherein the fluorescent label comprises Green Fluorescent Protein (GFP), eGFP, Red Fluorescent Protein (RFP), Teal Fluorescent Protein (TFP), Blue Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), miRFP, cerulean fluorescent protein (CFP), eCyanFP, mCherry, mVenus, mOrange, mTurquoise, tdTomato, aminocoumarin, fluorescein, texas red, Alexa Fluor dyes (e.g. Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 350, Alexa Fluor 532, and Alexa Fluor 700), Cy dyes (e.g. Cy3, Cy5), DyLight dyes, FITC, or Rhodamine, or functional variants thereof.

[0138] Aspect 110 is the method of aspect 73, wherein the steps (a) - (c) are conducted in-si tu.

[0139] Aspect 111 is the method of aspect 73, wherein the method further comprises imaging the biological sample.

[0140] Aspect 112 is the method of any one of aspects 73-111, wherein the biological sample comprises less than or equal to 1000, 750, 500, 100, 50, or 20 cells, or wherein the biological sample comprises a single cell, wherein the biological sample comprises less than 5 tissue sections, or wherein the biological sample comprises a single tissue section.

[0141] Aspect 113 is the method of any one of aspects 73-112, wherein the method does not comprise ultraviolet cross-linking.

[0142] Aspect 114 is the method any one of aspects 73-113, wherein the method does not comprise immunoprecipitation.

[0143] Aspect 115 is the method of any one of aspects 73-114, wherein the method does not comprise use of base editing proteins.301320428.1 - 24 -

[0144] Aspect 116 is the method any one of aspects 73-115, wherein the method does not comprise dissociating the one or more tissue section into single cells.

[0145] Aspect 117 is the method of any one of aspects 73-116, wherein the method detects transient and / or dynamic RNA-RBP interactions, optionally wherein the transient and / or dynamic RNA-RBP interactions occur on a timescale within 10 minutes.

[0146] Aspect 118 is the method of any one of aspects 73-117, wherein the method does not comprise oligo(dT) primer initiated reverse transcription.

[0147] Aspect 119 is the method of any one of aspects 73-118, wherein the method does not comprise Tn5 tagmentation.

[0148] Aspect 120 is the method of aspect 101, wherein the RBP is a splicing factor.

[0149] Aspect 121 is the method of aspect 120, wherein the method is used to determine splice variants between one or more biological samples.

[0150] Aspect 122 is the method of aspects 101, wherein the RBP is a YTH family reader protein, or wherein the RBP is G3BP1.

[0151] Aspect 123 is the method of any one of aspects 73-122, wherein the method can be used to determine one or more interaction sites of the RBP with RNA in the cytoplasm, or nucleus, or both.

[0152] Aspect 124 is the method of any one of aspects 73-123, wherein the method is used to measures relative binding strength of the RBP to the RNA in comparison to one or more other RBPs to the RNA.

[0153] Aspect 125 is the method of any one of aspects 73-124, wherein the cDNA is labeled.

[0154] Aspect 126 is the method of aspect 125, wherein the cDNA comprises one or more labeled nucleotides.

[0155] Aspect 127 is the method of aspect 126, wherein the nucleotides are labeled with a fluorescent label.

[0156] Aspect 128 is the method of aspect 126, wherein the labeled nucleotides comprises a biotinylated nucleotide.

[0157] Aspect 129 is the method of aspect 128, wherein the method further comprises purifying the cDNA with a streptavidin comprising agent, optionally wherein the streptavidin comprising agent comprises, a bead, a plate, a magnetic bead, an agarose bead, a microtiter plate, a nanoparticle, and / or a membrane.

[0158] Aspect 130 is the method of aspect 73, wherein two or more unique RBP targeting agents that interact with one or more RBPs are used in step (a).301320428.1 - 25 -

[0159] Aspect 131 is the method of aspect 130, wherein each of the two or more unique RBP targeting agents comprise a unique functionalized DNA barcode linked via an amino spacer.

[0160] Aspect 132 is the method of aspect 131, wherein the functionalized DNA barcode comprises an alkyne (3'-O-propargyl N 2'-5' linked) functionalized DNA barcode.

[0161] Aspect 133 is the method of aspect 132, wherein the alkyne functionalized barcodes comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs: 65-112, or a sequence at least 80% identical thereto.

[0162] Aspect 134 is a method of in-situ imaging of one or more RNA interaction sites of an RNA-binding Protein (RBP) in a biological sample bound to a solid surface, comprising: a) incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; b) incubating the first complex with one or more secondary binding agents that specifically binds the RBP-targeting agent, to form a secondary complex; c) incubating the first or the secondary complex with the composition of any one of aspects 34-66, and a transcriptase mix to obtain cDNA; and d) imaging the solid surface.

[0163] Aspect 135 is a method of in-situ imaging of one or more RNA interaction sites of an RNA-binding Protein (RBP) in a biological sample bound to a solid surface, comprising: a) incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; b) incubating the first complex with one or more secondary binding agents that specifically binds the RBP-targeting agent, to form a secondary complex; c) incubating the first or the secondary complex with a composition comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first or the secondary complex to form a tertiary complex; d) incubating the tertiary complex with a transcriptase mix and a composition comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; and e) imaging the solid surface.

[0164] Aspect 136 is the method of aspect 135, wherein the first fragment of the reverse transcriptase is a N-terminal fragment, a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof, or the C-terminal fragment, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase or any variant thereof.301320428.1 - 26 -

[0165] Aspect 137 is the method of aspect 135, wherein the second fragment of the reverse transcriptase is a N-terminal fragment, a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof, or the C-terminal fragment, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase or any variant thereof.

[0166] Aspect 138 is the method of any one of aspects 135-137, wherein the first dimerization domain is FKBP, or a variant thereof, or FRB or a variant thereof; and wherein the second dimerization domain is FKBP, or a variant thereof, or FRB or a variant thereof.

[0167] Aspect 139 is the method of any one of aspects 135-138, wherein the inducer of dimerization is rapamycin.

[0168] Aspect 140 is the method of any one of aspects 135-140, wherein the RBP-targeting agent or the one or more secondary binding agents or any combination thereof are labeled, optionally wherein the label comprises radioisotopes, a hapten, a fluorescent label, a fluorescent polypeptide, a phosphorescent molecule, a chemiluminescent molecule, a chromophore, a luminescent molecule, a photoaffinity molecule, a colored particle and / or a ligand.

[0169] Aspect 141 is the method of aspect 140, wherein the fluorescent label comprises Green Fluorescent Protein (GFP), eGFP, Red Fluorescent Protein (RFP), Teal Fluorescent Protein (TFP), Blue Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), miRFP, cerulean fluorescent protein (CFP), eCyanFP, mCherry, mVenus, mOrange, mTurquoise, tdTomato, aminocoumarin, fluorescein, texas red, Alexa Fluor dyes (e.g. Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 350, Alexa Fluor 532, and Alexa Fluor 700), Cy dyes (e.g. Cy3, Cy5), DyLight dyes, FITC, or Rhodamine, or functional variants thereof.

[0170] Aspect 142 is the method of any one of aspects 135-141, wherein the cDNA is labeled.

[0171] Aspect 143 is the method of aspect 142, wherein the cDNA comprises one or more labeled nucleotides optionally wherein the nucleotides are labeled with a fluorescent label and / or are biotinylated.

[0172] Aspect 144 is the method of any one of aspects 135-143, wherein the imaging is done using fluorescence microscopy.

[0173] Aspect 145 is the method of any one of aspects 135-144, wherein the biological sample is a RNA-protein complex, a cell, or a tissue section.301320428.1 - 27 -

[0174] Aspect 146 is the method of any one of aspects 135-145, further comprising fixing the biological sample with a fixing agent.

[0175] Aspect 147 is the method of aspect 146, wherein the fixing agent comprises formaldehyde, paraformaldehyde, and / or glutaraldehyde.

[0176] Aspect 148 is the method of aspect 147, wherein the fixing agent is paraformaldehyde at a concentration of about 0.5% to about 5% by volume or wherein the paraformaldehyde at a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5% by volume.

[0177] Aspect 149 is the method of any one of aspects 146-148, wherein the wherein the fixing comprises incubating the biological sample and the fixing agent for, or for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes.

[0178] Aspect 150 is the method of aspect 149, further comprising quenching of the fixing agent with a quenching agent, optionally wherein the quenching agent comprises glycine.

[0179] Aspect 151 is the method of aspect 150, wherein the quenching agent is a concentration of, of greater than, of at least, of at most, or of about 25, 50, 75, 100, 125, 150, 200, 225, or 250 mM.

[0180] Aspect 152 is the method of any one of aspects 135-151, further comprising permeabilizing the cell and / or the tissue section with a permeabilizing agent.

[0181] Aspect 153 is the method of aspect 152, wherein the permeabilizing agent comprises a detergent.

[0182] Aspect 154 is the method of aspect 153, wherein the detergent comprises Triton X-100, optionally wherein the Triton-X is at a concentration of, of greater than, of at least, of at most, or of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%.

[0183] Aspect 155 is the method of aspect 135, wherein the transcriptase mix further comprises an RNase.

[0184] Aspect 156 is the method of any one of aspects 135-155, wherein the RNA binding protein is a transcription factor, a splicing factor, RNA helicase, ribonuclease, RNA polymerase, translation initiation factor, or ribosomal protein.

[0185] Aspect 157 is the method of aspect 156, wherein the RBP comprises YTHDF1, YTHDF2, YTHDC1, HuR, PTB, Musashi, eIF4E, FMRP, LARP1, IMP, hnRNP family proteins, Lin28, AUF1, IGF2BP, FUBP1, LIN28B, RBM5, FUS, TIA1, TTP, QKI, MBNL, CELF, NONO, DDX5, RBM10, SAFB, TDP-43, Ataxin-2, hnRNP A / B, C9orf72, hnRNP H / F,301320428.1 - 28 -Matrin 3 (MATR3), Pur-alpha, TAF15, Huntingtin, RBFOX, SMN, ELAVL, Ro (SSA) and La (SSB) Proteins, hnRNP, Roquin, Staufenl, NF90 / NF110, ILF3, SF3B1, SRSF2, U2AF1, ZRSR2, PRPF8, PRPF31, SNRNP200, HNRNPA1, HNRNPA2B1, NELFE, CPEB1, SRSF1, NO VAI, NOVA2, G3BP1, PTBP1, RBFOX2, and / or HNRNPC.

[0186] Aspect 158 is the method of any one of aspects 135-157, wherein the RBP-targeting agent specifically binds the RBP.

[0187] Aspect 159 is the method of aspect 158, wherein the RBP-targeting agent is an antibody or a functional variant thereof, optionally wherein the antibody or the functional variant thereof comprises a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody variant, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, single chain antibody, single domain antibody, nanobodies, diabodies, a bi-specific antibody, a multispecific antibody, a DARPin, or a variant of each thereof.

[0188] Aspect 160 is the method of any one of aspects 135-159, wherein the one or more secondary binding agent is an antibody, or a functional variant thereof, optionally wherein the antibody, or the functional variant thereof comprises a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody variant, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, single chain antibody, single domain antibody, nanobodies, diabodies, a bi-specific antibody, a multi-specific antibody, a DARPin, or a variant of each thereof.

[0189] Aspect 161 is the method of any one of aspects 135-160, wherein the solid surface comprises a slide, a multi-well plate, a capillary, or the like.

[0190] Aspect 162 is the method of any one of aspects 135-161, further comprising sequencing the cDNA.

[0191] Aspect 163 is the method of aspect 162, wherein the sequencing is performed using Next Generation Sequencing (NGS) techniques.

[0192] Aspect 164 is the method of aspect 163, wherein the sequencing is done using a single cell genomic imaging techniques.

[0193] Aspect 165 is the method of aspect 164, wherein the single cell genomic imaging technique comprises, consists essentially of, or consists of spatial transcriptomics, MERFISH, SeqFISH, STARmap, Slide-Seq, Visium Spatial Gene Expression, or deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq).

[0194] Aspect 166 is the method of aspect 165, wherein the single cell genomic imaging technique is a microfluidic based technique comprising: ligating a first set and a second set of301320428.1 - 29 -spatial barcodes to the cDNA of step (c), prior to step (d), wherein the first set of spatial barcodes are contacted to the cDNA horizontally using a first multi-channel microfluidic chip, and wherein the second set of spatial barcodes are contacted to the solid surface vertically using a second multi-channel microfluidic chip.

[0195] Aspect 167 is the method of aspect 164, wherein the first set of spatial barcodes and second set of spatial barcodes form a 2D spatial barcode array.

[0196] Aspect 168 is a kit comprising the composition of any one of aspects 34-67, and a transcriptase mix.

[0197] Aspect 169 is a method of determining the one or more RBP binding sites on an mRNA throughout an mRNA cycle, the method comprising: a) incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; b) incubating the first complex with one or more secondary binding agents that specifically binds the RBP-targeting agent, to form a secondary complex; c) incubating the first or the secondary complex with the composition comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a first targeting moiety; wherein the first targeting moiety binds the first or the secondary complex to form a tertiary complex; d) incubating the tertiary complex with a transcriptase mix and a composition comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase, a second dimerization domain and a second targeting moiety, wherein the second dimerization domain binds the first dimerization domain, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA, and wherein the second targeting moiety binds a specific site on the mRNA; and e) sequencing the cDNA.

[0198] Aspect 170 is the method of aspect 169, wherein the second targeting moiety comprises a nucleoside analog.

[0199] Aspect 171 is the method of aspect 170, wherein the nucleoside analog is a 5-ethyluridine.

[0200] Aspect 172 is the method of aspect 169, wherein the first fragment of the reverse transcriptase is a N-terminal fragment, a fragment starting at least within the first 20 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof, or the C-terminal fragment, any variant thereof.

[0201] Aspect 173 is the method of aspect 169, wherein the second fragment of the reverse transcriptase is a N-terminal fragment, a fragment starting at least within the first 20 amino301320428.1 - 30 -acids of the N-terminal of the reverse transcriptase, or any variant thereof, or the C-terminal fragment, any variant thereof.

[0202] Aspect 174 is the method of any one of aspects 169-173, wherein the first dimerization domain is FKBP, or a variant thereof, or FRB or a variant thereof; and wherein the second dimerization domain is FKBP, or a variant thereof, or FRB or a variant thereof.

[0203] Aspect 175 is the method of any one of aspects 169-173, wherein the first dimerization domain is a Halo-tag, or a variant thereof, or a Halo-tag ligand or a variant thereof; and wherein the second dimerization is a Halo-tag, or a variant thereof, or a Halo-tag ligand or a variant thereof.

[0204] Aspect 176 is the method of aspect 174, wherein the binding of the first and the second dimerization domains can be induced by rapamycin.

[0205] Aspect 177 is the method of aspect 174, wherein the binding of the first and the second dimerization domains can be induced by alkyl chloride.

[0206] Aspect 178 is a method of determining one or more RNA interaction sites of a first RNA-binding Protein (RBP) and a second RBP, in a biological sample, comprising: a) incubating a first RBP-targeting agent comprising a functionalized first DNA barcode, with the first RBP, wherein the first RBP-targeting agent specifically binds the first RBP to form a first complex; b) incubating a second RBP-targeting agent comprising a functionalized second DNA barcode, with the second RBP, wherein the second RBP-targeting agent specifically binds the second RBP to form a second complex; b) incubating the first complex and the second complex with a composition comprising: (i) a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first complex; (ii) a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form a first barcoded cDNA library and a second barcoded cDNA library; d) amplifying and sequencing the first and the second barcoded cDNA library; and e) obtaining one or more interaction site of the first RBP and the second RBP by deconvoluting the sequenced cDNA library based on the first and the second DNA barcode.

[0207] Aspect 179 is the method of aspect 178, wherein the transcriptase composition comprise an RT primer sequence comprising a functional group and biotinylated dNTPs.

[0208] Aspect 180 is the method of aspect 179, wherein the functional group is an azide functional group.301320428.1 - 31 -

[0209] Aspect 181 is the method of aspect 179 or aspect 180, wherein the biotinylated dNTPs, and the RT primer sequence comprising the azide functional group, are incorporated into the cDNA to form proximal azide labeled biotinylated cDNAs during reverse transcription in step c.

[0210] Aspect 182 is the method of aspect 178, wherein the functionalized DNA barcode comprises an alkyne (3'-O-propargyl N 2'-5' linked) functionalized DNA barcode.

[0211] Aspect 183 is the method of aspect 178, wherein the alkyne functionalized barcodes comprise a nucleic acid sequence as set forth in any one of SEQ ID NO: 65-112, or a sequence at least 80% identical thereto.

[0212] Aspect 184 is the method of aspect 178, further comprising incorporating the alkyne functionalized first and / or second DNA barcode into the cDNA by reacting the alkyne functionalized first DNA barcode and / or second DNA barcode with the proximal azide labeled biotinylated cDNA of aspect 181, using in-situ copper catalyzed azide-alkyne cycloaddition (CuAAC), to obtain a first barcoded biotinylated cDNA library and / or the second biotinylated cDNA library.

[0213] Aspect 185 is the method of aspect 184, wherein the method further comprises purifying the first and / or the second barcoded biotinylated cDNA library over a streptavidin column prior to step (d).

[0214] Aspect 186 is the method of aspect 185, further comprising processing the CuAAC using a Klenow Fragment DNA polymerase for second strand synthesis.

[0215] Aspect 187 is the method of aspect 186, wherein the one or more interaction sites of the first RBP and the second RBP are obtained by deconvoluting the sequenced data based on the first and the second DNA barcodes incorporated into the cDNA.

[0216] Aspect 188 is a method of determining spatial distribution of a RNA modification sites on a biological sample bound to a solid surface, comprising: a) incubating a modificationtargeting agent that specifically binds the modification site on the RNA to form a primary complex; b) incubating the primary complex with a secondary binding agent that specifically bind the primary complex to form a secondary complex; c) incubating the first or the secondary complex with a composition comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a first targeting moiety; wherein the first targeting moiety binds the first or the secondary complex to form a tertiary complex; d) incubating the tertiary complex with a transcriptase mix and a composition comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase, a second dimerization domain and optionally a second targeting moiety, wherein301320428.1 - 32 -the second dimerization domain binds the first dimerization domain, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; e) optionally incorporating labelled barcodes into the cDNA; f) sequencing and imaging the biological sample using a single cell genomic imaging technique to determine the one or more modification sites.

[0217] Other objects, features and advantages of the present inventions will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the inventions described herein, are given by way of illustration only, since various changes and modifications within the spirit and scope of the inventions will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0218] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present inventions. The inventions can be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0219] FIG. 1 provides a comparison between ARTR-seq and split ARTR-seq. ARTR-seq can detect RBP binding by using pAG-RTase bound to primary and secondary antibodies, but there are some background signals generated by nonspecific binding of pAG-RTase. In split ARTR-seq, RTase is split into two halves (split-RTase-N and split-RTase-C) which show low reverse transcription activity alone. Split-RTase-C can be fused with pAG and can recognize primary and secondary antibodies. After washing steps to remove any unbound split-RTase-C, split-RTase-N is recruited to split-RTase-C by rapamycin, so that split-RTase will exhibit higher reverse transcription activity at RBP binding sites than in the nonspecific binding regions.

[0220] FIG. 2A provides a schematic of cut site screening workflow. Split-RTase-N and split-RTase-C are expressed in BL21(DE)3 E. coli. After prokaryotic expression, crude lysate of split-RTase-N and split-RTase-C expressing E. coli are used for reverse transcription and quantitative PCR (RT-qPCR).

[0221] FIG. 2B provides RT-qPCR analysis of relative enzymatic activity for separate split RTase (RT-N or RT-C), mixed split-MMLV-RTase with DMSO or 10 pM rapamycin at room temperature for 1 hour (RT-N+RT-C+DMSO or Rapamycin) compared with HaloTag-MMLV-RTase (full-length) evaluated based on RT-qPCR results.301320428.1 - 33 -

[0222] FIG. 2C provides a cartoon of protein structures of split-MMLV-RTases split from the cut site 123.

[0223] FIG. 2D provides RT-qPCR analysis of relative enzymatic activity for MMLV RTase (full length), HIV RTase p66, and HIV RTase p51.

[0224] FIG. 2E provides RT-qPCR analysis showing relative enzymatic activity of tested purified pAG-RTase fusion proteins. Two commercial RTases, SuperScript II and SuperScript III, serve as positive controls.

[0225] FIG. 2F provides a comparison of the amino acid sequence around MMLV-RTase cut site 123 (Full length sequence in SEQ ID NO: 1) and HIV-RTase cut site 85 (Full length sequence in SEQ ID NO: 5).

[0226] FIG. 2G provides normalized enzymatic activity of separate split-HI V-RTase (RT-N or RT-C), mixed split RTase with DMSO or 10 pM rapamycin at room temperature for 1 hour (RT-N+RT-C+DMSO or Rapamycin) compared with HIV-RTase (full-length) are evaluated based on RT-qPCR results.

[0227] FIG. 2H provides a cartoon of protein structures of split-HIV-RTases split from the cut site 85.

[0228] FIG. 21 provides the normalized enzymatic activity of lOOnM separate purified split MMLV-RTase (RT-N or RT-C), mixed purified split MMLV-RTase with DMSO or 10 pM rapamycin at room temperature for 1 hour (RT-N+RT-C+DMSO or Rapamycin).

[0229] FIG. 3A provides representative immuofluorescence images of Hela cells treated with the Halotag-ligand-labeled antibody (Antibody-Cl); split-RTase-C fused with protein A / G labeled with fluorophore (pAG-RT-C); split-RTase-N fused with HaloTag labeled with fluorophore (Halo-RT-N); nucleus (Hoechst); merged picture (Merge) of YTHDF2 split-RTase ARTR-seq in HeLa cells.

[0230] FIG. 3B provides the normalized cDNA amount computed based on the spike-in reads in RPS6 pAG-RTase ARTR-seq and no-antibody control sample in HEK293 cells on the left. Normalized cDNA amount computed based on the spike-in reads in YTHDF2 split- ARTR-seq and no-antibody control sample, in the presence of rapamycin or DMSO in HeLa cells are provided on the right.

[0231] FIG. 3C provides the signal profiles and heatmaps of read density in pAG-RTase ARTR-seq and split- ARTR-seq libraries at iCLIP-seq-identified YTHDF2 peaks (GEO: GSE78030, left) or iCLIP-seq-identified YTHDF2 peaks (GEO: GSE49339, right).

[0232] FIG. 3D provides snapshots from IGV showing overlap of YTHDF2 pAG-RTase ARTR-seq and split-RTase ARTR-seq signal in HeLa cells with reported YTHDF2 binding301320428.1 - 34 -sites from PAR-CLIP-seq data (GEO: GSE49339). The positions of reported YTHDF2 binding sites are indicated with arrows.

[0233] FIG. 4A provides representative immunofluorescence images of Hela cells treated with HaloTag-ligand-labeled antibody (Antibody-Cl); split-RTase-N fused with HaloTag labeled with fluorophore (Halo-RT-N), nucleus (Hoechst) of YTHDF1 and FMRP split-RTase ARTR-seq in HeLa cells.

[0234] FIG. 4B provides signal profiles and heatmaps of read density in YTHDF1 pAG-RTase ARTR-seq, FMRP pAG-RTase ARTR-seq and YTHDF1-FMRP split- ARTR-seq libraries at YTHDF1+FMRP coexisting sites, YTHDF1 -specific sites, and sites with strong FMRP binding and intermediate or weak YTHDF1 binding signals.

[0235] FIG. 4C provides representative snapshots from IGV showing YTHDF1 pAG-RTase ARTR-seq, FMRP pAG-RTase ARTR-seq and YTHDF1-FMRP split-RTase ARTR-seq signal at YTHDF1+FMRP coexisting sites and YTHDF1 -specific sites in HeLa cells.

[0236] FIG. 5A is a schematic summarizing a general scheme of the applications.

[0237] FIG. 5B is a schematic depicting that split ARTR-seq can be used to detect ribosome binding, and co-binding of two proteins of interest by fusing split-RTase with two antibodies. Split ARTR-seq can also be used to detect RBP binding on nascent RNAs when fusing one split-RTase with antibody and the other split-RTase with nucleoside analogs.

[0238] FIG. 6 is a schematic depicting an overview of split-ARTR-seq and a comparison between ARTR-seq and split ARTR-seq. ARTR-seq can detect RBP binding sites by using pAG-RTase bound to primary and secondary antibodies, but the background signals would be generated by non-specific binding of pAG-RTase (left). In split ARTR-seq, RTase is split into two halves (split-RTase-N and split-RTase-C), which show low reverse transcription activity alone. Split ARTR-seq can be used to detect the co-binding of two proteins of interest by fusing split-RTase with two antibodies (middle), or the binding of a single protein of interest with a low background noise signal (right). Nanobodies targeting the Fc or Fab fragment of rabbit or mouse antibody are fused with split-RTase to bind to specific antibodies.

[0239] FIG. 7A is a schematic depicting a design of split-ARTR-seq to detect RBPs cobinding sites. Nanobodies targeting the Fc or Fab fragment of rabbit or mouse antibody (TP897, bind to the Fc fragment of rabbit antibody; TP896, bind to the Fab fragment of rabbit antibody; TP1170, bind to the Fab fragment of mouse antibody (Kappa subtype); TP1107, bind to the Fc fragment of mouse antibody (IgGl subtype) are fused with split-RTases to bind to specific antibodies. FRB and FKBP are a pair of heterodimerization domains and can induce the split-301320428.1 - 35 -RTases to get closer under rapamycin induction, which will promote protein trans-splicing by intein-N and intein-C.

[0240] FIG. 7B is a schematic depicting a design of split-ARTR-seq to detect single RBPs co-binding sites with lower background. Nanobodies targeting the Fc or Fab fragment of rabbit or mouse antibody are fused with split-RTase to bind to specific parts of either a rabbit or a mouse antibody. FRB and FKBP are a pair of heterodimerization domains and can induce the split-RTases to get closer under rapamycin induction, which will promote protein trans-splicing by intein-N and intein-C.

[0241] FIG. 7C is a schematic depicting that under the treatment at 37°C for 2 hours in the presence of rapamycin, split-RTases can undergo protein trans-splicing to excise inteins and FKBP / FRB, and form the full-length RTase.

[0242] FIG. 8A provides a comparison of amino acid 120-131 between wild-type full-length RTases (FL) and full-length RTases with mutation T128C, V129W and P130N (FL-128CWN: SEQ IDNO: 156).

[0243] FIG. 8B provides relative reserve transcription activities of full-length RTases (FL), full-length RTases with mutation T128C (FL-128C), and full-length RTases with mutation T128C, V129W and P130N (FL-128CWN) normalized to wild-type full-length RTases (FL), evaluated based on RT-qPCR results.

[0244] FIG. 8C provides normalized enzymatic activities of separate split RTase split from split site 123 or 127 (RT-N or RT-C), mixed split RTase with DMSO or lOuM rapamycin at room temperature for 1 hour (RT-N+RT-C+DMSO or Rapamycin) compared with HaloTag-MMLV-RTase (full-length) evaluated based on RT-qPCR results.

[0245] FIG. 8D provides an image of a Coomassie brilliant blue-stained SDS-PAGE gel showing the split fragments and mixed fractions under the treatment at 37°C for 2 hours in the presence of rapamycin of split-RTases with inteins. The +1 positions of split-RTases are C90, T128C and S142C. The red asterisk indicates the band of full-length RTase formed by protein trans-splicing.

[0246] FIG. 8E provides an image Coomassie brilliant blue-stained SDS-PAGE gel showing the split fragments and mixed fractions (under the treatment at 37°C for 2 hours in the presence of rapamycin) of nanobody split-RTases with inteins (split from site 127, with the mutation T128C, V129W and P130N). The red asterisk indicates the band of full-length RTase formed by protein trans-splicing.

[0247] FIG. 8F provides normalized enzymatic activities of mixed nanobody split-RTases with DMSO at room temperature for 2 hours or lOuM rapamycin at 37°C for 2 hours (RT-301320428.1 - 36 -N+RT-C+DMSO or Rapamycin) compared with HaloTag-MMLV-RTase (full-length) evaluated based on RT-qPCR results.

[0248] FIG. 9A provides immunofluorescence images of YTHDF1; G3BP1; nucleus (Hoechst) and merged picture (Merge) in HeLa cells with or without treatment under 0.5mM sodium arsenite for 1 hour.

[0249] FIG. 9B provides immunofluorescence images of rabbit antibody targeting YTHDF1 (RbYTHDFl); split-RTase-C fused with nanobody TP896 labeled with fluorophore (4-C-Rb896); mouse antibody targeting G3BP1 (MsG3BPl); split-RTase-N fused with nanobody TP1170 labeled with fluorophore (5-N-Msll70), nucleus (Hoechst) and merged picture (Merge) of specific antibody and split-RTase in HeLa cells treated with 0.5mM sodium arsenite for 1 hour.

[0250] FIG. 9C provides immunofluorescence images of rabbit antibody targeting YTHDF1 (RbYTHDFl); split-RTase-N fused with nanobody TP896 labeled with fluorophore (3-N-Rb896); mouse antibody targeting G3BP1 (MsG3BPl); split-RTase-C fused with nanobody TP1170 labeled with fluorophore (6-C-Msll70), nucleus (Hoechst) and merged picture (Merge) of specific antibody and split-RTase in HeLa cells treated with 0.5mM sodium arsenite for 1 hour.

[0251] FIG. 10A provides signal profiles and heatmaps of read density of pAG-RTase ARTR-seq and split- ARTR-seq libraries at ARTR-seq-identified YTHDF1 peaks (GEO: GSE226161).

[0252] FIG. 10B provides signal profiles and heatmaps of read density in pAG-RTase ARTR-seq and split- ARTR-seq libraries at PAR-CLIP-seq-identified YTHDF1 peaks (GEO: GSE63591).

[0253] FIG. 10C provides snapshots from IGV showing overlap of YTHDF1 pAG-RTase ARTR-seq and split-RTase ARTR-seq signals in HeLa cells with reported YTHDF1 binding sites from PAR-CLIP-seq data (GEO: GSE63591). The positions of reported YTHDF1 binding sites are indicated below.

[0254] FIG. HA provides signal profiles and heatmaps of read density in G3BP1, YTHDF1 p AG- ARTR-seq and YTHDF1-G3BP1 split- ARTR-seq before or after sodium arsenite stress treatment at G3BP1 -binding sites and YTHDF1 -biding sites excluded from G3BP1 -binding sites (stress granule) identified from pAG-RTase ARTR-seq.

[0255] FIG. 11B provides a motif enrichment analysis on YTHDF1 -specific sites that are excluded from stress granules, and G3BP1 -binding sites and YTHDF1-G3BP1 co-binding sites (detected by split-ARTR-seq) under sodium arsenite stress.301320428.1 - 37 -

[0256] FIG. 11C provides representative snapshots from IGV showing YTHDF1, G3BP1, no-antibody pAG-RTase ARTR-seq and YTHDF1-G3BP1 split-RTase ARTR-seq signals on stress-granule enriched RNAs in HeLa cells before or after sodium arsenite stress treatment.

[0257] FIG. HD provides the number of genes detected by YTHDF1-G3BP1 split-RTase ARTR-seq before or after sodium arsenite stress treatment, and number of YTHDF1-G3BP1 split-RTase ARTR-seq detected genes overlapped with CAP-seq before or after sodium arsenite stress treatment.DETAILED DESCRIPTION

[0258] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense. It is specifically contemplated that any limitation discussed with respect to one aspect of the disclosure can apply to any other aspect of the disclosure. Furthermore, any composition of the disclosure can be used in any method of the disclosure, and any method of the disclosure can be used to produce or to utilize any composition of the disclosure. Aspects set forth in the Examples are also aspects that can be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.

[0259] In some aspects, the current disclosure is broadly related to methods for identifying RNA binding protein - RNA interaction sites, and / or RNA modification sites using Assay of Reverse Transcription-based RBP binding sites Sequencing (ARTR-seq), as described in the U.S. Application 18 / 726,353, which is incorporated by reference herein in its entirety. In some aspects, the ARTR-seq method, can generate background signals from the nonspecific binding of the secondary antibody and RTase in the input sample. Such signals would not be a concern for RBPs that have a known RNA binding motif and are mainly enriched in introns or 3' UTR regions. However, due to its great similarity with transcriptome, translatome is hard to distinguish from such background signals. Therefore, in the current disclosure, the inventors have attempted to engineer alternative methods that overcome this and other limitations of ARTR-seq.301320428.1 - 38 -

[0260] In some aspects, the current disclosure provides surprising and remarkable improvement over the previously disclosed ARTR-seq method. In some aspects, method disclosed herein uses RTase split into two parts (RTase-N-terminal also referred to as split-RTase-N or RT-N and RTase-C-terminal also referred to as split-RTase-C orRT-C), which exhibit low reverse transcription activity when staying apart but exhibit higher reverse transcription activity when in proximity with each other or induced to get assembled by some molecular inducer of dimerization like rapamycin (FIG. 1). This method will be referred herein as Split-ARTR-seq. Interestingly, in some aspects, use of a split RTase can reduce the background signal of ARTR-seq. Use of the split RTase also facilitated the development of several novel methods for single-cell spatial translatome profiling, temporal study of the translatome, simultaneous study of co-binding of two RBP proteins of interest, dynamic RBP binding, and several other applications.I. Definitions

[0261] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0262] The use of the word “a” or “an” when used in conjunction with the term “comprising” can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0263] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or. It is specifically contemplated that A, B, or C can be specifically excluded from an aspect.

[0264] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0265] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.301320428.1 - 39 -

[0266] As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, for example but not limited to, in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some aspects, a variant may also differ functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some aspects, a variant polypeptide or nucleic acid can differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups, fluorophores, small molecules) that are covalent components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone).

[0267] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antibody variant or antibody derivative) that it encodes. Mutations can be introduced using any technique known in the art. In some aspects, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another aspect, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. However it is made, a mutant polypeptide can be expressed and screened for a desired property.

[0268] Mutations can be introduced into a nucleic acid without significantly altering the sequence and / or the biological activity of a polypeptide that it encodes. For example, one can make silent mutations or nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. See, e.g., Romain Studer et al., Biochem. J. 449:581-594 (2013), incorporated herein by reference. For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody.

[0269] Variant polypeptides encoded by nucleic acids of the disclosure can contain amino acid changes that confer any of a number of desirable properties. Variant polypeptides can be301320428.1 - 40 -made using routine mutagenesis techniques and assayed as appropriate to determine whether they possess the desired property. The stability of protein(s) encoded by a variant nucleic acid can be measured by assaying thermal stability or stability upon urea denaturation or can be measured using in silico prediction. Methods for such experiments and in silico determinations are known in the art.

[0270] In some aspects, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is, is at least, is at most, or is between (inclusive or exclusive) any two of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%, or any percentage derivable therein. In some aspects, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some aspects, a reference polypeptide or nucleic acid has one or more biological activities. In some aspects, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some aspects, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some aspects, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid.

[0271] In some aspects, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Certain amino acids can be substituted for other amino acids in a protein or polypeptide sequence inserted, or deleted, as compared to the reference, with or without appreciable loss of interactive binding capacity with structures such as, for example, antigenbinding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines its functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties.

[0272] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. The variant polypeptide or nucleic acid sequence has at least one modification compared to the reference polypeptide or nucleic acid sequence, e.g., from 1 to about 50 modifications. A variation in a polypeptide of the disclosure can affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of the protein or polypeptide, as compared to wild-type. In some301320428.1 - 41 -aspects, the variant polypeptide or nucleic acid sequence has from 1 to about 50 modifications compared to the reference polypeptide or nucleic acid sequence. In some aspects, the variant polypeptide or nucleic acid sequence has from 1 to about 40 modifications compared to the reference polypeptide or nucleic acid sequence. In some aspects, the variant polypeptide or nucleic acid sequence has from 1 to about 30 modifications compared to the reference polypeptide or nucleic acid sequence. In some aspects, the variant polypeptide or nucleic acid sequence has from 1 to about 20 modifications compared to the reference polypeptide or nucleic acid sequence. In some aspects, the variant polypeptide or nucleic acid sequence has from 1 to about 10 modifications compared to the reference polypeptide or nucleic acid sequence. In some aspects, the variant polypeptide or nucleic acid sequence has from 1 to about 5 modifications compared to the reference polypeptide or nucleic acid sequence. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein.

[0273] It also will be understood that amino acid and nucleic acid sequences can include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' nucleic acid sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity. The addition of terminal sequences particularly applies to nucleic acid sequences that can, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0274] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0275] Deletion variants typically lack one or more residues of the native or wild type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon can be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.

[0276] Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This can include the insertion of one or more amino acid residues. Terminal additions can also be generated and can include fusion proteins which are301320428.1 - 42 -multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0277] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide and can be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions can be conservative, that is, one amino acid is replaced with one of similar chemical properties.

[0278] Conservative amino acid substitutions” can involve exchange of a member of one amino acid class with another member of the same class. Conservative replacements (also “conservative substitutions” or “conservative amino acid substitutions”) are those that take place within a family of amino acids that possess similar biochemical properties, including charge, hydrophobicity, and size. Genetically encoded amino acids are generally divided into families based on the chemical nature of the side chain, e.g., acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Thus, a conservative replacement can comprise replacement of an amino acid in one family for an amino acid in the same family (e.g., replacement of a lysine with an arginine, replacement of an aspartate for a glutamate, etc.). Alternatively, or in addition, amino acid similarity can be determined using a Blocks Substitution Matrix (BLOSUM), such as BLOSUM62 (Henikoff S and Henikoff JG, Proc. Natl. Acad. Sci. U.S.A 89(22): 10915-9 (1992)). In this case, a conservative replacement can be a substitution of amino acids having a non-negative value on a BLOSUM62 matrix. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Standard ELISA, Surface Plasmon Resonance (SPR), or other antibody binding assays can be performed by one skilled in the art to make a quantitative comparison of antigen binging affinity between the unmodified antibody and any polypeptide derivatives with conservative substitutions generated through any of several methods available to one skilled in the art. Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0279] Alternatively, substitutions can be “non-conservative” (also “nonconservative”). In some aspects, a non-conservative substitution affects a function or activity of the polypeptide. In some aspects, a non-conservative substitution does not affect a function or activity of the301320428.1 - 43 -polypeptide. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions can involve the exchange of a member of one of the amino acid classes for a member from another class.

[0280] In some aspects, a reference polypeptide or nucleic acid is a “wild type” or “WT” or “native” sequence found in nature, including allelic variations. A wild type polypeptide or nucleic acid sequence has a sequence that has not been intentionally modified. For the purposes of the present disclosure, “variants” of an amino acid sequence (peptide, protein, or polypeptide) comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. “Variants” of a nucleotide sequence comprise nucleotide insertion variants, nucleotide addition variants, nucleotide deletion variants and / or nucleotide substitution variants. The term “variant” includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular, those which are naturally occurring.

[0281] The term “fragment,” with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, e.g., a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3 '-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5 '-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A N-terminal fragment may further comprise one or more insertions, deletions or substitutions when compared to a wild-type N-terminal fragment in the region of overlap. In some aspects, one or more residues from the N-terminal of the fragment may be deleted, substituted, or additional residues inserted. A C-terminal fragment may further comprise one or more insertions, deletions or substitutions when compared to a wild-type C-terminal fragment in the region of overlap. In some aspects, one or more residues from the C-terminal of the fragment may be deleted, substituted, or additional residues inserted. As used herein, the term N-terminal fragment encompasses fragments that starts within the first 50 amino acids of the N-terminus of the reference polypeptide (for example the disclosed RTase), including a fragment that starts at, or within the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids of the N-terminus of the RTase. In some aspects, the N-terminal RTase fragment starts within the first301320428.1 - 44 -20 amino acids of the N-terminus of the RTase. An N-terminal fragment may further comprise one or more insertions, deletions, and / or substitutions relative to a wild-type N-terminal fragment, for example in a region of overlap. As used herein, the term C-terminal fragment encompasses fragments that ends within the last 50 amino acids of the C-terminus of the RTase, including a fragment that ends at, or within the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids of the C-terminus of the RTase, or any derivative thereof. A fragment of an amino acid sequence comprises at least, equal to or at most, e.g., 15%, or 20%, or 30%, or 40%, or 50 %, 60 %, 70 %, 80%, 90%, (or any range derivable therein) of the amino acid residues from an amino acid sequence. In the present disclosure, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least, at most, exactly, or between (inclusive or exclusive) any two of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any percentage derivable therein, with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived.

[0282] As used herein, the terms “region of overlap” or “overlapping sequence” or “overlapping fragment” with respect to two polypeptide sequences is used interchangeably to refer to a contiguous sequence of amino acids that are similar or identical in both proteins sequences. For example, a “region of overlap” of a fragment of reverse transcriptase has at least about 50%, 60%, 70%, 80%, 90%, or 100% sequence identity to the corresponding region of a wild-type reverse transcriptase. In some aspects, the overlap may arise from overlapping coding regions in nucleic acids. In some aspects, the overlapping sequences in the two polypeptides may be functionally and / or structurally similar.

[0283] In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 15% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 20% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 30% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least301320428.1 - 45 -40% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 50% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 60% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 70% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 80% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least 90% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived. In some aspects, the fragment may have reduced functionality compared to the wildtype protein from which it is derived. In some aspects, the fragment may be non-functional.

[0284] A variant of an amino acid sequence (peptide or protein) can be a “functional variant.” The term “functional variant” of an amino acid sequence relates to any variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, e.g., it is functionally equivalent. The term “functional variant,” as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence. In some aspects, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence.

[0285] In some aspects, a “functional variant” of a protein can retain at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, functionality with respect to the full length wild-type protein. Assays to determine the functionality of a given protein are well known in the art, for example, binding assays, cleavage assays, or enzyme assays. In some aspects, the protein can be an antibody, and a functional variant can be a variant that retains at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%,301320428.1 - 46 -8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, of its binding affinity and specificity for its antigen, as determined by any of the assays known in the art, for example ELISA, SPR, bilayer interferometry, flow cytometry, radioimmunoassay, isothermal titration calorimetry, affinity chromatography, or Western blotting. In some aspects, the protein can be a reverse transcriptase, and a functional variant can be a variant that retains at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, of its enzyme activity, either when measured alone or when measured when the functional variant is combined with a one or more other functional variants of the reverse transcriptase, as measured by any suitable techniques known in the art, for example, reverse transcription-quantitative PCR (RT-qPCR) assay to measure cDNA synthesis, the ELISA-based enzyme activity assay for detecting reverse transcriptase activity, and the radioactive nucleotide incorporation assay to quantify DNA polymerization by tracking radiolabeled nucleotides. In some aspects, the protein can be an enzyme, and a functional variant can be a variant that retains at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, of its enzymatic activity, either when measured alone or when measured when the functional variant is combined with a one or more other functional variants of the enzyme, as measured by any suitable techniques known in the art, for reverse transcription-quantitative PCR (RT-qPCR) assay to measure cDNA synthesis, ELISA-based enzyme activity assays, etc.

[0286] An amino acid sequence (peptide, protein, or polypeptide) “derived from” a designated amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is functionally equivalent, identical, essentially identical, or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence can be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the enzymes and / or antigens suitable for use herein can be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences (e.g., alone and / or when utilized in combination with one or more other polypeptides).301320428.1 - 47 -

[0287] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antigen or antibody or antibody derivative) that it encodes. Mutations can be introduced using any technique known in the art. In some aspects, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another aspect, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. In some aspects, however it is made, a mutant polypeptide can be expressed and screened for a desired property.

[0288] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody.

[0289] “Sequence similarity” indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. “Sequence identity” between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. The terms “% identical,” “% identity,” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences can be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison,” in order to identify local regions of corresponding sequences. The optimal alignment for a comparison can be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads. App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (FOGSAA, GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group). In some aspects, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website.301320428.1 - 48 -

[0290] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0291] In some aspects, the degree of similarity or identity is given for a region that is, is at least, is at most, or is between (inclusive or exclusive) any two of about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for, for at least, for at most, or for between any two of 100, 120, 140, 160, 180, or 200 nucleotides, or any range derivable therein, in some aspects, continuous nucleotides. In some aspects, the degree of similarity or identity is given for the entire length of the reference sequence.

[0292] Homologous amino acid sequences can exhibit at least, at most, or between (inclusive or exclusive) any two of 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity of the amino acid residues. In some aspects, homologous amino acid sequences exhibit at least 95% identity of the amino acid residues. In some aspects, homologous amino acid sequences exhibit at least 98% identity of the amino acid residues. In some aspects, homologous amino acid sequences exhibit at least 99% identity of the amino acid residues.

[0293] The terms “antibody” refers to an intact immunoglobulin of any class or isotype, or a fragment thereof, or a variant that can compete with the intact antibody for specific binding to the target antigen. An isotype refers to the genetic variations or differences in the constant regions of the heavy and light chains of an antibody. In humans, there are five heavy chain isotypes: IgA, IgD, IgG, IgE, and IgM and two light chain isotypes: kappa and lambda. The IgG class is divided into four isotypes: IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a, IgG2b and IgG3 in mice. They share more than 95% homology in the amino acid sequences of the Fc regions but show major differences in the amino acid composition and structure of the hinge region. The term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody derivative, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, single chain antibody, single domain antibody, nanobodies, diabodies, a bi-specific antibody, a multi-specific antibody, a DARPin, or a variant of each thereof. Also contemplated are antibodies having specificity for more than one antigen or target, including bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and other multispecific antibodies. As used herein, an “antibody” includes whole antibodies and301320428.1 - 49 -any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule. As used herein, the terms “antibody” or “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of an animal, including IgM, IgD, IgG, IgA, IgE, and related proteins, as well as polypeptides comprising antibody CDR domains that retain antigen-binding activity. Examples of such include but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region or any portion thereof or at least one portion of a binding protein.

[0294] The term “polypeptide construct” as used herein, refers to a polypeptide engineered by combining a amnio acid sequence with two or more different moi eties (for example targeting moieties selected from proteins, peptides, oligonucleotides, aptamers, ligands, small molecules, or any combination thereof. The term “fusion polypeptide” as used herein, refers to a polypeptide engineered by combining sequences from two or more different proteins or peptides into a single polypeptide chain. These combined sequences typically retain one or more of their functional domains, allowing the fusion protein to exhibit multiple properties or activities from its constituent parts. The term polypeptide construct encompasses fusion polypeptides as well as polypeptides (polypeptides or fusion polypeptides) comprising non-protein / peptide moieties. In some aspects, the terms “polypeptide construct” or “fusion polypeptide” can be used interchangeably to refer to an engineered polypeptide comprising two or more protein sequences. In some aspects, the terms “polypeptide construct” or “fusion polypeptide” cannot be interchangeable, wherein the polypeptide construct can comprise one or more proteins and a non-protein / peptide component. In some aspects, the polypeptide construct or fusion polypeptide can further comprise one or more additional sequences, for example for example a leader sequence, one or more purification tags, one or more solubility tags, one or more linker sequences, one or more protease cleavage tags, fluorophores, fluorescent proteins or peptides, or any combination thereof. The one or more protein sequences that are incorporated into the polypeptide construct can be wild-type protein sequences, or variants thereof, or engineered protein sequences, or variants thereof.

[0295] As used herein, the term “promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more genes (or coding sequence), located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase,301320428.1 - 50 -transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active under most physiological and developmental conditions. An “inducible” promoter is a promoter that is regulated depending on physiological or developmental conditions. A “tissue specific” promoter is preferentially active in specific types of differentiated cells / tissues.

[0296] As used herein the term “affinity interactions” refer to the specific, non-covalent binding between two molecules, for example a ligand and its receptor, based on complementary shapes, charge distribution, and molecular interactions such as hydrogen bonding, van der Waals forces, and hydrophobic effects. These interactions are fundamental to numerous biological processes, including enzyme-substrate binding, antigen-antibody recognition, hormone-receptor binding, and DNA-protein interactions. The strength of these interactions, often measured as the affinity constant (Kd), determines the stability and specificity of the binding event. High-affinity interactions, like the biotin-streptavidin pair, which is among the strongest known in nature, are utilized in molecular biology for techniques like affinity purification, immunoprecipitation, and diagnostics.

[0297] As used herein, the terms “primary”, “secondary” and similar descriptors are meant to simply indicate a first composition or event, a second composition or event respectively, and do not imply any relative importance or priority between them.

[0298] “Reconstitute” (and “reconstituted”) in the context of reverse transcriptase means to restore, generate, or measurably increase reverse transcriptase (RTase) enzymatic function from separate RTase fragments or variants by bringing those fragments into proximity and / or causing them to associate or assemble — whether non covalently (e.g., via dimerization domains, electrostatic interactions, hydrophobic contacts) or covalently (e.g., by peptide ligation, chemical crosslinking, or splicing / intein mediated fusion) — such that the resulting complex exhibits higher reverse transcription activity than one or more of the fragments alone. Reconstitution includes induced proximity (e.g., heterodimerization of the first and second dimerization domains), self assembly, templated assembly, conditional assembly (e.g., ligand-, light-, or environment triggered), and post translational joining or processing (e.g., intein mediated protein splicing) that functionally restores the RTase active site, binding sites, or structural integrity required for catalysis. Reconstitution is satisfied when the assembled complex reduces background signal by maintaining low or negligible RT activity in the separated state and higher RT activity upon association / assembly, regardless of whether the301320428.1 - 51 -activity equals, exceeds, or falls below the activity of the full length wild type enzyme. Reconstitution may be transient or stable, and does not require permanent fusion, provided that the association yields a detectable, quantifiable increase in RTase activity (e.g., cDNA yield, processivity, primer extension, or strand synthesis metrics) relative to the fragments when not associated.II. Polypeptide constructs and polynucleotides encoding the same

[0299] In some aspects, the current disclosure encompasses a polypeptide construct comprising: a) a fragment of a reverse transcriptase enzyme, or a variant thereof; b) a dimerization domain; and c) a targeting moiety. In some aspects, a targeting moiety is covalently linked to the RTase fragment, which allows site-specific delivery of the RTase to the biological target of interest. In some aspects, the polypeptide construct is a fusion protein, comprising sequences from two or more polypeptides. In some aspects, the polypeptide construct is not a fusion protein, and comprises at least one non-polypeptide moiety. In some aspects, the polypeptide construct can further comprise additional peptide sequences, for example a leader sequence, intein sequences, one or more purification tags, one or more solubility tags, one or more linker sequences, one or more protease cleavage tags, or any combination thereof.

[0300] In some aspects, disclosed herein is a first polypeptide construct and a second polypeptide construct that together provide reverse transcriptase activity upon association and / or splicing, wherein each construct comprises a fragment of a reverse transcriptase enzyme (RTase) and may further comprise a dimerization moiety, a targeting moiety, and / or optional elements such as linkers, tags, or fluorophores. In some aspects, the first polypeptide construct comprises an N-terminal RTase fragment, and the second polypeptide construct comprises a C-terminal RTase fragment, although the assignment of fragments to “first” and “second” polypeptides may be reversed. In some aspects, the RTase fragments have low reverse transcription activity when separated but higher activity when brought into proximity and / or induced to assemble and / or spliced, thereby reducing background signal.

[0301] In some aspects, the first and second polypeptide constructs comprise first and second dimerization moieties that specifically bind each other, or can be induced to bind each other, to promote association of the RTase fragments. Suitable dimerization moieties include, without limitation, rapamycin-mediated dimerization domains (FKBP and FRB), as well as other protein-based, small-molecule-induced, or mixed-partner dimerization systems. In some301320428.1 - 52 -aspects, dimerization may be driven by proximity alone or may require induction by a small molecule, drug, or other cue.

[0302] In certain aspects, the dimerization moieties are FKBP and FRB such that dimerization (and corresponding split-RTase assembly) can be induced in the presence of rapamycin or an analog. In some aspects, either construct may carry FKBP or FRB, provided the pair supports induced association (reconstitution). In some aspects, rapamycin is provided during a method step as an inducer of dimerization to promote assembly of the split RTase fragments and initiation of reverse transcription. In additional aspects, dimerization may be induced by other ligand-dependent systems.

[0303] In some aspects, one or both constructs further comprise a targeting moiety that directs the construct to a biological target, including by binding to a primary and / or secondary antibody used in a targeting strategy. Targeting moieties may include Fc-binding proteins and other affinity reagents, and may be present on one construct or both constructs (and may be the same or different). Non-limiting examples include protein A, protein G, protein A / G (pAG), and protein L, as well as nanobodies that bind antibody domains (e.g., Fab and / or Fc). In some aspects, the system is implemented without inteins, such that induced dimerization promotes proximity-based activity restoration. In other aspects, split inteins are included such that, upon induced dimerization, protein trans-splicing covalently joins the RTase fragments to form a full-length RTase, optionally improving signal -to-background. In any of the foregoing aspects, constructs may further include one or more linkers, purification / solubilization tags, leader sequences, and / or fluorophores arranged in orientations that preserve the functional interactions described herein.

[0304] Individual components that may form part of the polypeptide constructs of the current disclosure are further described below. Also, described are compositions comprising these polypeptides and methods using these polypeptides.A. Reverse transcriptase and fragments thereof

[0305] In some aspects, the polypeptide construct comprises a fragment of a reverse transcriptase (RTase) enzyme, or a variant thereof. In some aspects, the fragment of the reverse transcriptase enzyme is aN-terminal fragment. In some aspects, the fragment of the RT is a C-terminal fragment. As used herein, a RTase fragment shortened at the C-terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment)301320428.1 - 53 -is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5 '-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. In some aspects, the fragment of the reverse transcriptase enzyme is a N-terminal fragment, or a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof. A N-terminal RTase fragment may further comprise one or more insertions, deletions or substitutions when compared to a wild-type N-terminal fragment in the region of overlap. In some aspects, one or more residues from the N-terminal of the RTase fragment may be deleted, substituted, or additional residues inserted. In some aspects the fragment of the reverse transcriptase enzyme is a C-terminal fragment, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase, or any variant thereof. A C-terminal fragment may further comprise one or more insertions, deletions or substitutions when compared to a wild-type C-terminal fragment in the region of overlap. In some aspects, one or more residues from the C-terminal of the fragment may be deleted, substituted, or additional residues inserted. A fragment of the RT amino acid sequence comprises at least, equal to or at most, e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50 %, 55%, 60 %, 65%, 70 %, 75%, 80%, 85%, 90%, or any percentage derivable therein, of the amino acid residues from the full-length RT amino acid sequence. In some aspects, the fragment of the reverse transcriptase enzyme may comprise an amino acid sequence with at least, equal to, or at most 50%, 60%, 70%, 80%, 90%, 100% or any percentage derivable therein, amino acid sequence identity to the overlapping fragment of the reverse transcriptase. The region of overlap can be ascertained by a person of ordinary skill in the art, for example by using various sequence alignment and / or bioinformatics tools available in the field.

[0306] In some aspects, the fragment of the reverse transcriptase enzyme comprises at least, equal to, or at most 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,301320428.1 - 54 -269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914,301320428.1 - 55 -915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000 consecutive amino acids from the N-terminal, C-terminal or full-length wild-type RTase protein, or any variant thereof. In some aspects, the fragment of the reverse transcriptase comprises about 25-50, or about 50-100, or about 100-150, or about 150-200, or about 200-250, or about 250-300, or about 300-350, or about 350-400, or about 400-450, or about 450-500 consecutive amino acid residues from the N-terminal, C-terminal, or full-length wild-type RTase protein, or any variant thereof.

[0307] In some aspects, the fragment of the reverse transcriptase enzyme is partially or completely non-functional, as can be determined by a suitable RT assay. In some aspects, the RTase fragment is less than, or equal to, or about 0%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% functional as compared to the wild-type RT enzyme, as measured using a suitable reverse transcriptase enzyme assay. Suitable RT assays are well known in the art, for example RT-PCR which combines reverse transcription and PCR to amplify and quantify specific RNA targets; RT-qPCR which combines reverse transcription and quantitative PCR to amplify and quantify specific RNA targets.

[0308] In some aspect, the RTase from which the fragment is derived may be any RTase known in the art, or functional variant thereof. These include RTases found in viruses, bacteria, plants and animals. Non-limiting examples include, Human T-Cell Leukemia Virus RTase, Hepadnavirus RTase, Moloney murine leukemia virus (MMLV) RTase, avian myeloblastosis virus (AMV) RTase, human immunodeficiency virus (HIV) RTase-p51, HIV RTase-p66. In some aspects, the RTase is not HIV RTase. In some aspects, the RTase is not HIV RTase-p51. In some aspects, the RTase is not HIV RTase-p66. In some aspects, the RTase is not AMV RTase. In some aspects, the RTase is not Human T-Cell Leukemia Virus RTase. In some aspects, the RTase is not Hepadnavirus RTase. In some aspects, a polypeptide construct comprises a fragment of an RTase, for example, Moloney murine leukemia virus (MMLV) RTase, avian myeloblastosis virus (AMV) RTase, human immunodeficiency virus (HIV) RTase or a functional variant thereof. In some aspects, a polypeptide construct comprises a fragment from an RTase that is enzymatically active at a temperature below 46 °C, 45 °C, 44 °C, 43 °C, 42 °C, 41 °C, or 40 °C. In some aspects, the RTase is enzymatically active at 37 °C.

[0309] In some aspects, the fragment of the RTase comprises at least about 25-50, or about 50-100, or about 100-150, or about 150-200, or about 200-250, or about 250-300, or about 300-301320428.1 - 56 -350, or about 350-400, or about 400-450, or about 450-500 consecutive amino acid residues from an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 6, or 147 or a sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity thereto.

[0310] In some aspects, the fragment of the RTase is encoded by a nucleic acid sequence comprising at least about 75-150, or about 150-300, or about 300-450, or about 450-600, or about 600-750, or about 750-900, or about 900-1050, or about 1050-1200, or about 1200-1350, or about 1350-1500 consecutive nucleic acids from a polynucleotide sequence as set forth in any one of SEQ ID NOs: 1, 3 5, or 146 or a sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity thereto.

[0311] In some aspects, the fragment of the RTase is encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOs: 7-30, 148, and 150 or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0312] In some aspects, the fragment of the RTase is an N-terminal fragment of MMLV RTase encoded by a nucleic acid sequence as set for in any one of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 148 or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0313] In some aspects, the fragment of the RTase is an C-terminal fragment of MMLV RTase encoded by a nucleic acid sequence as set for in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 150 a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.301320428.1 - 57 -

[0314] In some aspects, the fragment of the RTase comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 117-142, 149 or 151 or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0315] In some aspects, the fragment of the RTase is an N-terminal fragment of MMLV RTase comprising an amino acid sequence as set for in any one of SEQ ID NOs: 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, or 137 or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0316] In some aspects, the fragment of the RTase is an C-terminal fragment of MMLV RTase comprising an amino acid sequence as set for in any one of SEQ ID NOs: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138, or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0317] In some aspects, the fragment of the RTase is an N-terminal fragment of HIV RTase encoded by a nucleic acid sequence as set for in any one of SEQ ID NOs: 27, or 29, or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0318] In some aspects, the fragment of the RTase is an C-terminal fragment of HIV RTase encoded by a nucleic acid sequence as set for in any one of SEQ ID NOs: 28, or 30, or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0319] In some aspects, the fragment of the RTase is an N-terminal fragment of HIV RTase comprising an amino acid sequence as set for in any one of SEQ ID NOs: 139, or 141, or a301320428.1 - 58 -sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0320] In some aspects, the fragment of the RTase is an C-terminal fragment of HIV RTase encoded by a nucleic acid sequence as set for in any one of SEQ ID NOs: 141, or 142, or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0321] In some aspects, a polypeptide construct disclosed herein comprises a fragment of an RTase as disclosed herein, a dimerization moiety and a targeting moiety. In some aspects, the one or more dimerization moieties on corresponding fragments (N and C-terminal) of the RTases facilitates the interaction between the two corresponding fragments to form a complex, wherein the interaction results in the complex having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or any percentage derivable therein, functionality of the reverse transcriptase from which the fragments were derived. Exemplary dimerization moieties are provided herein below.

[0322] In some aspects, the polypeptide construct disclosed herein comprises a fragment of an RTase, a dimerization moiety, a split intein and a targeting moiety. Inteins and protein transsplicing domains are described further below. In some aspects, the reverse transcriptase (RTase) or RTase fragment(s) used in the split-RTase systems (including split-RTase systems that incorporate inteins and protein trans-splicing domains) comprise one or more mutations selected to improve compatibility with the intein-mediated trans-splicing reaction. In certain aspects, the RTase comprises a T128C mutation (e.g., to provide a cysteine at the intein +1 extein position), which can promote efficient protein trans-splicing in split-RTase constructs designed to be reconstituted via intein excision and ligation. In further aspects, the RTase comprises a set of mutations at positions 128-130, such as T128C, V129W, and P130N (collectively, “CWN”), which has been reported as a prevalent extein sequence associated with efficient intein-mediated trans-splicing and is contemplated herein as a variant for improved trans-splicing performance. In some aspects, full-length RTases comprising T128C and / or CWN mutations retain reverse transcription activity comparable to a wild-type full-length RTase, and thus these mutations can be incorporated into RTases and RTase fragments disclosed herein without materially compromising reverse transcription function.301320428.1 - 59 -

[0323] In some aspects, the fragment of the RTase is an C-terminal fragment of MMLV RTase comprising a T128C mutation, wherein the fragment is encoded by a nucleic acid sequence as set for in SEQ ID NO: 184 or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto. In some aspects, the fragment of the RTase is an C-terminal fragment of MMLV RTase comprising an amino acid sequence as set forth in SEQ ID NO: 185, or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0324] In some aspects, the fragment of the RTase is an C-terminal fragment of MMLV RTase comprising a T128C, V129W, and P130N mutation, wherein the fragment is encoded by a nucleic acid sequence as set for in SEQ ID NO: 150 or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto. In some aspects, the fragment of the RTase is an C-terminal fragment of MMLV RTase comprising an amino acid sequence as set forth in SEQ ID NO: 151, or a sequence that is, is at least, or is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0325] In some aspects, the targeting moiety may comprise for example, an Fc binding protein, or a variant thereof; an antibody, or a variant thereof (for example, a nanobody), an oligonucleotide, a peptide, a receptor, an aptamer, a ligand, a small molecule, or any combination thereof. In some aspects, a polypeptide construct comprises a RTase, or a functional variant thereof; and a Fc / Fab binding protein, or a functional variant thereof.

[0326] In some aspects, a polypeptide construct and / or polynucleotide construct disclosed herein may further comprise a fluorophore. In some aspects, a polypeptide construct and / or polynucleotide construct disclosed herein does not comprise a fluorophore. In some aspects, the fluorophore comprises, consists essentially of, or consists of Green Fluorescent Protein (GFP), eGFP, Red Fluorescent Protein (RFP), Teal Fluorescent Protein (TFP), Blue301320428.1 - 60 -Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), miRFP, cerulean fluorescent protein (CFP), eCyanFP, mCherry, m Venus, mOrange, mTurquoise, tdTomato, aminocoumarin, fluorescein, texas red, Alexa Fluor dyes (e.g. Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 350, Alexa Fluor 532, and Alexa Fluor 700), Cy dyes (e.g. Cy3, Cy5), DyLight dyes, FITC, or Rhodamine, or functional variants thereof. In some aspects, polypeptide construct and / or polynucleotide constructs described herein do not comprise a fluorophore. In some aspects, a polypeptide construct and / or polynucleotide construct does not comprise Green Fluorescent Protein (GFP), eGFP, Red Fluorescent Protein (RFP), Teal Fluorescent Protein (TFP), Blue Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), miRFP, cerulean fluorescent protein (CFP), eCyanFP, mCherry, mVenus, mOrange, mTurquoise, tdTomato, aminocoumarin, fluorescein, texas red, Alexa Fluor dyes (e.g. Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 350, Alexa Fluor 532, and Alexa Fluor 700), Cy dyes (e.g. Cy3, Cy5), DyLight dyes, FITC, or Rhodamine, or functional variants thereof.B. Dimerization domains

[0327] In some aspects, a polypeptide construct and / or polynucleotide construct as disclosed herein, also comprises a dimerization moiety. In some aspects, a polypeptide construct and / or a polynucleotide construct as disclosed herein comprises a multimerization moiety. In some aspects, In some aspects, dimerization may comprise interaction of two fragments of a polypeptide, each fragments comprising a dimerization moiety, wherein the dimerization moieties are capable of specifically binding to each other, or wherein the dimerization moieties can be induced to bind each other. In some aspects, the polypeptide construct may comprise a first dimerization moiety which is functional in binding to a corresponding second dimerization moiety on a second polypeptide construct. In some aspects, the first and the second dimerization moieties are both polypeptides, referred herein as a first and second dimerization domains (for example, a FRB, an FKBP domain, a SH3 domain, leucine zipper). The dimerization domain typically includes specific amino acid sequences and structural motifs that promote binding through non-covalent interactions such as hydrogen bonds, ionic interactions, van der Waals forces, and hydrophobic effects. In some cases, disulfide bonds can also stabilize the dimer. In some aspects, the first and / or the second dimerization moiety is not a polypeptide (for example, biotin, chelators). Non-limiting examples of polypeptide dimerization domain and corresponding binding partners include FRB-FKBP / FK506, Maltose-binding protein(MBP) / amylose, leucine zipper / leucine zipper,301320428.1 - 61 -Fos / Jun dimerization domains, HA-tag / anti-HA antibodies, RGD peptide / integrins, PDZ domain / PDZ-ligand, Protein A / IgG, streptavidin / Strep-tag, or FLAG tag / anti-FLAG-tag antibody. In some aspects, the dimerization may be caused by proximity of the two dimerization partners. In some aspects, the dimerization may need to be induced by a small molecule, a drug, a change in environment or any combination thereof. In some aspects, the first and second dimerization domains are FRB and FKBP respectively, wherein the dimerization can be induced in the presence of a small molecule rapamycin or rapamycin analog FK506. In some aspects, the dimerization domain is FRB, wherein the dimerization domain comprises an amino acid sequence as set forth in SEQ ID NO: 34, or a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto. In some aspects, the dimerization domain is FKBP, wherein the dimerization domain comprises an amino acid sequence as set forth in SEQ ID NO: 32, or a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0328] In some aspects, the current disclosure also encompasses a polynucleotide sequence comprising a nucleic acid sequence encoding the dimerization domains as disclosed herein. In some aspects, the polynucleotide sequence comprises a nucleic acid sequence encoding a fragment of the RTase operably linked, with or without a sequence encoding a linker sequence, to a nucleic acid sequence encoding the dimerization domain. In some aspects, the dimerization domain is FRB, and the polynucleotide sequence encoding the dimerization domain can comprise a nucleic acid sequence as set forth in any one of SEQ ID NO: 33, or a nucleic acid sequence with at least 60% identity thereto. In some aspects, the dimerization domain in FKBP, and the polynucleotide sequence encoding the dimerization domain can comprise a nucleic acid sequence as set forth in any one of SEQ ID NO: 31, or a nucleic acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto. The polynucleotide can be an isolated DNA, a plasmid, a transposon, a viral vector, a genome integrated polynucleotide, or a chromosome.

[0329] Similar to dimerization domains disclosed herein, other non-polypeptide dimerization moieties, or dimerization moieties comprising one polypeptide and one non-polypeptide dimerization moiety may comprise structural motifs that promote binding through non-covalent interactions such as hydrogen bonds, ionic interactions, van der Waals forces,301320428.1 - 62 -and hydrophobic effects, or through covalent interactions. Non-limiting examples of peptide-non-polypeptide dimerization partners include, but are not limited to avidin / biotin, streptavidin / biotin, SNAP-tag / benzylguanine, CLIP-tag / benzylcytosine derivatives, GST-tag / glutathione, HisTag / Nickel and Halotag / HaloTag ligand.

[0330] In some aspects, the dimers are heterodimers, formed by dimerization of the N-terminal and the C-terminal fragment of a RTase as disclosed herein.C. Inteins or protein trans-splicing domains

[0331] In some aspects, the polypeptide constructs disclosed herein further comprise an intein (also referred to as an intervening protein sequence) or split intein system configured to mediate protein trans-splicing between two separate polypeptide constructs (e.g., between a first split-RTase polypeptide and a second split-RTase polypeptide). In certain aspects, intein-mediated trans-splicing is used in combination with the dimerization domains described above (e.g., FRB and FKBP) such that induced proximity (e.g., via rapamycin) promotes formation of an active enzyme through covalent reconstitution.

[0332] In some aspects, the current disclosure describes positioning a split intein between a split-RTase fragment and a dimerization domain (e.g., between split-RTase and FRB / FKBP), such that, upon induction of dimerization, the two split-RTase fragments undergo protein trans-splicing, the intein fragments are excised, and the split-RTase fragments are joined by a peptide bond to form a full-length reverse transcriptase. In some aspects, this intein-containing arrangement is used to improve reverse transcription activity relative to constructs lacking inteins.

[0333] In certain aspects, a split intein comprises the Npu DnaE split intein, including an intein N fragment and an intein C fragment (also referred to herein as intein-N and intein-C). In some aspects, the first and the second polypeptide of the current disclosure may comprise an intein N fragment and an intein C fragment respectively. Exemplary sequences for an intein N fragment and an intein C fragment are provided herein. In some aspects, the intein-N can comprise an amino acid sequence as set forth in SEQ ID NO: 161, or an amino acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity thereto. In some aspects, the current disclosure also encompasses polynucleotide sequences encoding the disclosed intein N fragment. In some301320428.1 - 63 -aspects, the polynucleotide sequence encoding the nanobody comprises a nucleic acid sequence as set forth in SEQ ID NO: 160, or a nucleic acid sequence with at least 60% identity thereto. In some aspects, the polynucleotide sequence encoding the intein N fragment comprises a nucleic acid sequence as set forth in SEQ ID NO: 160, or a nucleic acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity thereto.

[0334] In some aspects, the intein-C can comprise an amino acid sequence as set forth in SEQ ID NO: 163, or an amino acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity thereto. In some aspects, the current disclosure also encompasses polynucleotide sequences encoding the disclosed intein N fragment. In some aspects, the polynucleotide sequence encoding the nanobody comprises a nucleic acid sequence as set forth in SEQ ID NO: 162, or a nucleic acid sequence with at least 60% identity thereto. In some aspects, the polynucleotide sequence encoding the intein N fragment comprises a nucleic acid sequence as set forth in SEQ ID NO: 162, or a nucleic acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity thereto.

[0335] In some aspects, the split-RTase constructs are configured so that the +1 extein position for the intein system is a residue compatible with efficient protein trans-splicing, and in certain aspects the +1 position is cysteine (C) or another similar amino acid. In some aspects, to improve trans-splicing efficiency, one or more mutations are introduced at or near the split junction, including mutation of the amino acids corresponding to T128, V129, and P130 to C128, W129, and N130 (i.e., “CWN”) corresponding to the MMLV RTase sequence, as “CWN” is a prevalent sequence for efficient intein-mediated trans-splicing. In some aspects, a full-length RTase containing a T128C mutation and / or a CWN mutation at the 128-130 sites exhibits reverse transcription activity similar to wild-type full-length RTase.

[0336] In some aspects, the reverse transcriptase is split at or near a site selected to provide suitable extein residues for the intein system, and in certain aspects the RTase is split from the 127 site and includes the T128C mutation (and optionally V129W and P130N), which is301320428.1 - 64 -described as being selected because T128 is close to a previously used split site and is a nearby residue with similar properties to cysteine. In some aspects, split-RTases split from site 127 with inteins can undergo protein trans-splicing and form a full-length RTase band in the presence of rapamycin under 37°C treatment for 2 hours. In some aspects, other +1 position designs (e.g., using C90 or S142C) may also be tested, and in certain aspects such designs may exhibit lower trans-splicing efficiency relative to the T128C version (split from site 127 and having the T128C mutation).

[0337] In some aspects, the intein-containing split-RTase constructs are further combined with targeting moieties (including, for example, antibody-binding nanobodies) to recruit each split-RTase construct to targets of interest, and upon recruitment and rapamycin treatment, the split constructs undergo trans-splicing around the target and reverse transcribe RNA around RBP binding sites with higher specificity and lower background relative to pAG-RTase ARTR-seq. In some aspects, the disclosure contemplates that multiple combinations of such split-RTase constructs can be assembled (including constructs fused to different antibody -binding nanobodies) and validated for trans-splicing and / or rapamycin-dependent increases in reverse transcription activity. In some aspects, one or more linker sequences may be included adjacent to intein fragments to promote proper folding, spacing, and / or activity of the fusion proteins.D. Targeting moiety

[0338] A targeting moiety, as used herein, can be any specific molecule or structure that directs an RTase to a particular biological target. In some aspects, the biological target can be an RNA binding protein (RBP), an RNA modification site, a nucleic acid or an antibody. In some aspects, the biological target can be in proximity, or part of an RNA molecule, which can act as a template for a reverse transcription reaction. In some aspects, wherein the biological target is a RBP, the targeting moiety may be referred to as a RBP-targeting moiety.

[0339] In some aspects, the targeting moiety can comprise an Fc binding protein, or a variant thereof; an antibody, or a variant thereof, an oligonucleotide or a variant thereof, a peptide, a receptor, an aptamer, a ligand, a small molecule, a nucleoside, or any combination thereof. In some aspects, the targeting moiety is an Fc binding protein or peptide. An Fc binding protein or peptide binds specifically to an Fc (Fragment crystallizable) region of an immunoglobulin (an antibody, or an antibody like polypeptides, for example, polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody variant, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, single chain antibody, single domain antibody,301320428.1 - 65 -nanobodies, diabodies, a bi-specific antibody, a multi-specific antibody, a DARPin, or any variant of each thereof).

[0340] Non-limiting examples of targeting moi eties that bind the Fc region of an antibody include, protein A, protein G, protein A / G (pAG), protein L, anti-rabbit IgG, and / or anti-mouse IgG. In some aspects, a targeting moiety that binds the Fc region of an antibody expressly does not include protein A, protein G, protein A / G (pAG), protein L, anti-rabbit IgG, and / or antimouse IgG. In some aspects, the Fc binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 54, 56, 58, or 60 or an amino acid sequence at least 60% identical thereto. In some aspects, the Fc binding protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 54, 56, 58, or 60, or an amino acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto. In some aspects, the current disclosure also encompasses a polynucleotide sequence comprising a nucleic acid sequence encoding the targeting moiety as disclosed herein. In some aspects, the polynucleotide sequence comprises a nucleic acid sequence encoding a fragment of the RTase operably linked, with or without a sequence encoding a linker sequence, to a nucleic acid sequence encoding the fragment of the RTase or the dimerization domain. In some aspects, the current disclosure also encompasses nucleic acid sequences encoding the disclosed Fc binding protein. In some aspects, the nucleic acid sequence encoding the Fc binding protein can comprise a sequence as set forth in any one of SEQ ID NOs: 53, 55, 57, or 59 or a nucleic acid sequence with at least 60% identity thereto. In some aspects, the polynucleotide sequence encoding the Fc binding protein can comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs: 53, 55, 57, or 59 or a nucleic acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto. The polynucleotide can be an isolated DNA, a plasmid, a transposon, a viral vector, a genome integrated polynucleotide, or a chromosome.

[0341] In some aspects, the Fc binding targeting moiety can bind the Fc region of an antibody, wherein the antibody specifically binds a RNA binding protein (RBP). In some aspects, the RBP can be a transcription factor, a splicing factor, RNA helicase, ribonuclease, RNA polymerase, translation initiation factor, or a ribosomal protein. Non-limiting examples301320428.1 - 66 -of RBPs include YTHDF1, YTHDF2, YTHDC1, HuR, PTB, Musashi, eIF4E, FMRP, LARP1, IMP, hnRNP family proteins, Lin28, AUF1, IGF2BP, FUBP1, LIN28B, RBM5, FUS, TIA1, TTP, QKI, MBNL, CELF, NONO, DDX5, RBM10, SAFB, TDP-43, Ataxin-2, hnRNP A / B, C9orf72, hnRNP H / F, Matrin 3 (MATR3), Pur-alpha, TAF15, Huntingtin, RBFOX, SMN, ELAVL, Ro (SSA) and La (SSB) Proteins, hnRNP, Roquin, Staufenl, NF90 / NF110, ILF3, SF3B1, SRSF2, U2AF1, ZRSR2, PRPF8, PRPF31, SNRNP200, HNRNPA1, HNRNP A2B1, NELFE, CPEB1, SRSF1, NO VAI, NOVA2, G3BP1, PTBP1, RBFOX2, HNRNPC, or any variants thereof, or any combinations thereof. In some aspects, the RBP is G3BP1, PTBP1, RBFOX2, HNRNPC, YTHDF1, YTHDF2, YTHDC1, or any or any variants thereof, or any combinations thereof. In some aspects, RBPs expressly do not include YTHDF1, YTHDF2, YTHDC1, HuR, PTB, Musashi, eIF4E, FMRP, LARP1, IMP, hnRNP family proteins, Lin28, AUF1, IGF2BP, FUBP1, LIN28B, RBM5, FUS, TIA1, TTP, QKI, MBNL, CELF, NONO, DDX5, RBM10, SAFB, TDP-43, Ataxin-2, hnRNP A / B, C9orf72, hnRNP H / F, Matrin 3 (MATR3), Pur-alpha, TAF15, Huntingtin, RBFOX, SMN, ELAVL, Ro (SSA) and La (SSB) Proteins, hnRNP, Roquin, Staufenl, NF90 / NF110, ILF3, SF3B1, SRSF2, U2AF1, ZRSR2, PRPF8, PRPF31, SNRNP200, HNRNPA1, HNRNP A2B1, NELFE, CPEB1, SRSF1, NOVAI, NOVA2, G3BP1, PTBP1, RBFOX2, HNRNPC, or any variants thereof, or any combinations thereof. In some aspects, the RBP expressly does not include G3BP1, PTBP1, RBFOX2, HNRNPC, YTHDF1, YTHDF2, YTHDC1, or any or any variants thereof, or any combinations thereof. In some aspects, the Fc binding targeting moiety can bind an antibody that specifically targets a RNA modification site. Non-limiting examples of RNA modifications that can be targeted include m6C, m5C, nVA, m7G, or a pseudouridine modification. In some aspects, Fc binding protein can specifically bind a secondary binding agent, for example a secondary antibody, that binds a primary antibody targeting a RBP, or a RNA modification site.

[0342] In some aspects, the targeting moiety can be an antibody. As indicated, the term antibody is applied broadly here to comprise a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a humanized antibody, an antibody variant, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, single chain antibody, single domain antibody, nanobodies, diabodies, a bi-specific antibody, a multi-specific antibody, a DARPin, a polypeptide comprising the CDRs of an antibody, or a variant of each thereof. In some aspects, the antibody can specifically bind an RBP. In some aspects, any RBP can be used as a bait for the targeting moiety. The RBP can be a transcription factor, a splicing factor, RNA helicase, ribonuclease, RNA polymerase, translation initiation factor, or a ribosomal protein. Non-limiting examples of RBPs include YTHDF1, YTHDF2,301320428.1 - 67 -YTHDC1, HuR, PTB, Musashi, eIF4E, FMRP, LARP1, IMP, hnRNP family proteins, Lin28, AUF1, IGF2BP, FUBP1, LIN28B, RBM5, FUS, TIA1, TTP, QKI, MBNL, CELF, NONO, DDX5, RBM10, SAFB, TDP-43, Ataxin-2, hnRNP A / B, C9orf72, hnRNP H / F, Matrin 3 (MATR3), Pur-alpha, TAF15, Huntingtin, RBFOX, SMN, ELAVL, Ro (SSA) and La (SSB) Proteins, hnRNP, Roquin, Staufenl, NF90 / NF110, ILF3, SF3B1, SRSF2, U2AF1, ZRSR2, PRPF8, PRPF31, SNRNP200, HNRNPA1, HNRNP A2B1, NELFE, CPEB1, SRSF1, NOVAI, NOVA2, G3BP1, PTBP1, RBFOX2, HNRNPC, or any variants thereof, or any combinations thereof. In some aspects, the RBP is G3BP1, PTBP1, RBFOX2, HNRNPC, YTHDF1, YTHDF2, YTHDC1, or any variants thereof, or any combinations thereof. In some aspects the antibody can specifically bind a RNA modification site. Non-limiting examples of RNA modifications that can be targeted include m6C, m5C, mxA, m7G, or a pseudouridine modification. In some aspects, the antibody (e.g., a secondary antibody) can specifically bind the Fc region of another antibody, that specifically binds a RBP, or a RNA modification. In some aspects, the secondary antibody can be an anti-rabbit IgG, and / or anti-mouse IgG. Both anti-rabbit IgG and anti-mouse IgG are categorized under the IgG class of antibodies. Each IgG antibody is composed of two identical heavy chains, approximately 50 kDa each, and two identical light chains, approximately 25 kDa each. In some aspects, the secondary antibody can comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 62 or 64, or an amino acid sequence at least 60% identical thereto. In some aspects, the secondary antibody can comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 62, or 64, or an amino acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity thereto. In some aspects, the current disclosure also encompasses polynucleotide sequences encoding the disclosed secondary antibody. In some aspects, the polynucleotide sequence encoding the secondary antibody comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 61 or 63, or a nucleic acid sequence with at least 60% identity thereto. In some aspects, the polynucleotide sequence encoding the secondary antibody comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 61 or 63, or a nucleic acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity thereto.301320428.1 - 68 -

[0343] In some aspects, the targeting moiety comprises a nanobody (single-domain antibody fragment) that is configured to bind an immunoglobulin used to target an RNA-binding protein (RBP) or other target of interest, thereby localizing the split reverse transcriptase (RTase) activity to the target-bound complex. In some aspects, such nanobodybased targeting moieties are provided as fusions with split RTase fragments in Split-ARTR-seq to reduce background and / or enable detection of co-binding by recruiting split RTase fragments to one or more primary antibodies.

[0344] In some aspects, provided herein are nanobodies that specifically bind a primary or a secondary antibody. In some aspects, the nanobody specifically binds the constant region of an antibody. In some aspects, the nanobody may bind the Fc region or the Fab region of the relevant antibody. In some aspects, the nanobody may specifically bind a rabbit antibody or a mouse antibody. In some aspects, the nanobody is fused (directly or via one or more linkers) to the disclosed split RTase fragment such that, when the nanobody binds an antibody that itself binds a target (e.g., an RBP), the split RTase fragment is recruited to the target region.

[0345] In some aspects, the nanobody-split RTase fusions are used such that one split RTase fragment is recruited via a nanobody to a first antibody (e.g., a rabbit antibody), and a second split RTase fragment is recruited via a nanobody to a second antibody (e.g., a mouse antibody), thereby enabling recruitment of the two split fragments to two different antibody-labeled targets in a sample. In some aspects, this configuration enables detection of co-binding by using, for example, a rabbit antibody to target a first protein of interest and a mouse antibody to target a second protein of interest, while fusing rabbit- and mouse-reactive nanobodies to corresponding split RTase fragments.

[0346] Non-limiting examples of nanobodies that may be included in the polypeptide constructs of the current disclosure, and in the methods disclosed herein include RbNbTP897, RbNbTP896, MsNbTP1170, and MsNbTP1107. In some aspects, the nanobody can comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 153, 155, 157 and 159, or an amino acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity thereto. In some aspects, the current disclosure also encompasses polynucleotide sequences encoding the disclosed nanobodies. In some aspects, the polynucleotide sequence encoding the nanobody comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 152, 154, 156 and 158, or a nucleic acid sequence with at least 60% identity thereto. In some aspects, the polynucleotide301320428.1 - 69 -sequence encoding the nanobody comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 152, 154, 156 and 158, or a nucleic acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity thereto.

[0347] In some aspects, the nanobody-split RTase constructs further incorporates a dimerization domain (e.g. a rapamycin-inducible heterodimerization domains FRB and FKBP) to bring the split RTase fragments into proximity under, for example, rapamycin induction. In some aspects, the nanobody-split RTase constructs additionally include split intein components (for example, Npu DnaE intein-N and intein-C) positioned between the split RTase fragments and the dimerization domains to support protein trans-splicing, such that, upon dimerization, or induction of dimerization (for example, by the addition of rapamycin) the two separate split RTases can undergo trans-splicing and form a complete RTase via a peptide bond. In some aspects, such trans-splicing is performed at about 37°C for about 2 hours in the presence of rapamycin to form a full-length RTase.

[0348] In some aspects, the targeting moiety can be a small molecule or a ligand. In some aspects, the small molecule can specifically bind a RBP. The RBP can be a transcription factor, a splicing factor, RNA helicase, ribonuclease, RNA polymerase, translation initiation factor, or a ribosomal protein. Non-limiting examples of RBPs include YTHDF1, YTHDF2, YTHDC1, HuR, PTB, Musashi, eIF4E, FMRP, LARP1, IMP, hnRNP family proteins, Lin28, AUF1, IGF2BP, FUBP1, LIN28B, RBM5, FUS, TIA1, TTP, QKI, MBNL, CELF, NONO, DDX5, RBM10, SAFB, TDP-43, Ataxin-2, hnRNP A / B, C9orf72, hnRNP H / F, Matrin 3 (MATR3), Pur-alpha, TAF15, Huntingtin, RBFOX, SMN, ELAVL, Ro (SSA) and La (SSB) Proteins, hnRNP, Roquin, Staufenl, NF90 / NF110, ILF3, SF3B1, SRSF2, U2AF1, ZRSR2, PRPF8, PRPF31, SNRNP200, HNRNPA1, HNRNP A2B1, NELFE, CPEB1, SRSF1, NOVAI, NOVA2, G3BP1, PTBP1, RBFOX2, HNRNPC, or any variant thereof, or any combinations thereof. In some aspects, the RBP is G3BP1, PTBP1, RBFOX2, HNRNPC, YTHDF1, YTHDF2, YTHDC1, or any variant thereof, or any combinations thereof. Non-limiting examples of ligands that can bind RBPs include certain quinoline derivatives, small molecule inhibitors like isoxazole, indole-based compounds, benzothiazole derivatives, and pyrimidine analogs.

[0349] In some aspects, the targeting moiety is an oligonucleotide or a variant thereof, wherein the oligonucleotide specifically binds a RNA or a DNA sequence of interest. In some301320428.1 - 70 -aspects the oligonucleotide binds a nucleic acid sequence at or in proximity of the RNA that can act as a template for the RT enzyme. In some aspects, the oligonucleotide comprises, consists essentially of, consists an RNA molecule, DNA molecule, LNA (locked nucleic acid), PNA (peptide nucleic acid), morpholino oligonucleotide, phosphorothioate oligonucleotide, gapmers, 2'-Fluoro-modified RNA, or an aptamer sequence. In some aspects the oligonucleotide binds a nucleic acid sequence in proximity of the RNA that can act as a template for the RT enzyme. In some aspects, the oligonucleotide or variant thereof further comprise a label, a barcode, a modified nucleotide, indices, or an affinity tag sequences. Nonlimiting examples of labels and tags include streptavidin and Avitag™, biotin, or a fluorophore. Non-limiting examples of modifications that can be incorporated into the oligonucleotide include modified bases, modified sugar moieties, and modified phosphate backbones. Examples of modified base moieties which can be incorporated at any position on its structure include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N~6-sopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methyl cytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, methoxyarninomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-S-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, 2,6-diaminopurine and biotinylated analogs, amongst others. Examples of modified sugar moieties which may be used to modify nucleotides at any position on its structure include, but are not limited to arabinose, 2-fluoroarabinose, xylose, and hexose, or a modified component of the phosphate backbone, such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof.

[0350] In some aspects, the RT enzyme may bind the tag sequence, for example a streptavidin-conjugated RT enzyme may bind the biotinylated oligonucleotide. In some aspects, the RT enzyme may be covalently linked to the oligonucleotide. In some aspects, the oligonucleotide specifically binds an RNA modification site or an RBP as disclosed herein.301320428.1 - 71 -

[0351] In some aspects, the targeting moiety can be an aptamer. Aptamers are oligomers of artificial ssDNA, RNA, XNA (Xeno nucleic acids), or peptides that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities, with variable levels of off-target binding and are sometimes classified as chemical antibodies. Peptide aptamers can include a peptide loop (which is specific for a target protein) attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody. The variable loop length is typically 8 to 20 amino acids (e.g., 8 to 12 amino acids), and the scaffold can be any protein which is stable, soluble, small, and non-toxic (e.g., thioredoxin-A, stefin A triple mutant, green fluorescent protein, eglin C, and cellular transcription factor Spl). Peptide aptamer selection can be made using different systems, such as the yeast two-hybrid system (e.g., Gal4 yeast-two-hybrid system) or the LexA interaction trap system. In some aspects, the peptide aptamer specifically binds the RBP as provided herein or a RNA modification site as provided herein.

[0352] Nucleic acid aptamers are single-stranded nucleic acid (DNA or RNA) ligands that function by folding into a specific globular structure that dictates binding to target proteins, nucleic acids, or other molecules with high affinity and specificity, as described by Osborne et al., Curr. Opin. Chem. Biol. 1:5-9, 1997; and Cerchia et al., FEBS Letters 528:12-16, 2002. In particular aspects, aptamers are small (15 KD; or between 15-80 nucleotides or between 20-50 nucleotides). Aptamers are typically isolated from libraries consisting of 1014-1015random oligonucleotide sequences by a procedure termed SELEX (systematic evolution of ligands by exponential enrichment). Further methods of generating aptamers are described in, for example, U.S. Pat. Numbers 6,344,318; 6,331,398; 6,110,900; 5,817,785; 5,756,291; 5,696,249; 5,670,637; 5,637,461; 5,595,877; 5,527,894; 5,496,938; 5,475,096; and 5,270,16. Spiegelmers are similar to nucleic acid aptamers except that at least one P-ribose unit is replaced by P-D-deoxyribose or a modified sugar unit selected from, for example, P-D-ribose, a-D-ribose, P-L-ribose. In some aspects, an aptamer for use in the current disclosure specifically binds a RBP, or a RNA modification site. Non-limiting examples of aptamers that bind RBPs, for example transcription factors, include aptamers to NF-KB, Spl, AP-1, c-Myc, STAT3, or TATA-binding protein.

[0353] Some aspects of the present disclosure are directed to and / or comprise polypeptide constructs comprising the targeting moiety fused to a RTase.301320428.1 - 72 -E. Other elements of the polypeptide construct

[0354] In some aspects, the polypeptide construct may further comprise a linker sequence. Suitable linker sequences are well known in the art, and can comprise a glycine-serine linker. The term “linker” according to the disclosure relates to a peptide between two protein domains to connect said domains. There is no particular limitation regarding the linker sequence. However, it is preferred that the linker sequence reduces steric hindrance between the two peptide domains, and is well translated. The linker can comprise 3 or more, 6 or more, 9 or more, 10 or more, 15 or more, 20 or more and in some aspects, up to 100, up to 90, up to 80, up to 70 or up to 60, up to 50, up to 45, up to 40, up to 35, or up to 30 amino acids. The linker may be enriched in glycine and / or serine amino acids. In some aspects, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or more of the amino acids of the linker are glycine and / or serine. In some aspects, a linker is substantially composed of the amino acids glycine and serine. In some aspects, the linker is a Glycine / Serine linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n or (Gly-Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=l, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10. In some aspects, the linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3. In another aspect, the linkers include multiple repeats of (GlyxSer)n, where x=l, 2, 3, 4 or 5 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some aspects, the linker comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 42, 44, 46, 48, or 50, or an amino acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, sequency identity thereto. In some aspects, the linker sequence can be encoded by a polynucleotide sequence comprising a nucleic acid sequence as set forth in SEQ ID NOs: 41, 43, 45, 47, or 49, or a sequence at least 60% identical thereto. In some aspects, the linker sequence can be encoded by polynucleotide sequence comprising a nucleic acid sequence as set forth in any one of SEQ ID NOs: 41, 43, 45, 47, or 49 or a nucleic acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, sequency identity thereto.

[0355] In some aspects, the polypeptide construct can comprise one or more peptide tag sequences. Peptide tag sequences are well known in the art and can comprise purification tag, solubilization tags, and cleavage tags. Non-limiting examples of peptide tags include, the a301320428.1 - 73 -maltose binding protein (MBP) tag, a GST-tag, a FLAG tag, an HA tag, a His-tag, a SUMO-tag, a Trx-tag, or a Halo-tag. In some aspects, the purification tag comprises an amino acid sequence as set forth in SEQ ID NO: 36, 38, or 40, or an amino acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, sequency identity thereto. In some aspects, the peptide tag sequence can be encoded by a polynucleotide sequence comprising a nucleic acid sequence as set forth in SEQ ID NOs: 35, 37 or 39, or a sequence at least 60% identical thereto. In some aspects, the peptide tag sequence can be encoded by polynucleotide sequence comprising a nucleic acid sequence as set forth in any one of SEQ ID NOs: 35, 37 or 39 or a nucleic acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, sequency identity thereto.

[0356] In some aspects, the polypeptide construct as disclosed herein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 143-145, 169, 171, 173, 175, 177, 179, 181, or 183, or an amino acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, sequency identity thereto.

[0357] In some aspects, the first polypeptide construct comprising the N-terminal RTase, has an amino acid sequence as set for in any one of SEQ ID NOs: 145, 169, 173, 177, or 181, or an amino acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, sequency identity thereto. In some aspects, the second polypeptide construct, comprising the C-terminal fragment of an RTase comprises an amino acid sequence as set for in any one of SEQ ID NOs: 143, 144, 171, 175, 179, 183, or 185, or an amino acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, sequency identity thereto.301320428.1 - 74 -F. Polynucleotides

[0358] In some aspects, the current disclosure also encompasses a polynucleotide encoding the polypeptides disclosed herein. In some aspects, the polynucleotide encoding disclosed polypeptide constructs can be an isolated DNA, a plasmid, a transposon, a viral vector, a genome integrated polynucleotide, or a chromosome. In some aspects, a polynucleotide sequence can further comprise one or more regulatory sequences. A regulatory sequence refers to any genetic element that is known to drive or otherwise regulate expression of nucleic acids. Non-limiting examples include promoters, transcription terminators, enhancers, repressors, silencers, kozak sequences, polyA sequences, ribosome skipping sequences (for example sequences encoding P2A and T2A peptides) and the like. In some aspects, a regulatory sequence can, for example, be inducible, non-inducible, constitutive, cell-cycle regulated, metabolically regulated, and the like. A regulatory sequence may comprise a promoter. In some aspects, the nucleic acid sequence encoding the fusion polypeptide can be operably linked to the promoter. In some aspects, the promoter can be an inducible promoter, a constitutive promoter, a tissue specific promoter, a weak promoter, a strong promoter, or combinations thereof. In some aspects, the promoter may also comprise an enhancer sequence.

[0359] In some aspects, the polynucleotide sequence is an plasmid expression vector. Nonlimiting examples of plasmid expression vectors include the pET Series (e.g., pET-28a-c(+), pET-21a-d(+)); the pUC series (e.g., pUC18, pUC19), pGEX Series (e.g., pGEX-4T-l), pCMV Series (e.g., pCMV-Tag), pCDNA Series (e.g., pcDNA3.1), pYES2, and the pBAD Series (e.g., pB AD / My c-Hi s) .

[0360] In some aspects, the polynucleotide sequence may comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs: 114, 115, or 116 or a nucleic acid sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, sequency identity thereto.III. Compositions and systems

[0361] In some aspects, the current disclosure also encompasses a composition and systems comprsing said compositions, wherein the composition comprises a first polypeptide construct as disclosed herein, wherein the first polypeptide construct comprises a N-terminal fragment of a reverse transcriptase, or a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof, a first dimerization moiety, and301320428.1 - 75 -optionally a first targeting moiety; and / or a second polypeptide construct as disclosed herein, wherein the second polypeptide construct comprises a C-terminal fragment of the reverse transcriptase, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase, or any variant thereof, a second dimerization moiety that specifically binds to the first dimerization moiety, and optionally a second targeting moiety.

[0362] In some aspects, the disclosed compositions comprise a first polypeptide construct and a second polypeptide construct that together provide reverse transcriptase activity upon reconstitution, wherein each construct comprises a fragment of a reverse transcriptase enzyme (RTase) and may further comprise a dimerization moiety, a targeting moiety, and / or optional elements such as linkers, tags, or fluorophores as disclosed above. In some aspects, the first polypeptide construct comprises an N-terminal RTase fragment and the second polypeptide construct comprises a C-terminal RTase fragment, although the assignment of fragments to “first” and “second” polypeptides may be reversed. In some aspects, the RTase fragments have low reverse transcription activity when separated but higher activity when brought into proximity and / or induced to assemble, thereby reducing background signal.

[0363] In some aspects, fragments of the RTase, the dimerization domains and the targeting moieties are as disclosed herein above. In some aspects, the composition may further comprise one or more agents that help stabilize the polypeptide constructs. Non-limiting examples of agents that may be included in the composition include, but are not limited to glycerol, trehalose, sucrose, polyethylene glycol (PEG), bovine serum albumin (BSA), ammonium sulfate, dithiothreitol (DTT), reducing agents, EDTA, magnesium ions, potassium chloride, sodium azide, buffering agents and cryoprotectants.

[0364] In some aspects, the compositions and systems disclosed herein may comprise pairs of polypeptide constructs comprising a first polypeptide construct as disclosed herein comprising the N-terminal fragment of an RTase and a second polypeptide construct as disclosed herein comprising the C-terminal fragment of an RTase, which on dimerization and / or reconstitutionform a functional RTase. Non-limiting examples of such pairs are provided in the Table 1. In some aspects, the pairs of polypeptide constructs and / or polynucleotide constructs may be part of separate compositions, to be used simultaneously or in tandem in a method disclosed herein.301320428.1 - 76 -Table 1: Pairs of RTase fragments for use together in a transcriptase composition or a method of the current disclosureIV. Transcriptase mix

[0365] Non-limiting examples of molecules that can be incorporated into the transcriptase mix include primers (for example, an adapter RT -primer comprising an adapter primer and an RT primer sequence), dNTPs, buffer solutions, magnesium ions (Mg2+), and RNase inhibitor. In some aspects, the transcriptase composition comprises one or more polypeptide constructs disclosed herein, and transcriptase mix comprising one or more adapter RT primers, wherein the one or more adapter RT primer each comprises an adapter primer sequence and an RT primer sequence. An adapter primer in sequencing is a short, synthetic oligonucleotide used to facilitate the attachment of DNA fragments to a sequencing platform. It typically contains two parts: a complementary sequence that binds to the target DNA or RNA fragment and an adapter sequence that is recognized by the sequencing machinery. Without being bound by theory, adapter primers are in some aspects crucial in next-generation sequencing (NGS) workflows,301320428.1 - 77 -where they enable DNA fragments to be amplified and sequenced. They also allow for the incorporation of additional sequences like barcodes or indexes, which help identify different samples within a sequencing run. In some aspects, the adapter primers comprises a barcode.

[0366] In some aspects, the adapter RT primer of the current disclosure comprises an adapter sequence that is a short, synthetic oligonucleotide used to facilitate the attachment of DNA fragments to a sequencing platform, and which can further comprise barcodes, indexes etc , and aRT primer. RT primer sequences are short, single-stranded sequences of nucleotides used to initiate the synthesis of complementary DNA (cDNA) from an RNA template during reverse transcription. There are three common types of primers used for reverse transcription: oligo(dT) primers, random RT primers (for example, random 6-mers, 7-mers, 8-mers, 9-mers, 10-mers, 11-mers, 12-mers), and gene-specific primers. Oligo(dT) primers are used to bind the poly-A tail of eukaryotic mRNA, ensuring that only mRNA is reverse transcribed. Random RT primers are short, random sequences that bind to multiple locations on the RNA, allowing for the reverse transcription of all RNA species, including non-polyadenylated RNA. Genespecific primers are designed to bind to a specific region of a target RNA, facilitating the reverse transcription of particular genes or transcripts.

[0367] In some aspects, any type of suitable RT primer may be used, or may be expressly excluded from the transcriptase composition. In some aspects, the RT primers do not comprise oligo(dT) primers. In some aspects, the RT primer comprises a gene-specific primer. In some aspects, the RT primer does not comprise a gene specific primer. In some aspects, the RT primer is a random RT primer. In some aspects, the random RT primer is a hexamer. In some aspects, the random RT primer is not a hexamer, but oligonucleotide greater than six nucleotides in length. In some aspects, the random RT primer is at least 8 nucleotides in length, at least 9 nucleotides in length, or at least 10 nucleotides in length, at least 11 nucleotides in length, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, or more nucleotides in length. In some aspects, the random RT primer is between 8 and 20 nucleotides in length. In some aspects, the random RT primer is less than 20 nucleotides in length. In some aspects, the random RT primers comprise, consist, or consist essentially of at least septamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers, octadecamers, nonadecamers, or eicosamers.

[0368] In some aspects, the random RT primer may further comprise a reactive moiety such that it can react with a barcoded oligonucleotide comprising an antibody or targeting moiety, wherein the barcode oligonucleotide comprises a corresponding reactive moiety for301320428.1 - 78 -click chemistry. A reactive moiety of a random RT primer may be selected from the nonlimiting group consisting of azides, alkynes, nitrones (e.g., 1,3 -nitrones), strained alkenes (e.g., trans-cycloalkenes such as cyclooctenes or oxanorbomadiene), tetrazines, tetrazoles, iodides, thioates (e.g., phorphorothioate), acids, amines, and phosphates. For example, the first reactive moiety of the RT primer may comprise an azide moiety, and a second reactive moiety of the barcode oligonucleotide may comprise an alkyne moiety. The first and second reactive moieties may react to form a linking moiety. A reaction between the first and second reactive moieties may be, for example, a cycloaddition reaction such as a strain-promoted azide-alkyne cycloaddition, a copper-catalyzed azide-alkyne cycloaddition, a strain-promoted alkyne-nitrone cycloaddition, a Diels- Alder reaction, a [3+2] cycloaddition, a [4+2] cycloaddition, or a [4+1] cycloaddition; a thiol-ene reaction; a nucleophilic substation reaction; or another reaction. In some cases, reaction between the first and second reactive moieties may yield a triazole moiety or an isoxazoline moiety. A reaction between the first and second reactive moieties may involve subjecting the reactive moieties to suitable conditions such as a suitable temperature, pH, or pressure and providing one or more reagents or catalysts for the reaction. For example, a reaction between the first and second reactive moieties may be catalyzed by a copper catalyst, a ruthenium catalyst, or a strained species such as a difluorooctyne, dibenzylcyclooctyne, or biarylazacyclooctynone. In some aspects, the random RT primer disclosed herein may further comprise a azide functional group (NNNN-N3).

[0369] In some aspects, the transcriptase composition comprises, consists, or consists essentially of at least one polypeptide construct, and transcriptase mix comprising the adapter-RT primers, dNTPs, and other components for reverse transcription. In some aspects, the adapter-RT primers comprise, consist, or consists essentially of an adapter primer fused to a random RT primer. In some aspects, an adapter-RT primer is encoded by a polynucleotide sequence with, with at least, or with about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity to SEQ ID NO: 51.SEQ ID NO: 51 - Adapter-RT primer polynucleotide coding sequence AGACGTGTGCTCTTCCGATCTNNNNNNNNNN (SEQ ID NO: 51 )

[0370] In some aspects, the adapter RT primer comprises a nucleic acid sequence reverse complementary to a cDNA adaptor. In some aspects, the cDNA adapter is encoded by a polynucleotide sequence, with greater than, equal to, at least, at most, or about 60%, 61%, 62%,301320428.1 - 79 -63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identity to SEQ ID NO: 52.SEQ ID NO: 52 - cDNA Adaptor 5Phos / NNNNNNNNAGATCGGAAGAGCGTCGTGT / 3SpC3 / (SEQ ID NO: 52 )

[0371] In some aspects, the Transcriptase mix comprises adapter-RT primers at a concentration of at greater than, equal to, at least, at most, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 pM / mM, including any range or value derivable therein. In some aspects, the adapter-RT primer comprises, consists, or consists essentially of a sequence at least 80% identical to SEQ ID NO: 51.

[0372] In some aspects, the transcriptase mix further comprises nucleotides, for example dNTPs. In some aspects, the dNTPs comprise, consist, or consist essentially of dCTPS, dTTPs, dATPs, and dGTPs. In some aspects, the dNTPs comprise, consist, or consist essentially of at least one labeled dNTP. In some aspects, the labeled dNTP is labeled with biotin. In some aspects, the labeled dNTP is labeled with biotin- 16. In some aspects, the labeled dNTP comprise, consist, or consist essentially of biotin- 16-dUTP, or biotin- 16-dCTP. In some aspects, the labeled dNTP is mixed with a corresponding non-labeled dNTP. In some aspects, the labeled dNTP is mixed with a non-labeled dNTP at a ratio of greater than, equal to, at least, at most, or about 0.5:1, 0.6: 1, 0:7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2:1. In some aspects, the dNTPs comprise, consist, or consist essentially of a combination of biotin- 16-dUTP, biotin- 16-dCTP, dTTP, dCTP, dATP, or dGTP, or any combination thereof. In some aspects, the biotin- 16-dUTP is at a concentration of greater than, equal to, at least, at most, or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 pM / mM, including any range or value derivable therein. In some aspects, the biotin- 16-dCTP is at a concentration of greater than, equal to, at least, at most, or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 pM / mM, including any range or value derivable therein. In some aspects, the dTTP is at a concentration of greater than, equal to, at least, at most, or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 pM / mM, including any range or301320428.1 - 80 -value derivable therein. In some aspects, the dCTP is at a concentration of greater than, equal to, at least, at most, or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 pM / mM, including any range or value derivable therein. In some aspects, the dATP is at a concentration of greater than, equal to, at least, at most, or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 pM / mM, including any range or value derivable therein. In some aspects, the dGTP is at a concentration of greater than, equal to, at least, at most, or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 pM / mM, including any range or value derivable therein.

[0373] In some aspects, one of more nucleotides comprise modified bases, modified sugar moieties, and modified phosphate backbones. Examples of modified base moieties which can be incorporated at any position on its structure include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N~6-sopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methyl cytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5 '-methoxy carboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-S-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, 2,6-diaminopurine and biotinylated analogs, amongst others. Examples of modified sugar moieties which may be used to modify nucleotides at any position on its structure include, but are not limited to arabinose, 2-fluoroarabinose, xylose, and hexose, or a modified component of the phosphate backbone, such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or a formacetal or analog thereof.

[0374] In some aspects, the transcriptase mix may further comprise an RNAse inhibitor (for example, a non-competitive inhibitor of pancreatic-type ribonucleases). In some aspects, the non-competitive inhibitor of pancreatic-type ribonucleases comprises, consists, or consists301320428.1 - 81 -essentially of at greater than, equal to, at least, at most, or about 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 U / pl RNaseOUT, including any range or value derivable therein.

[0375] In some aspects, the transcriptase mix may further comprise a buffer. In some aspects, any suitable buffer may be used. Non-limiting examples include Tris-HCl, MOPS, phosphate buffered saline (PBS), or Dulbecco’s phosphate buffered saline. In some aspects, the pH of the reaction mixture ranges from 5 to 9, such as from 7 to 9, including from 8 to 9, e.g., 8 to 8.5. In some instances, the reaction mixture includes or expressly does not include a pH adjusting agent. pH adjusting agents of interest include, but are not limited to, sodium hydroxide, hydrochloric acid, phosphoric acid buffer solution, citric acid buffer solution, Tris-HCl, MOPS, phosphate buffered saline (PBS), or Dulbecco’s phosphate buffered saline (DPBS), and the like. In some aspects, the buffer comprises, consists, or consists essentially of greater than, equal to, at least, at most, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 pl of DPBS, including any range or value derivable therein. In some aspects, the MgCh is at a concentration of, of greater than, equal to, at least, at most, or about 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 mM, including any range or value derivable therein.

[0376] In some aspects, the transcriptase composition comprising one (single composition comprising a pair of polypeptide constructs) or a pair of compositions, each comprising a member of the pair of polypeptide constructs as disclosed herein and the transcriptase mix is provided to the sample for of greater than, equal to, at least, or at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, including any range or value derivable therein to obtain a cDNA. In some aspects, the transcriptase mix is provided to the sample at less than, equal to, about or more than 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, or 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, or any range derivable there. In some aspects, the transcriptase mix is provided to the sample at less than, equal to, about, or more than 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C. In some aspects, the transcriptase mix is provided to the sample at 37 °C - 42 °C.

[0377] In some aspects, the transcriptase, on dimerization is enzymatically active at a temperature below 42 °C. In some aspects, the transcriptase comprises one or more mutations in one or both fragments to render transcriptase enzymatically active at a temperature below 42 °C.301320428.1 - 82 -

[0378] In some aspects, the cDNA comprises, consists, or consists essentially of the dNTPs. In some aspects, the cDNA comprises, consists, or consists essentially of unlabeled dNTPs. In some aspects, the cDNA comprises, consists, or consists essentially of labeled dNTPs. In some aspects, the cDNA comprises both labeled and unlabeled dNTPs. In some aspects, the cDNA is biotinylated. In some aspects, the cDNA comprises biotin-16.V. RNA binding proteins (RBP) and RBP targeting agents

[0379] In some aspects, the current disclosure encompasses methods for determining one or more interaction sites of a RNA-binding protein (RBP) in a biological sample. In some aspect, the current disclosure also encompasses methods for determining one or more interaction sites of more than one RBP in a biological sample. In some aspects, an RBP of the current disclosure is any protein that interacts with RNA molecules through RNA-binding domains or motifs. RBPs are essential regulators of gene expression, playing critical roles in almost every aspect of RNA metabolism, including transcription, splicing, transport, localization, translation, and degradation. By binding to specific RNA sequences or structures, RBPs control the fate and function of various types of RNAs, such as mRNA, rRNA, and noncoding RNAs. Their activity is vital for cellular processes like differentiation, development, and response to stress. Dysregulation of RBPs is associated with various diseases, including neurodegenerative disorders and cancers. Therefore, in some aspects, the current disclosure also encompasses using the compositions and methods disclosed herein, for the study of RBPs and application of such studies for clinical and non-clinical developments.

[0380] Any RBP of interest, or variant thereof, is suitable for study using the current disclosure. In an aspect, the RBP may be a eukaryotic RBP, or a eukaryotic RBP. In an aspect, the RBP may be RBP commonly found in the eukaryotic class Animalia, Plantae, Fungi, or Protista. In an aspect, the RBP is a mammalian RBP. In an aspect, the RBP is from a laboratory animal, for example a mouse, a rat, a gerbil, a nematode, or a fruit fly. In an aspect, the RBP is a human RBP. The RBP can be a wild-type RBP, or a natural variant thereof, or an engineered RBP. In some aspects, a disclosed RBP comprises, consists essentially of, consists of a transcription factor, a splicing factor, RNA helicase, ribonuclease, RNA polymerase, translation initiation factor, or ribosomal protein. Non-limiting examples of RBPs include YTHDF1, YTHDF2, YTHDC1, HuR, PTB, Musashi, eIF4E, FMRP, LARP1, IMP, hnRNP family proteins, Lin28, AUF1, IGF2BP, FUBP1, LIN28B, RBM5, FUS, TIA1, TTP, QKI, MBNL, CELF, NONO, DDX5, RBM10, SAFB, TDP-43, Ataxin-2, hnRNP A / B, C9orf72, hnRNP H / F, Matrin 3 (MATR3), Pur-alpha, TAF15, Huntingtin, RBFOX, SMN, ELAVL, Ro301320428.1 - 83 -(SSA) and La (SSB) Proteins, hnRNP, Roquin, Staufenl, NF90 / NF110, ILF3, SF3B1, SRSF2, U2AF1, ZRSR2, PRPF8, PRPF31, SNRNP200, HNRNPA1, HNRNPA2B1, NELFE, CPEB1, SRSF1, NOVAI, NOVA2, G3BP1, PTBP1, RBFOX2, and / or HNRNPC.

[0381] In some aspects, the current disclosure also encompasses targeting agents that specifically bind to a RBP. The some aspects the RBP-targeting agent may comprise any molecule that specifically binds the RBP. Non-limiting examples include antibodies, and functional variants thereof, oligonucleotides or variants thereof, peptides, ligands, small molecules, or aptamers. In an aspect, the RBP-targeting agent is an antibody. The term antibody is used broadly here and comprises monoclonal antibodies, polyclonal antibodies, recombinant antibodies, IgG, Fv, single chain antibodies, single domain antibodies, nanobodies, diabodies, bispecific and / or multispecific antibodies, scFv, Fab, F(ab')2, Fab, or variants thereof.

[0382] In some aspects, the RBP-targeting agent can comprise an oligonucleotide comprising a DNA-barcode. In some aspects, the oligonucleotide is linked to the RBP-targeting agent via an amino spacer. In some aspects, the amino spacer is a 7 C6 amino spacer, wherein a non-nucleoside modification adds a primary amino group to an oligo's internal position. The amino group is separated from the 5' end nucleotide base by a 6-carbon spacer arm to reduce steric interaction.

[0383] The DNA-barcode can be unique for each RBP being studied. In some aspects, use of multiple barcoded antibodies, wherein each barcode is specific to a RBP, allows for studying more than one RBP using the methods disclosed herein. In some aspects, the oligonucleotide may further comprise a reactive moiety that is operable in attaching the barcode to a cDNA of the disclosed method. A reactive moiety of a barcoded antibody may be selected from the nonlimiting group consisting of azides, alkynes, nitrones (e.g., 1,3 -nitrones), strained alkenes (e.g., trans-cycloalkenes such as cyclooctenes or oxanorbomadiene), tetrazines, tetrazoles, iodides, thioates (e.g., phorphorothioate), acids, amines, and phosphates. For example, the first reactive moiety of the RT primer may comprise an azide moiety, and a second reactive moiety of the barcode oligonucleotide may comprise an alkyne moiety. The first and second reactive moieties may react to form a linking moiety. A reaction between the first and second reactive moieties may be, for example, a cycloaddition reaction such as a strain-promoted azide-alkyne cycloaddition, a copper-catalyzed azide-alkyne cycloaddition, a strain-promoted alkyne-nitrone cycloaddition, a Diels- Alder reaction, a [3+2] cycloaddition, a [4+2] cycloaddition, or a [4+1] cycloaddition; a thiol-ene reaction; a nucleophilic substation reaction; or another reaction. In some cases, reaction between the first and second reactive moieties may yield a triazole moiety or an isoxazoline moiety. A reaction between the first and second reactive301320428.1 - 84 -moi eties may involve subjecting the reactive moi eties to suitable conditions such as a suitable temperature, pH, or pressure and providing one or more reagents or catalysts for the reaction. For example, a reaction between the first and second reactive moi eties may be catalyzed by a copper catalyst, a ruthenium catalyst, or a strained species such as a difluorooctyne, dibenzylcyclooctyne, or biarylazacyclooctynone.

[0384] Table 2 provides a list of some exemplary oligonucleotides that may be linked to the RBP binding agent via an amino spacer, and that comprise an alkyne group reactive moiety.Table 2: List of exemplary oligonucleotides with barcodes that can be linked to RBP-targeting agent via an amino spacer 7 C6 ([AmC6](5' Amino C6 linker) and comprises a reactive alkyne group ([PPG-3-O-N]: 3'-0-propargyl N 2’-5' linked)301320428.1 - 85 -301320428.1 - 86 -

[0385] In some aspects, the oligonucleotide comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 65-112, or a nucleic acid sequence greater than, equal to, at least, at most, or about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any percentage derivable therein, identical thereto.

[0386] In some aspects, the RBP-targeting agent is labeled. In some aspects, the label comprises, consists essentially of, or consists of a radioisotopes, a hapten, a fluorescent label, a fluorescent polypeptide, a phosphorescent molecule, a chemiluminescent molecule, a chromophore, a luminescent molecule, a photoaffinity molecule, a colored particle, and / or a ligand. In some aspects, the RBP-targeting agent comprises a fluorescent label. In some aspects, the fluorescent label comprises, consists essentially of, or consists of Green Fluorescent Protein (GFP), eGFP, Red Fluorescent Protein (RFP), Teal Fluorescent Protein (TFP), Blue Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), miRFP, cerulean fluorescent protein (CFP), eCyanFP, mCherry, mVenus, mOrange, mTurquoise, tdTomato, aminocoumarin, fluorescein, texas red, Alexa Fluor dyes (e.g. Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 350, Alexa Fluor 532, and Alexa Fluor 700), Cy dyes (e.g. Cy3, Cy5), DyLight dyes, FITC, or Rhodamine, or functional variants thereof.301320428.1 - 87 -VI. Methods

[0387] In some aspects, the current disclosure encompasses methods of determining one or more RNA interaction sites of a RNA-binding protein (RBP) in a biological sample, comprising the steps: a) incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; b) incubating the first complex with one or more secondary binding agents that specifically bind the RBP-targeting agent, to form a secondary complex; c) incubating the first or the secondary complex with a composition comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first or the secondary complex to form a tertiary complex; d) incubating the tertiary complex with a transcriptase mix and a composition comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; e) sequencing the cDNA to determine the one or more RNA interaction sites of the RBP.

[0388] Also disclosed herein are methods of in-situ imaging of one or more RNA interaction sites of an RNA-binding Protein (RBP) in a biological sample bound to a solid surface, comprising the steps of: a) incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; b) incubating the first complex with one or more secondary binding agents that specifically binds the RBP-targeting agent, to form a secondary complex; c) incubating the first or the secondary complex with the one or more compositions as disclosed herein, and a transcriptase mix to obtain cDNA; and d) imaging the solid surface. In some aspects, step c comprises: i) incubating the first or the secondary complex with a composition comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first or the secondary complex to form a tertiary complex; and ii) incubating the tertiary complex with a transcriptase mix and a composition comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA. In some aspects, the method disclosed herein may also be adapted for use to determine the one or more RBP binding sites on an mRNA throughout an mRNA cycle,301320428.1 - 88 -mapping co-binding of a first and a second RBP, for deciphering the translatome of a cell, determining spatial distribution of RNA modification sites on a biological sample. The changes needed to adapt the method disclosed herein to various applications may be easily determined by a person with ordinary skill in the art. Some exemplary variations to the method are schematically depicted in FIG. 5B.

[0389] In some aspects, one or more incubating steps as described herein are sufficient in conditions (e.g., time, temperature, pH, etc.) to promote one or more desired results. For example (but without limitation), one or more incubating steps described herein are sufficient to facilitate binding of a targeting moiety to a target antigen, dimerization of one or more polypeptide constructs, transcription to initiate and / or proceed, complexes to form and / or be detected, detectable signal to be produced, etc. For example, in some aspects, the incubation step is sufficient in conditions (e.g., time, temperature, pH, etc.) to promote facilitation of binding between a targeting moiety and a target antigen. More specifically, as an example, an antibody based targeting moiety may be incubated with the primary and / or the secondary complex such that the conditions for incubation, (time, temperature, pH and ionic strength), are sufficient and / or optimal to ensure high-affinity binding while preventing degradation and minimize nonspecific interactions. The critical factors influencing incubation include but are not limited to time, temperature, pH, ionic strength, and the presence of necessary cofactors. The duration of incubation must be sufficient to allow the desired interactions or reactions to occur while avoiding nonspecific binding or degradation. Temperature is often set to physiological levels, such as 37°C, but may be adjusted for specific reactions, such as higher temperatures to enhance enzyme activity or lower temperatures to stabilize complexes. Similarly, pH is maintained within a range that preserves the activity and stability of the molecules involved, while ionic strength and cofactors are fine-tuned to mimic physiological conditions or to stabilize interactions.

[0390] In some aspects, also provided herein are methods for determining the RNA interactions sites of more than one RNA binding protein. In some aspects, the method may be used to map the RNA binding sites for greater than, equal to, at least, at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 RBPs. In some aspects, the methods disclosed herein may be used to map the RNA binding sites of all the RBPs in a cell.

[0391] In some aspects, also provided herein are modifications of the methods (split ARTR-seq and multiplex split ARTR-seq) for determining RNA binding sites with spatial resolution. These method are broadly referred to herein as spatial split ARTR-seq. In some301320428.1 - 89 -aspects, any of the methods disclosed herein (for example, split ARTR-seq, multiple split ARTR-seq, spatial split ARTR-seq) may be modified to study RNA modification sites.

[0392] The aspects provided herein are in no way limiting, and additional aspects with obvious modifications of the disclosed methods may be envisaged by a person of ordinary skill in the art. Some of these aspects are described in detail herein. Any one or more of the preceding steps of each of the methods disclosed can be excluded from certain aspects of the disclosure. A person of skill in the art is well aware of common techniques to accomplish each of the preceding steps.1. Biological sample

[0393] The term biological sample, as used herein encompasses any sample obtained from an organism or prepared in vitro to mimic a sample of biological origin. Non-limiting examples of biological samples include isolated or assembled RNA-protein complexes, biological fluid, cells, tissue samples, or biological materials derived from cells or tissue samples. In some aspects, the biological sample may be obtained from a prokaryotic, or a eukaryotic organism. In some aspects, the eukaryotic organism may be from the kingdoms Animalia, Plantae, Fungi, Protista. In an aspect, the eukaryotic organism is a mammal. In an aspect, the eukaryotic organism is a laboratory animal, for example a primate, a rodent - a mouse, a rat, a gerbil, a nematode, or a fruit fly. In some aspects, the laboratory animal is a genetically engineered animal. In some aspects, the mammal is a human. In some aspects, the mammal has, or is at a risk of having a disease.

[0394] In certain aspects, the disclosed methods comprise obtaining a sample (also a “biological sample”) from a subject wherein the subject has, or is at a risk of having a disease or disorder. In some aspects, the methods of obtaining a biological sample can include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy. In other aspects the sample can be obtained from any of the tissues provided herein that include but are not limited to non-cancerous or cancerous tissue and non-cancerous or cancerous tissue from the serum, gall bladder, mucosal, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue. Alternatively, the sample can be obtained from any other source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician can obtain a301320428.1 - 90 -biological sample for testing. Yet further, the biological sample can be obtained without the assistance of a medical professional.

[0395] A sample can include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject. The biological sample can be a heterogeneous or homogeneous population of cells or tissues. The biological sample can be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein. The sample can be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.

[0396] The sample can be obtained by methods known in the art. In certain aspects the samples are obtained by biopsy. In other aspects the sample is obtained by swabbing, endoscopy, scraping, phlebotomy, or any other methods known in the art. In some cases, the sample can be obtained, stored, or transported using components of a kit of the present methods. In some cases, multiple samples, such as multiple esophageal samples can be obtained for diagnosis by the methods described herein. In other cases, multiple samples, such as one or more samples from one tissue type (for example esophagus) and one or more samples from another specimen (for example serum) can be obtained for diagnosis by the methods. In some cases, multiple samples such as one or more samples from one tissue type (e.g. esophagus) and one or more samples from another specimen (e.g. serum) can be obtained at the same or different times. Samples can be obtained at different times are stored and / or analyzed by different methods. For example, a sample can be obtained and analyzed by routine staining methods or any other cytological analysis methods.

[0397] In some aspects the biological sample can be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist. The medical professional can indicate the appropriate test or assay to perform on the sample. In certain aspects a molecular profiling business can consult on which assays or tests are most appropriately indicated. In further aspects of the current methods, the patient or subject can obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[0398] In other cases, the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, endoscopy, or phlebotomy. The method of needle aspiration can further include fine needle aspiration, core needle biopsy, vacuum assisted301320428.1 - 91 -biopsy, or large core biopsy. In some aspects, multiple samples can be obtained by the methods herein to ensure a sufficient amount of biological material.

[0399] General methods for obtaining biological samples are also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, which is herein incorporated by reference in its entirety, describes general methods for biopsy and cytological methods. In some aspects, the sample is a fine needle aspirate of a esophageal or a suspected esophageal tumor or neoplasm. In some cases, the fine needle aspirate sampling procedure can be guided by the use of an ultrasound, X-ray, or other imaging device.

[0400] In some aspects of the present methods, a molecular profiling business can obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party. In some cases, the biological sample can be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business. In some cases, the molecular profiling business can provide suitable containers, and excipients for storage and transport of the biological sample to the molecular profiling business.

[0401] In some aspects of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. An individual can alternatively obtain a sample through the use of an over the counter (OTC) kit. An OTC kit can contain a means for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit. In some cases, molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately. A sample suitable for use by the molecular profiling business can be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and / or adequacy are provided.

[0402] In some aspects, the subject can be referred to a specialist such as an oncologist, surgeon, or endocrinologist. The specialist can likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample. In some cases the medical professional can refer the subject to a testing center or laboratory for submission of the biological sample. In other cases, the subject can provide the sample. In some cases, a molecular profiling business can obtain the sample.301320428.1 - 92 -2. Sample preparation

[0403] In some aspects, the current disclosure also encompasses methods of preparing the biological sample, as disclosed herein for further processing. Methods for preparing the samples are well known in the art and can comprise use of common laboratory equipment, for example centrifuges, perfusion equipment, dissection equipment, cryostats, mounting equipment, mounting media, solid surface, for example slides, multi-well plates, capillaries etc, microscopes, staining equipment etc. In an exemplary set up, once a tissue sample is obtained, the tissue may be placed in O.C.T, and frozen in liquid nitrogen, and sliced using a cryostat (for example, Leica CM1900). The tissue sections may then be mounted on a suitable solid surface, and further fixed and permeabilized. In another exemplary set up, a tissue obtained may be further dissected into cells, diluted and mounted on a solid surface. In yet another aspect, one or more cells from a cell line may be obtained, and processed. In yet another exemplary aspect, a ribosome, a polysome, or other RNA-protein complexes may be isolated used in the disclosed methods.

[0404] In some aspects, the processed biological sample may be fixed. A person of skill in the art is familiar with common techniques to accomplish fixation of a sample. In some aspects, the fixing step can comprise, consist, or consist essentially of rapidly freezing the sample, or can comprise, consist, or consist essentially of treating the sample with formaldehyde and / or paraformaldehyde (PF A).

[0405] A cellular sample can be fixed by treatment with a fixing agent. A fixing agent can comprise, consist, or consist essentially of a crosslinking agent, including aldehydes like formalin, glutaraldehyde, formaldehyde, PF A, or a precipitating agent, including organic solvents like methanol, acetone, or piric acid, or any combination thereof. In some aspects, the fixing step is quenched, for example with glycine. A person of skill in the art is familiar with common techniques to accomplish quenching of a fixing reaction, including addition of sodium borohydride, or addition of exogenous amine-containing reagents like ammonium chloride and / or glycine. In some aspects, the fixing step comprises, consists, or consists essentially of treating the sample with formaldehyde. In some aspects, the fixing step comprises, consists, or consists essentially of treating the sample with paraformaldehyde (PF A). In some aspects, the fixing step comprises, consists, or consists essentially of treating the sample with greater than, equal to, at least, at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or 2.5% PF A. In some aspects, the fixing step occurs for greater than, equal to, at least, or at most 1, 2,301320428.1 - 93 -3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, including any range or value derivable therein. In some aspects, the fixing step occurs at room temperature.

[0406] In some aspects, the fixing step is quenched. In some aspects, the fixing step is quenched with glycine. In some aspects, the quenching glycine is greater than, equal to, at least, at most 25, 50, 75, 100, 125, 150, 200, 225, or 250 mM, including any range or value derivable therein. In some aspects, the quenching step occurs for greater than, equal to, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, including any range or value derivable therein. In some aspects, the quenching step occurs at room temperature.

[0407] In some aspects, the sample is permeabilized. In some aspects, a cell permeabilizing agent may comprise a detergent, an enzyme, a solvent, a small molecule, a buffer or any combination thereof. In some aspects, the cell permeabilizing agent comprises a detergent. In some aspects, the agent that permeabilizes cell membranes comprises, consists, or consists essentially of greater than or equal to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%, including any range or value derivable therein, Triton X-100. In some aspects, the sample is contacted with the permeabilizing agent greater than, equal to, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, including any range or value derivable therein. In some aspects, the contacting (e.g., incubating together) step occurs on ice.

[0408] In some aspects, the at least one RNase is optionally provided to the sample following the permeabilizing step or further downstream. In some aspects, the providing of the at least one RNase improves resolution during the sequencing step. In some aspects, the at least one RNase comprises, consists, or consists essentially of ribonuclease I (RNase I, via Thermo Fisher Scientific), RNase A, and / or RNase Tl. In some aspects, the RNase is provided to the sample for greater than, equal to, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, including any range or value derivable therein. In some aspects, the at least one RNase is provided to the sample at 37 °C.3. Primary and secondary complex formation

[0409] In some aspects, the current disclosure provides a split ARTR-seq method for determining one or more RNA interaction sites of a RNA-binding Protein (RBP) in a biological sample, comprising: contacting a RBP-targeting agent to the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a first complex; contacting the first complex with one or more secondary binding agents that specifically bind the RBP-targeting agent, to form a second complex; incubating the first or the secondary complex with the composition301320428.1 - 94 -comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first or the secondary complex to form a tertiary complex; incubating the tertiary complex with a transcriptase mix and a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; sequencing the cDNA to determine the one or more RNA interaction sites of the RBP. A schematic of an exemplary split ARTR-seq procedure in comparison with the ARTR-seq method is provided in FIG. 1 A. In some aspects, an incubating step is for a duration and / or under conditions sufficient to facilitate RBP-targeting agent and RBP complex formation.

[0410] In some aspects, the method may further comprise one or more of a sample preparation step, a fixing step, a quenching step, a permeabilizing step, RNAse treatment, blocking step or any combination thereof, as disclosed herein and / or known in the art. In some aspects, any one or more of these steps may be omitted from the method. In some aspects, the sample is blocked before the RBP targeting agent is provided to the sample to form a first complex. A person of skill in the art is familiar with common techniques to accomplish sample blocking, which reduce background or non-specific staining of the sample. As is known to a person of skill in the art, agents like hydrogen peroxide, levamisole, avidin / biotin blocking reagents, and / or protein blocking solutions like BSA, gelatin, and / or non-fat dry milk. In an aspect, the blocking agent may comprise BSA. In some aspects, the sample may be blocked using greater than, equal to, at least, at most 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5.0 mg / mL blocking agent in any suitable buffer. In some aspects, the blocking may be done for greater than, equal to, at least, or at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or more minutes at RT. In some aspects, the samples can be blocked for greater than, equal to, at least, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hrs at 4 °C. In some aspects, practicing one or more of these steps comprising sample preparation step, a fixing step, a quenching step, a permeabilizing step, blocking step steps provide a processed sample for use in downstream steps of the method.

[0411] In an aspect, the method comprises contacting one or more RBP-targeting agents disclosed herein, with RBPs in the processed sample. In some aspects, the RBP-targeting agent may comprise any molecule as disclosed here, that specifically binds the RBP. Non-limiting examples include antibodies, and functional variants thereof, oligonucleotides or variants301320428.1 - 95 -thereof, peptides, ligands, small molecules, or aptamers. In some aspects, the RBP-targeting agent is an antibody. In an aspect, the contacting step may be carried out in any suitable buffer composition, for example Tris-HCl, MOPS, phosphate buffered saline (PBS), or Dulbecco’s phosphate buffered saline. In some aspects, the buffer composition may further comprise a blocking agent as disclosed herein. In some aspects, the RBP-targeting agent is incubated with the processed sample for greater than, equal to, at least, or at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or more minutes at RT. In some aspects, the samples can be incubated with the RBP -targeting agent for greater than, equal to, at least, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hrs at 4°C. In some aspects, contacting of the RBP binding agent with the RBP forms a primary complex.

[0412] In some aspects, primary complex may optionally be incubated with a secondary binding agent, which specifically binds the RBP-targeting agent. In some aspects, the secondary binding agent may comprise any molecule as disclosed here, that specifically binds the RBP binding agent. Non-limiting examples include antibodies, and functional variants thereof, oligonucleotides or variants thereof, peptides, ligands, small molecules, or aptamers. In some aspects, the secondary binding agent is an antibody, for example an antibody that specifically binds the primary complex. In some aspects, the secondary binding agent is incubated with the sample for greater than, equal to, at least, or at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or more minutes at RT. In some aspects, the samples can be incubated with the secondary binding agent for greater than, equal to, at least, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hrs at 4 °C. In some aspects, contacting of the secondary binding agent with the primary complex forms a secondary complex. In some aspects, the RBP-binding agent, or the secondary binding agent may be labeled as provided herein above. In some aspects, an incubating step is for a duration and / or under conditions sufficient to facilitate complex formation.

[0413] In some aspects, the sample may be washed between any or after any of the steps disclosed herein. In some aspects, the sample is washed for greater than, equal to, at least, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, after the at least one RNase is provided to the sample, the RBP targeting step, after the primary or secondary complex formation and / or after the blocking step. In some aspects, the washing step comprises, consists, or consists essentially of washing the sample with a suitable buffer, for example Tris-HCl, MOPS, phosphate buffered saline (PBS), or Dulbecco’s phosphate buffered saline. In some aspects, the washing buffer may further comprise a blocking agent, as disclosed herein, a RNase inhibitor, and additional301320428.1 - 96 -ingredients, as well known in the art. In some aspects, the washing step comprises, consists, or consists essentially of shaking the sample with DPBS. In some aspects, the washing step occurs for greater than, equal to, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, including any range or value derivable therein. In some aspects, the washing step occurs at room temperature.4. Reverse transcription and cDNA synthesis

[0414] In some aspects, the disclosed method further comprises incubating the primary or the secondary complex, or both with a composition as disclosed herein and a suitable transcriptase mix as disclosed herein. As provided, in some aspects, the composition comprises a first polypeptide construct and / or a second polypeptide construct and the transcriptase mix. In some aspects, the first and / or the second polypeptide construct comprises a targeting moiety as disclosed herein; and a fragment of the reverse transcriptase enzyme as disclosed herein. In some aspects, the method comprises incubating the first or the secondary complex with the composition comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first or the secondary complex to form a tertiary complex; and further incubating the tertiary complex with a transcriptase mix and a composition comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA. In some aspects, the first and the second polypeptide constructs may be added simultaneously, or the addition may be temporarily staggered. In some aspects, one or more wash steps as disclosed herein may be included between the incubating with the first and the second polypeptide construct. In some aspects, an incubating step is for a duration and / or under conditions sufficient to reverse transcription.

[0415] As an aspect, the transcriptase mix comprises one or more ingredients for initiation and synthesis of cDNA. In an aspect, the transcriptase mix comprises one or more adapter-RT primer, wherein the one or more adapter RT-primer each comprises an adapter primer sequence and an RT primer sequence. In some aspects, the RT primer comprises random RT primers as disclosed herein. In some aspects, the adapter primer comprises one or more of a barcode sequence, indexes etc. In some aspects, the transcriptase mix may further comprise components known in the art, for example labeled and / or unlabeled dNTPs as disclosed herein, RNase inhibitor, salts, reducing agents, buffers, solvents, osmotic agents etc. In some aspects, any one301320428.1 - 97 -or more of the aforementioned components may be expressly excluded from a transcriptase mix.

[0416] A person of skill in the art is familiar with conditions capable of producing cDNA. As noted above, in some aspects, the conditions to produce cDNA can comprise, consist, or consist essentially of providing the sample with at least one primer (random, oligo(dT) or gene specific), dNTPs, and other components in order to conduct reverse transcription (RT) before halting the reaction. In an aspect, the primer is an adapter RT primer as disclosed herein. The other components can comprise, consist, or consist essentially of a non-competitive inhibitor of pancreatic-type ribonucleases, a buffer or buffers, MgCh, a reducing reagent, and / or water. In some aspects, the transcriptase composition is provided to the sample for greater than, equal to, at least, or at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, including any range or value derivable therein to obtain a cDNA. In some aspects, the transcriptase mix provided to the sample at less than, equal to, about or more than 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, or 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C. In some aspects, the transcriptase mix is provided to the sample at less than, equal to, about or more than 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C. In some aspects, the transcriptase mix is provided to the sample at 37 °C - 42 °C.

[0417] A person of skill in the art is aware of standard conditions and protocols with which to conduct reverse transcription. For example, primers with which to conduct reverse transcription can comprise, consist, or consist essentially of oligo(dT) primers, random primers, and / or gene-specific primers. A person of skill in the art can select random primers to improve cDNA synthesis for detection. These random primers can comprise, consist, or consist essentially of at least septamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers, octadecamers, nonadecamers, or eicosamers. As a further example, the dNTPs with which to conduct reverse transcription can be labelled or not labelled; as known to a person in the art a dNTP label can comprise, consist, or consist essentially of biotin, biotin-16, a-32P, fluorescein, a fluorescent dye, and / or another label that facilitates detection and / or purification. The labeled and label-free dNTPs can be mixed at different ratios, for example 2:1, 1:1, 1:2, or any range or value derivable therein. In some aspects, the dNTPs can comprise, consist, or consist essentially of a combination of labelled dUTP, labelled dCTP, labelled dGTP, labelled dATP, dTTP, dCTP, dATP, and / or dGTP.301320428.1 - 98 -

[0418] A non-competitive inhibitor of pancreatic-type ribonucleases suitable for conducting reverse transcription can comprise, consist, or consist essentially of RNase inhibitor, RNAseOUT, and / or another agent which prevents RNA degradation by RNase. Buffers with suitable for conducting reverse transcription can comprise, consist, or consist essentially of a phosphate buffer solution like PBS and / or DPBS, and / or another buffer providing a favorable pH and ionic strength for the reaction. A reducing reagent suitable for conducting reverse transcription can comprise, consist, or consist essentially of dithiothreitol (DTT), and / or another agent suitable for reducing disulfide bonds in RNases. Water suitable for conducting reverse transcription can comprise, consist, or consist essentially of nuclease-free water, water treated with diethylpyrocarbonate, and / or water treated with another agent that eliminates any RNases.

[0419] In some aspects, the disclosed method does not comprise oligo(dT) primer initiated reverse transcription. In some aspects, the method does not comprise Tn5 tagmentation.

[0420] A person of skill in the art is familiar with methods for halting RT. For example, a chelating agent can be added to the sample to halt RT. As known to a person of skill in the art, chelating agents can comprise, consist, or consist essentially of EDTA and / or EGTA. In some aspects, halting RT comprises, consists, or consists essentially of providing at least one chelating agent to the sample. In some aspects, the at least one chelating agent comprises, consists, or consists essentially of EDTA and / or EGTA. In some aspects, the EDTA is at a concentration of greater than, equal to, at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mM, including any range or value derivable therein. In some aspects, the EGTA is at a concentration of greater than, equal to, at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM, including any range or value derivable therein. In some aspects, the at least one chelating agent is provided to the sample for greater than, equal to, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, including any range or value derivable therein. In some aspects, the at least one chelating agent is provided to the sample at room temperature.

[0421] As noted above, the present methods can further comprise, consist, or consist essentially steps which permit recovery of DNA and / or cDNA from a sample. For example, an optional cell digestion step can be included after the incubating step or optional in-situ imaging step. A person of skill in the art can also use alternative DNA extraction protocols, such as treatment with chemical extractants, physical disruption, treatment with proteases, and / or treatment with other cellular lysis agents. As is known in the art, chemical extractants can301320428.1 - 99 -comprise, consist, or consist essentially of sodium dodecyl sulfate (SDS), chloroform, phenol, Chelex 100, and / or guanadinium isothiocyanate. As is known in the art, physical disruption methods can comprise, consist, or consist essentially of bead mill homogenization and / or freeze-thaw lysis. As is known in the art, proteases or other cellular lysis agents can comprise, consist, or consist essentially of a lysozyme, a proteinase K, achromopeptidase, and / or pronase E.5. cDNA Sequencing

[0422] As noted above, in some aspects, the cDNA sequencing step produces a binding profile for the RBP of interest. As is commonly known in the art, DNA sequencing can comprise, consist, or consist essentially of amplifying the cDNA, purifying the amplified cDNA, and sequencing the purified cDNA. A person of skill in the art is familiar with common sequencing methods, which can include high-throughput sequencing.

[0423] In some aspects, the methods of the disclosure include a sequencing method. In certain aspects, methods involve sequencing the cDNA produced by incubation step. The cDNA can be prepared for sequencing by any method known in the art, such as library preparation, hybrid capture, sample quality control, product-utilized ligation-based library preparation, or a combination thereof. The cDNA can be prepared for any sequencing technique. In some aspects, sequencing, can be performed to cover approximately 70%, 75%, 80%, 85%, 90%, 95%, 99%, or greater percentage of targets at more than 20x, 25x, 30x, 35x, 40x, 45x, 50x, or greater than 50x coverage. In some aspects, sequencing further comprises the use of at least one bioinformatic too including VarScan2, any R package (including CopywriteR) and / or Annovar.

[0424] Non-limiting examples of suitable sequencing methods include MPSS captures adapter-ligated cDNA fragments on microbeads and decodes them by iterative hybridization / ligation to quantify transcript tags at high throughput; 454 (pyrosequencing) uses emulsion PCR on beads with light-based pyrophosphate detection to generate moderate-length reads suitable for isoform and junction resolution; Illumina (sequencing-by-synthesis) employs flow-cell bridge amplification with reversible-terminator chemistry to yield accurate short paired-end reads for high-depth quantification; SOLiD (ligation-based) performs sequencing by oligonucleotide ligation in color space to provide high accuracy for variant detection in short-read libraries; Ion Torrent (semiconductor) uses emulsion PCR with pH-based nucleotide incorporation detection for rapid turnaround in targeted cDNA panels; PacBio SMRT (long-read) produces single-molecule real-time long reads and high-fidelity circular consensus for301320428.1 - 100 -isoform-level mapping and structural variation; Oxford Nanopore (long-read / direct RNA) measures nanopore current disruptions to enable ultra-long reads and direct RNA sequencing for splice isoforms and modification profiling; and Sanger (capillary) applies chain-termination chemistry for targeted validation of specific variants or junctions identified in discovery datasets. Sequencing outputs are then processed using standard pipelines for quality control, alignment, quantification, and detection of sequence and structural features appropriate to the assay.6. Sample Imaging

[0425] As noted above, in some aspects, present methods can further comprise, consist, or consist essentially of an optional in-situ imaging step after the incubating step. As is known in the art, imaging can be performed by light microscopy, fluorescence microscopy, confocal microscopy, and / or other commonly known microscopy techniques.

[0426] A person of skill in the art can use an imaging moiety, for example a fluorophore such aminocoumarin, fluorescein, Texas red, Alexa Fluor dyes (e.g. Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 350, Alexa Fluor 532, and Alexa Fluor 700), Cy dyes (e.g. Cy3, Cy5), DyLight dyes, FITC, or Rhodamine, or functional variants thereof to target certain aspects of the sample of interest, for example the biotin-tagged cDNA. Suitable moieties are known to a person of skill, and can consist, comprise, or consist essentially of a biotinylated monoclonal antibody like Alexa Fluor dye. A person of skill in the art can use a nuclear counterstain to indicate live cells with intact, nonpermeable plasma membranes in the sample. A nuclear counterstain can consist, comprise, or consist essentially of a cell-permanent nuclear counterstain which emits fluorescence when bound to dsDNA, like Hoechst stains and / or SYTO stains.B. Spatial split ARTR-seq

[0427] In some aspects, the disclosed method may be modified to obtain spatial information with respect to RNA binding sites. By introducing single-cell and / or spatial barcodes, split ARTR-seq can achieve single-cell or spatial resolution. These barcodes can be seamlessly incorporated either through the use of barcoded RT primers during the reverse transcription process or through ligation. They can be subsequently employed to assign singlecell identity or spatial localization during data analysis.

[0428] In spatial barcoding-based split ARTR-seq, resolution can be fine-tuned by adjusting the density of barcode primers, allowing for cellular and / or subcellular resolution.301320428.1 - 101 -Apart from spatial barcoding strategy, the in-situ sequencing method may be used in spatial split ARTR-seq to achieve subcellular resolution.

[0429] Spatial split ARTR-seq offers compatibility with imaging techniques, such as FISH or variations on FISH, microfluidics imaging techniques, or any other single-cell profiling techniques. This compatibility provides additional information alongside sequencing data, such as subcellular structure identification and / or cell stage determination. In an aspect, the disclosed methods may be combined with advanced single cell imaging techniques to provide spatially resolved binding sites and expression date. Commonly used techniques are provided herein.

[0430] Spatial Transcriptomics: This technique combines gene expression analysis with spatial information, allowing researchers to map RNA molecules in a tissue sample. It involves capturing gene expression data while preserving the spatial context, often using barcoded slides or arrays.

[0431] MERFISH (Multiplexed Error-Robust Fluorescence In Situ Hybridization): A highly multiplexed method for visualizing the spatial distribution of thousands of RNA molecules within cells. It uses fluorescent probes to detect RNA and generate a spatially resolved map of gene expression at the single-cell level.

[0432] SeqFISH (Sequential Fluorescence In Situ Hybridization): Similar to MERFISH, SeqFISH sequentially labels and images RNA molecules within cells using different fluorescent probes, enabling the spatial resolution of hundreds to thousands of genes within 3D tissue sections.

[0433] STARmap (Spatially Resolved Transcript Amplicon Readout Mapping): A technique that preserves the 3D structure of tissues while performing RNA sequencing. It uses hydrogel-tissue chemistry to encode RNA spatial information, allowing for highly multiplexed in situ transcriptomics.

[0434] Slide-Seq: A method that uses barcoded beads on a slide to capture RNA transcripts from tissue sections. This technique maps gene expression across the tissue with single-cell resolution, while maintaining spatial context.

[0435] Visium Spatial Gene Expression: Developed by lOx Genomics, this technique captures mRNA from tissue sections using spatially barcoded microarrays. It provides a spatial map of gene expression, linking molecular data with histological information.

[0436] Laser Capture Microdissection (LCM): A technique that physically isolates specific regions or cells from a tissue sample using a laser. These cells are then analyzed for301320428.1 - 102 -gene expression or other molecular features, allowing for spatially resolved insights, though in a more manual and targeted way.

[0437] Imaging Mass Cytometry (IMC): Combines high-resolution imaging with mass cytometry to map the spatial distribution of proteins, DNA, or RNA in tissue sections. It allows multiplexed detection of dozens of markers at a time, preserving spatial and cellular context.

[0438] DBiT-seq (Deterministic Barcoding in Tissue for Spatial Omics Sequencing) is a method for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via nextgeneration sequencing (NGS). Parallel microfluidic channels are used to deliver DNA barcodes to the surface of a tissue slide, and crossflow of two sets of barcodes, Al-50 and Bl-50, followed by ligation in situ, yielding a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Gene expression profiles in 10-pm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions.C. Multiplex split ARTR-seq

[0439] In some aspects, also provided herein are methods for determining the RNA interactions sites of more than one RNA binding protein. In an aspect, the methods comprise modification of the split ARTR-seq method, such that each RBP-targeting agent is tagged with a separate barcode, and wherein the barcode may be incorporated into the cDNA using click chemistry. In some aspects, the method may be used to map the RNA binding sites for greater than, equal to, at least, or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 40, 48, 50, 60, 70, 72, 80, 84, 90, 96, 100, 108, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 RBPs, or any range derivable therein. In some aspects, the methods disclosed herein may be used to map the RNA binding sites of all the RBPs in a cell.

[0440] In some aspects, provided herein is method of determining one or more RNA interaction sites of a first RNA-binding Protein (RBP) in a biological sample, comprising: a) contacting a first RBP-targeting agent comprising an alkyne functionalized first DNA barcode, to the first RBP, wherein the first RBP-targeting agent specifically binds the first RBP to form a first complex; b) contacting the first complex with one or more secondary binding agents that specifically binds the first RBP-targeting agent, to form a second complex; c) incubating the first or the secondary complex with a composition comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase as disclosed herein, a first dimerization domain as disclosed herein and a targeting moiety as disclosed herein; wherein the targeting301320428.1 - 103 -moiety binds the first or the secondary complex to form a tertiary complex; d) incubating the tertiary complex with a transcriptase mix and a composition comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase, as disclosed herein and a second dimerization domain as disclosed herein, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; e) amplifying and sequencing the first barcoded cDNA library; f) obtaining one or more interaction site of the first RBP by deconvoluting the sequenced cDNA library based on the first DNA barcode. In some aspects, the transcriptase composition for use in the method may comprise RT primers comprising a reactive moiety such that it can react with a barcoded oligonucleotide containing antibody or targeting moiety, wherein the barcode oligonucleotide comprises a corresponding reactive moiety for click chemistry. A reactive moiety of a random RT primer may be selected from the non-limiting group consisting of azides, alkynes, nitrones (e.g., 1,3 -nitrones), strained alkenes (e.g., trans-cycloalkenes such as cyclooctenes or oxanorbomadiene), tetrazines, tetrazoles, iodides, thioates (e.g., phorphorothioate), acids, amines, and phosphates. For example, the first reactive moiety of the RT primer may comprise an azide moiety, and a second reactive moiety of the barcode oligonucleotide may comprise an alkyne moiety. The first and second reactive moieties may react to form a linking moiety. A reaction between the first and second reactive moieties may be, for example, a cycloaddition reaction such as a strain-promoted azide-alkyne cycloaddition, a copper-catalyzed azide-alkyne cycloaddition, a strain-promoted alkyne-nitrone cycloaddition, a Diels- Alder reaction, a [3+2] cycloaddition, a [4+2] cycloaddition, or a [4+1] cycloaddition; a thiol-ene reaction; a nucleophilic substation reaction; or another reaction. In some cases, reaction between the first and second reactive moieties may yield a triazole moiety or an isoxazoline moiety. A reaction between the first and second reactive moieties may involve subjecting the reactive moieties to suitable conditions such as a suitable temperature, pH, or pressure and providing one or more reagents or catalysts for the reaction. For example, a reaction between the first and second reactive moieties may be catalyzed by a copper catalyst, a ruthenium catalyst, or a strained species such as a difluorooctyne, dibenzylcyclooctyne, or biarylazacyclooctynone. In some aspects, the random RT primer disclosed herein may further comprise a azide functional group (NNNN-N3).

[0441] In some aspects, the method comprises RBP-targeting agents that comprise an oligonucleotide comprising a DNA-barcode. In some aspects, the oligonucleotide is linked to the RBP-targeting agent via an amino spacer. In some aspects, the amino spacer is a 7 C6 amino301320428.1 - 104-spacer, wherein a non-nucleoside modification adds a primary amino group to an oligo's internal position. The amino group is separated from the 5' end nucleotide base by a 6-carbon spacer arm to reduce steric interaction.

[0442] In some aspects, the DNA-barcode can be unique for each RBP being studied. In some aspects, use of multiple barcoded antibodies, wherein each barcode is specific to a RBP, allows for studying more than one RBP using the methods disclosed herein. In some aspects, the oligonucleotide may further comprise a reactive moiety that is operable in attaching the barcode to a cDNA of the disclosed method. A reactive moiety of a barcoded antibody may be selected from the non-limiting group consisting of azides, alkynes, nitrones (e.g., 1,3 -nitrones), strained alkenes (e.g., trans-cycloalkenes such as cyclooctenes or oxanorbomadiene), tetrazines, tetrazoles, iodides, thioates (e.g., phosphorothioate), acids, amines, and phosphates. For example, the first reactive moiety of the RT primer may comprise an azide moiety, and a second reactive moiety of the barcode oligonucleotide may comprise an alkyne moiety. The first and second reactive moieties may react to form a linking moiety. A reaction between the first and second reactive moieties may be, for example, a cycloaddition reaction such as a strain-promoted azide-alkyne cycloaddition, a copper-catalyzed azide-alkyne cycloaddition, a strain-promoted alkyne-nitrone cycloaddition, a Diels-Alder reaction, a [3+2] cycloaddition, a [4+2] cycloaddition, or a [4+1] cycloaddition; a thiol-ene reaction; a nucleophilic substation reaction; or another reaction. In some cases, reaction between the first and second reactive moieties may yield a triazole moiety or an isoxazoline moiety. A reaction between the first and second reactive moieties may involve subjecting the reactive moieties to suitable conditions such as a suitable temperature, pH, or pressure and providing one or more reagents or catalysts for the reaction. For example, a reaction between the first and second reactive moieties may be catalyzed by a copper catalyst, a ruthenium catalyst, or a strained species such as a difluorooctyne, dibenzylcyclooctyne, or biarylazacyclooctynone. Table 2 provides a list of some exemplary oligonucleotides that may be linked to the RBP binding agent via an amino spacer, and that comprise an alkyne group reactive moiety.

[0443] In some aspects, the method further comprises incorporation of the biotinylated dNTPs, and the RT primer sequence comprising azide functional group, into the cDNA to form proximal azide labeled biotinylated cDNAs during reverse transcription. In some aspects, the method further comprises incorporating the alkyne functionalized first DNA barcode into the cDNA by reacting the alkyne functionalized first DNA barcode with the proximal azide labeled biotinylated cDNA of claim, using in-situ copper catalyzed azide-alkyne cycloaddition (CuAAC), to obtain a first barcoded biotinylated cDNA library. In some aspects, the method301320428.1 - 105 -further comprises purifying the barcoded biotinylated cDNA library over a streptavidin column prior to step (d). In some aspects, the method further comprises processing the CuAAC using a Klenow Fragment DNA polymerase for second strand synthesis prior to sequencing. In some aspects the one or more interaction sites of the first RBP are obtained by deconvoluting the sequenced data based on the first DNA barcode incorporated into the cDNA. In some aspects, the method further comprises similarly determining the one or more RNA-interaction sites of a second RNA-binding Protein (RBP) in a biological sample, comprising: a) contacting a second RBP-targeting agent comprising a alkyne functionalized second DNA barcode, to the second RBP, wherein the RBP-targeting agent specifically binds the second RBP to form a second primary complex; b) contacting the second primary complex with one or more secondary binding agents that specifically binds the first RBP-targeting agent, to form a second secondary complex; c) incubating the second primary or the second secondary complex with the transcriptase composition, to obtain a second barcoded cDNA library; d) amplifying and sequencing the second barcoded cDNA library; and e) obtaining one or more interaction site of the second RBP by deconvoluting the sequenced cDNA library based on the second DNA barcode.

[0444] In some aspects, this process can be simultaneously conducted for, for greater than, for equal to, for at least, or for at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 40, 48, 50, 60, 70, 72, 80, 84, 90, 96, 100, 108, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 RBPs, or any range derivable therein.D. Advanced split ARTR-seq and spatial split ARTR-seq for RNA modifications

[0445] In some aspects, any of the methods disclosed herein may be modified to determine RNA modification sites, either at sequence level or spatial level. In an aspect, suitable modification sites may comprise, consist of, consist essentially of m6C, m5C, mxA, m7G, or a pseudouridine modification. In an aspect, the method may comprise using a modification targeting agent instead of a RBP targeting agent in the split ARTR-method. In some aspects, suitable modification targeting agents comprise, consist of, consist essentially of an antibody or variant thereof, an oligonucleotide or variant thereof, a receptor, a ligand, a small molecule, an aptamer, or any combination thereof. In some aspects, the modification targeting agent specifically binds to a modification site. The method may be used with split ARTR-seq,301320428.1 - 106-multiplex split ARTR-seq or spatial split ARTR-seq as provided herein, with suitable adjustments as will be known to a person of skill in the art with the disclosure herein.

[0446] Thus, in some aspects, the current disclosure encompasses method of determining spatial distribution of a RNA modification site on a biological sample bound to a solid surface, comprising: a) contacting a modification-targeting agent that specifically binds the modification site on the RNA to form a primary complex; b) contacting the primary complex with a secondary binding agent that specifically bind the primary complex to form a secondary complex; c) incubating the first or the secondary complex with a composition comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first or the secondary complex to form a tertiary complex; d) incubating the tertiary complex with a transcriptase mix and a composition comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; e) and sequencing and / or imaging the biological sample using a single cell genomic imaging technique to determine the one or more modification sites.

[0447] In some aspects, the modification-targeting agent is an oligonucleotide, or a variant thereof, or a small molecule. In some aspects, the oligonucleotide comprises, consists essentially of, or consists of fluorescent NTPs, or a fluorescent probe. In some aspects, modification-targeting agent is an antibody or a functional variant thereof. In some aspects, the antibody or the functional variant thereof comprises monoclonal antibodies, polyclonal antibodies, recombinant antibody, IgG, Fv, single chain antibody, single domain antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), scFv, Fab, F(ab')2, Fab, or variants thereof. In some aspects, modification targeting agent specifically binds to a modification comprising, consisting essentially of, or consisting of m6C, m5C, mlA, m7G, or a pseudouridine modification. In some aspects, the sequencing and imaging is done using a single cell genomic imaging technique as disclosed herein. In some aspects, the single cell genomic imaging technique comprises, consists essentially of, consists of deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) comprising: ligating a first set and a second set of spatial barcodes to the cDNA of step (c), prior to step (d), wherein the first set of spatial barcodes are contacted to the cDNA horizontally using a first multi-channel microfluidic chip, and the second set of spatial barcodes are contacted to the solid surface vertically using a second multi-channel microfluidic chip.301320428.1 - 107 -E. Temporal determination of RBP binding site through mRNA cycle

[0448] In some aspects, the current disclosure also encompasses a method of determining the one or more RBP binding sites on an mRNA throughout an mRNA cycle, the method comprising: a) incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex; b) incubating the first complex with one or more secondary binding agents that specifically binds the RBP-targeting agent, to form a secondary complex; c) incubating the first or the secondary complex with the composition comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a first targeting moiety; wherein the first targeting moiety binds the first or the secondary complex to form a tertiary complex; d) incubating the tertiary complex with a transcriptase mix and a composition comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase, a second dimerization domain and a second targeting moiety, wherein the second dimerization domain binds the first dimerization domain, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA, and wherein the second targeting moiety binds a specific site on the mRNA; e) sequencing the cDNA.VII. Kits

[0449] Certain aspects of the present disclosure also concern kits containing compositions of the disclosure and / or compositions to implement methods disclosed herein. In some aspects, the current disclosure encompasses a kit comprising at least two polypeptide constructs as disclosed herein, or compositions thereof. In some aspects, the at least 2 polypeptide constructs and / or polynucleotide constructs comprises (or encodes) a first polypeptide construct, wherein the first polypeptide construct comprises a N-terminal fragment of a reverse transcriptase, or a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof, a first dimerization moiety, and optionally a first targeting moiety; and b) a second polypeptide construct, wherein the second polypeptide construct comprises a C-terminal fragment, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase, a second dimerization moiety that specifically binds to the first dimerization moiety, and optionally a second targeting moiety.

[0450] In some aspects, the kit further comprises a transcriptase mix as disclosed herein. In some aspects, the current disclosure encompasses a kit comprising in one or more suitable container(s), an RBP-targeting agent that specifically binds to an RBP, one or more secondary301320428.1 - 108 -binding agents, at least a pair of polypeptide construct as disclosed herein, and / or the transcriptase composition as disclosed herein, and a transcriptase mix as disclosed herein.

[0451] In some aspects, disclosed are kits that can be used to prepare a sample for RBP-RNA binding site and / or RNA modification site identification. In some aspects, disclosed are kits that can be used to identify RBP-RNA binding sites via split ARTR-seq, spatial split ARTR-seq, multiplexed split ARTR-seq, advanced split ARTR-seq techniques for determining RNA modification sites and for translatome analysis.

[0452] The kit can optionally provide additional components that are useful in the procedure. These optional components include buffers, capture reagents, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information. In certain aspects, a kit contains, contains at least, or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therein. In some ...

Claims

CLAIMSWhat is claimed is:

1. A system for profiling RNA-protein interactions comprising:a) a first polypeptide construct comprising an N-terminal fragment of a reverse transcriptase (RTase), or a variant thereof, and a first dimerization domain;andb) a second polypeptide construct comprising a C-terminal fragment of the reverse transcriptase, or a variant thereof, and a second dimerization domain that specifically binds to the first dimerization domain;and wherein the dimerization of the first polypeptide construct and the second polypeptide at the dimerization domain reconstitutes the reverse transcriptase.

2. The system of claim 1, wherein the first polypeptide construct comprises a first targeting moiety.

3. The system of claim 1 or 2, wherein the second polypeptide construct comprises a second targeting moiety.

4. The system of any one of claims 1-4, wherein the reverse transcriptase comprises Moloney murine leukemia virus (MMLV) RTase, human immunodeficiency virus (HIV) RTase, Avian Myeloblastosis Virus (AMV) RTase or a variant thereof.

5. The system of claim 4, wherein the N-terminal fragment of the reverse transcriptase enzyme is encoded by a nucleic acid sequence as set forth is any one of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 116, 148, 168, 172, 176, or 180, or a nucleic acid sequence at least 60% identical thereto.

6. The system of claim 4, wherein the C-terminal fragment of the reverse transcriptase enzyme is encoded by a nucleic acid sequence as set forth is any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 114, 115, 150, 170, 174, 178, 182, or 184, or a nucleic acid sequence at least 60% identical thereto.

7. The system of claim 5, wherein the N-terminal fragment comprises an amino acid sequence as set for in any one of SEQ ID NOs: 117, 119, 121, 123, 125, 127, 129, 131, 133,301320428.1 - 190-135, 137, 139, 141, 145, 149, 169, 173, 177, or 181, an amino acid sequence at least 60% identical thereto.

8. The system of claim 6, the fragment of the RTase is a C-terminal fragment comprising an amino acid sequence as set for in any one of SEQ ID NOs: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 143, 144, 151, 171, 175, 179, 183, or 185, or an amino acid sequence at least 60% identical thereto.

9. The system of any one of claims 2-8, wherein the first and / or the second targeting moiety is a protein; an oligonucleotide, a nucleoside analog, a nucleotide, a receptor, a ligand, an antibody, a nanobody, a small molecule, or any combination thereof.

10. The system of any one of claims 2-9, wherein the first and the second targeting moiety target different targets.

11. The system of claim 9, wherein the first and / or the second targeting moiety comprises a Fc or Fab binding protein.

12. The system of claim 9, wherein the first and / or the second targeting moiety comprises an oligonucleotide or a variant thereof.

13. The system of claim 12, wherein the oligonucleotide comprises a barcode, indices, affinity tag, label, a modified nucleotide, or any combination thereof.

14. The system of claim 13, wherein the affinity tag comprises a streptavidin, or an avidin tag.

15. The system of claim 9, wherein the first and / or the second targeting moiety comprises a small molecule.

16. The system of claim 11, wherein the Fc or Fab binding protein comprises protein A, protein G, protein A / G (pAG), protein L, anti-rabbit IgG, anti-mouse IgG, or a nanobody.

17. The system of claim 16, wherein the Fc or Fab binding protein comprises an anti-mouse or an anti-rabbit nanobody which specifically binds the Fc, or the Fab region of an antibody.301320428.1 - 191 -18. The system of claim 16 or 17, wherein the nanobody is encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOS: 152, 154, 156 or 158, or a sequence at least about 90% identical thereto.

19. The system of claim 16 or 17, wherein the nanobody comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 153, 155, 157, or 159 or a sequence at least about 90% identical thereto.

20. The system of claim 16, wherein the Fc binding protein is encoded by a a nucleic acid sequence as set forth in any one of SEQ ID NOS: 53, 55, 57, or 59, or a sequence at least about 90% identical thereto.

21. The system of claim 16, wherein the Fc binding protein comprises an amino acid sequence as set forth in SEQ ID NOS: 54, 56, 58, or 60, or a or a sequence at least about 90% identical thereto.

22. The system of any one of claims 1-21, wherein the first and / or the second dimerization moieties comprise binding regions of a rapamycin mediated dimerization domain , a leucine zipper, an SH3 domain, a PDZ domain, an Fc domain, an Fc binding domain, a basic helixloop-helix domain (bHLH), a zinc finger domain, a tet repressor dimerization domain, or a Halo-tag ligand, a Halo-tag, or any combination of.

23. The system of any one of claims 1-22, wherein the first and the second dimerization domain comprises, consists essentially of, or consists of a rapamycin mediated dimerization domain.

24. The system of claim 23, wherein the first polypeptide construct comprises an FRB domain, and the second polypeptide comprises an FKBP domain.

25. The system of claim 23, wherein the second polypeptide construct comprises an FRB domain, and the first polypeptide comprises a FKBP domain.

26. The system of claim 24 or 25, wherein:(i) the FRB domain is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 33, or a sequence at least about 90% identical thereto, and comprises an amino acid sequence as set forth in SEQ ID NO: 34, or an amino acid sequence at least about 90% identical thereto;301320428.1 - 192 -(ii) the FKBP domain is encoded by a nucleic acid sequence as set forth in SEQ ID NOS: 31, or a sequence at least about 90% identical thereto, and comprises an amino acid sequence as set forth in SEQ ID NO: 32, or an amino acid sequence at least about 90% identical thereto.

27. The system of any one of claims 1-26, wherein the first polypeptide further comprises a split N-intein (Npu DnaE-N) and the second polypeptide further comprises a split C-intein (Npu DnaE-C).

28. The system of claim 27, wherein the split N-intein is encoded by a polynucleotide comprising a nucleic acid sequence as set forth in SEQ ID NO: 160, or a sequence at least about 90% identical thereto, and comprises an amino acid sequence as set forth in SEQ ID NOS: 161, or a sequence at least about 90% identical thereto; and the split C-intein is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 162, or a sequence at least about 90% identical thereto, and comprises an amino acid sequence as set forth in SEQ ID NOS: 163, or a sequence at least about 90% identical thereto.

29. The system of any one of claims 1-28, wherein the first and / or the second polypeptide construct further comprises one or more linker sequences.

30. The system of claim 29, wherein the one or more linker sequences are 2-100 amino acids in length.

31. The system of claims 29 or 30, wherein the linker is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 41, 43, 45, 47, 49, 164 or 166 or a sequence at least about 80% identical thereto, and comprises an amino acid sequence as set forth in SEQ ID NOs: 42, 44, 46, 48, 50, 165, or 167 or a sequence at least 90% identical thereto.

32. The system of any one of claims 1-31, wherein the first and / or the second polypeptide construct further comprises a fluorophore.

33. The system of claim 32, wherein the fluorophore comprises Green Fluorescent Protein (GFP), eGFP, Red Fluorescent Protein (RFP), Teal Fluorescent Protein (TFP), Blue Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), miRFP, cerulean fluorescent protein (CFP), eCyanFP, mCherry, m Venus, mOrange, mTurquoise, tdTomato,301320428.1 - 193 -aminocoumarin, fluorescein, texas red, Alexa Fluor dyes (e.g. Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 350, Alexa Fluor 532, and Alexa Fluor 700), Cy dyes (e.g. Cy3, Cy5), DyLight dyes, FITC, or Rhodamine, or functional variants thereof.

34. The system of any one of claims 1-33, wherein the first and / or the second polypeptide construct further comprises a purification and / or a solubilization tag.

35. The system of claim 34, wherein the purification and / or a solubilization tag comprises a maltose binding protein (MBP) tag, a GST-tag, a FLAG tag, an HA tag, a His-tag, a SUMO-tag, a Trx-tag, a Halo-tag ligand, a Halo-tag, or any combination of.

36. The system of any one of claims 1-35, wherein the first and / or the second polypeptide construct further comprises a peptide leader sequence.

37. The system of any one of claims 1-8, wherein the first polypeptide is encoded by a a nucleic acid sequence as set forth in any one of SEQ ID NOS: 168, 172, 176, or 180, or a nucleic acid sequence at least 60% identical thereto.

38. The system of any one of claims 1-8, wherein the second polypeptide is encoded by a nucleic acid sequence as set forth in any one of SEQ ID NOS: 170, 174, 178, 182, or 184, or a nucleic acid sequence at least 60% identical thereto.

39. The system of any one of claims 1-8, wherein the first polypeptide comprises an amino acid sequence as set for in any one of SEQ ID NOs: 145, 169, 173, 177, or 181, or an amino acid sequence at least 60% identical thereto.

40. The system of any one of claims 1-8, wherein the second polypeptide comprises an amino acid sequence as set for in any one of SEQ ID NOs: 143, 144, 171, 175, 179, 183, or 185, or an amino acid sequence at least 60% identical thereto.

41. A method of determining one or more RNA interaction sites of a RNA-binding Protein (RBP) in a biological sample, comprising:a) incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex;b) incubating the primary complex with one or more secondary binding agents that specifically bind the RBP-targeting agent, to form a secondary complex;301320428.1 - 194-c) incubating the first or the secondary complex with the system of any one of claims 1-36 to form a tertiary complex;d) adding a transcriptase mix to obtain cDNAs; ande) sequencing the cDNA to determine the one or more RNA interaction sites of the RBP.

42. A method of determining one or more RNA interaction sites of a RNA-binding Protein (RBP) in a biological sample, comprising:a) incubating a RBP-targeting agent with the biological sample, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex;b) incubating the primary complex with one or more secondary binding agents that specifically bind the RBP-targeting agent, to form a secondary complex;c) incubating the first or the secondary complex with a system comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first or the secondary complex to form a tertiary complex;d) incubating the tertiary complex with a transcriptase mix and a system comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; ande) sequencing the cDNA to determine the one or more RNA interaction sites of the RBP.

43. The method of claim 42, wherein the first fragment of the reverse transcriptase is a N-terminal fragment, or a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof; or the C-terminal fragment, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase or any variant thereof.

44. The method of claim 42, wherein the second fragment of the reverse transcriptase is a N-terminal fragment, or a fragment starting at least within the first 50 amino acids of the N-terminal of the reverse transcriptase, or any variant thereof; or the C-terminal fragment, or a fragment ending at least within the last 50 amino acids of the C-terminal of the reverse transcriptase or any variant thereof.301320428.1 - 195-45. The method of claim 41 or claim 42, wherein the method further comprises adding an inducer of dimerization, wherein the addition of the inducer of dimerization brings the first polypeptide construct and the second polypeptide construct in proximity to form a functional reverse transcriptase.

46. The method of claim 45, wherein the addition of the inducer of dimerization brings the first polypeptide construct and the second polypeptide construct in proximity allowing the splicing together of the N-terminal fragment of the reverse transcriptase to the C-terminal fragment of the reverse transcriptase, thereby forming a functional reverse transcriptase.

47. The method of claim 41 or claim 42, wherein the method further comprises one or more wash steps between any of the steps of the method.

48. The method of any one of claims 41-47, wherein the first dimerization domain is FKBP, or a variant thereof and the second dimerization domain is FRB, or a variant thereof, or the second dimerization domain is FKBP, or a variant thereof and the first dimerization domain is FRB, or a variant thereof, and wherein the inducer of dimerization is rapamycin.

49. The method of any one of claims 41-47, wherein the first dimerization domain is a Halo-tag, or a variant thereof, or a Halo-tag ligand or a variant thereof; wherein the second dimerization is a Halo-tag, or a variant thereof, or a Halo-tag ligand or a variant thereof and wherein the binding of the first and the second dimerization domains can be induced by alkyl chloride.

50. The method of any one of claims 41 or 42, wherein the transcriptase mix comprises one or more adapter-RT primer, wherein the one or more adapter RT -primer each comprises an adapter primer sequence and an RT primer sequence.

51. The method of claim 50, wherein at least one of the one or more RT primer sequence is a random RT primer.

52. The method of claim 51, wherein the random RT primer comprises at least 7 nucleotides.

53. The method of claim 52, wherein the random RT primer is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more, nucleotides in length.301320428.1 - 196-54. The method of claim 50, wherein the adapter primer sequence comprises a sequencing barcode.

55. The method of any one of claims 50-54, wherein the transcriptase mix further comprises non-labeled dNTPs, labeled dNTPs, or any combination thereof.

56. The method of claim 55, wherein the labeled dNTPs are biotinylated dNTPs, wherein the biotinylated dNTPs comprises biotin- 16-dUTP, or biotin- 16-dCTP, or both.

57. The method of claim 55 or 56, wherein the labeled dNTP and the non-labeled dNTP are at a ratio of at least 0.5:1, 1:1, or 2:1.

58. The method of any one of claims 50-57, wherein the RT primer sequence further comprises an azide functional group.

59. The method of any one of claims 50-57, wherein the adapter-RT primer comprises a nucleotide sequence as set forth in as set forth in SEQ ID NO: 51, or a sequence at least 80% identical thereto.

60. The method of any one of claims 41-59, wherein the biological sample is a RNA-protein complex, a cell, or a tissue section.

61. The method of claim 60, further comprising one or more of permeabilizing the biological sample, fixing the biological sample with a fixing agent, and quenching the fixing agent.

62. The method of claim 61, wherein the fixing agent comprises formaldehyde, paraformaldehyde, and / or glutaraldehyde, and the quenching agent comprises glycine.

63. The method of any one of claims 41-62, wherein the RBP is a transcription factor, a splicing factor, RNA helicase, ribonuclease, RNA polymerase, translation initiation factor, or ribosomal protein.

64. The method of claim 63, wherein the RBP comprises YTHDF1, YTHDF2, YTHDC1, HuR, PTB, Musashi, eIF4E, FMRP, LARP1, IMP, hnRNP family proteins, Lin28, AUF1, IGF2BP, FUBP1, LIN28B, RBM5, FUS, TIA1, TTP, QKI, MBNL, CELF, NONO, DDX5, RBM10, SAFB, TDP-43, Ataxin-2, hnRNP A / B, C9orf72, hnRNP H / F, Matrin 3 (MATR3), Pur-alpha, TAF15, Huntingtin, RBFOX, SMN, ELAVL, Ro (SSA) and La (SSB) Proteins,301320428.1 - 197-hnRNP, Roquin, Staufenl, NF90 / NF110, ILF3, SF3B1, SRSF2, U2AF1, ZRSR2, PRPF8, PRPF31, SNRNP200, HNRNPA1, HNRNPA2B1, NELFE, CPEB1, SRSF1, NOVAI, NOVA2, G3BP1, PTBP1, RBFOX2, and / or HNRNPC.

65. The method of any one of claims 41-64, wherein the RBP-targeting agent and / or the secondary binding agent comprises an antibody or functional variant thereof, wherein the antibody or functional variant thereof comprises a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody variant, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, single chain antibody, single domain antibody, nanobodies, diabodies, a bi-specific antibody, a multi-specific antibody, or a DARPin.

66. The method of any one of claims 41-65, wherein the RBP-targeting agent and / or the secondary binding agent is labeled, wherein the label comprises a radioisotopes, a hapten, a fluorescent label, a fluorescent polypeptide, a phosphorescent molecule, a chemiluminescent molecule, a chromophore, a luminescent molecule, a photoaffinity molecule, a colored particle, and / or a ligand.

67. The method of any one of claims 41-65, wherein the RBP-targeting agent is linked to a functionalized DNA barcode via an amino spacer, optionally wherein the functionalized DNA barcode comprises an alkyne (3'-O-propargyl N 2'-5' linked) functionalized DNA barcode.

68. The method of claim 67, wherein the alkyne functionalized barcodes comprise a nucleic acid sequence as set forth in any one of SEQ ID NOs: 65-112, or a nucleic acid sequence at least 80% identical thereto.

69. The method of claim 41 or claim 42, wherein the steps (a) - (c) are conducted in-situ.

70. The method of claim 42, wherein the method further comprises imaging the biological sample after steps (a)-(c).

71. The method of any one of claims 41-70, wherein the biological sample comprises less than or equal to 1000, 750, 500, 100, 50, or 20 cells, or wherein the biological sample comprises a single cell, wherein the biological sample comprises less than 5 tissue sections, or wherein the biological sample comprises a single tissue section.301320428.1 - 198-72. The method of any one of claims 41-71, wherein the method does not comprise any one or more of ultraviolet cross-linking, immunoprecipitation, use of base editing proteins, dissociating the one or more tissue section into single cells, oligo(dT) primer initiated reverse transcription, Tn5 tagmentation.

73. The method of any one of claims 42-72, wherein the method detects transient and / or dynamic RNA-RBP interactions, wherein the transient and / or dynamic RNA-RBP interactions occur on a timescale within 10 minutes.

74. The method of any one of claims 42-73, wherein the method can be used to determine one or more interaction sites of the RBP with RNA in the cytoplasm, or nucleus, or both.

75. The method of any one of claims 42-74, wherein the method is used to measures relative binding strength of the RBP to the RNA in comparison to one or more other RBPs to the RNA.

76. The method of any one of claims 42-75, wherein the cDNA comprises one or more labeled nucleotides, wherein the one or more labeled nucleotides are labeled with a fluorescent label, and / or are biotinylated.

77. The method of any one of claims 42-76, wherein the method further comprises purifying the cDNA with a streptavidin comprising agent, wherein the streptavidin comprising agent comprises, a bead, a plate, a magnetic bead, an agarose bead, a microtiter plate, a nanoparticle, and / or a membrane.

78. The method of claim 42, wherein two or more unique RBP targeting agents that interact with one or more RBPs are used in step (a).

79. The method of claim 78, wherein each of the two or more unique RBP targeting agents comprise a unique functionalized DNA barcode linked via an amino spacer.

80. The method of claim 79, wherein the functionalized DNA barcode comprises an alkyne (3'-O-propargyl N 2'-5' linked) functionalized DNA barcode.

81. The method of claim 80, wherein the alkyne functionalized barcodes comprise a nucleic acid sequence as set forth in any one of SEQ ID NO: 65-112, or a sequence at least 80% identical thereto.301320428.1 - 199-82. A method of in-situ imaging of one or more RNA interaction sites of an RNA-binding Protein (RBP) in a biological sample bound to a solid surface, comprising:a) incubating an RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex;b) incubating the first complex with one or more secondary binding agents that specifically binds the RBP-targeting agent, to form a secondary complex;c) contacting the first or the secondary complex with the system of any one of claims 1-36 to form a tertiary complex;d) adding a transcriptase mix to obtain cDNAs; ande) imaging the solid surface.

83. A method of in-situ imaging of one or more RNA interaction sites of an RNA-binding Protein (RBP) in a biological sample bound to a solid surface, comprising:a) incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex;b) incubating the first complex with one or more secondary binding agents that specifically binds the RBP-targeting agent, to form a secondary complex;c) incubating the first or the secondary complex with a system comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first or the secondary complex to form a tertiary complex;d) incubating the tertiary complex with a transcriptase mix and a system comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA; ande) imaging the solid surface.

84. The method of claim 82 or claim 83, wherein the imaging is done using fluorescence microscopy.

85. The method of any one of claims 82-84, wherein the solid surface comprises a slide, a multi-well plate, a capillary, or a microfluidic chamber.

86. The method of any one of claims 82-85, further comprising sequencing the cDNA.301320428.1 - 200 -87. The method of claim 86, wherein the sequencing is performed using Next Generation Sequencing (NGS) techniques.

88. The method of claim 87, wherein the sequencing is done using a single cell genomic imaging techniques.

89. The method of claim 88, wherein the single cell genomic imaging technique comprises, consists essentially of, or consists of spatial transcriptomics, MERFISH, SeqFISH, STARmap, Slide-Seq, Visium Spatial Gene Expression, or deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq).

90. The method of claim 89, wherein the single cell genomic imaging technique is a microfluidic based technique comprising: ligating a first set and a second set of spatial barcodes to the cDNA of step (c), prior to step (d), wherein the first set of spatial barcodes are contacted to the cDNA horizontally using a first multi-channel microfluidic chip, and wherein the second set of spatial barcodes are contacted to the solid surface vertically using a second multi-channel microfluidic chip.

91. The method of claim 88, wherein the first set of spatial barcodes and second set of spatial barcodes form a 2D spatial barcode array.

92. A kit comprising the system of any one of claims 1-36.

93. The kit of claim 92, wherein the kit further comprises a transcriptase mix.

94. A method of determining the one or more RBP binding sites on an mRNA throughout an mRNA cycle, the method comprising:a) incubating a RBP-targeting agent with the RBP, wherein the RBP-targeting agent specifically binds the RBP to form a primary complex;b) incubating the first complex with one or more secondary binding agents that specifically binds the RBP-targeting agent, to form a secondary complex;c) incubating the first or the secondary complex with the system comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a first targeting moiety; wherein the first targeting moiety binds the first or the secondary complex to form a tertiary complex;d) incubating the tertiary complex with a transcriptase mix and a system comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase, a301320428.1 - 201 -second dimerization domain and a second targeting moiety, wherein the second dimerization domain binds the first dimerization domain, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA, and wherein the second targeting moiety binds a specific site on the mRNA; ande) sequencing the cDNA.

95. The method of claim 94, wherein the second targeting moiety comprises a nucleoside analog.

96. The method of claim 95, wherein the nucleoside analog is a 5-ethyluridine.

97. A method of determining one or more RNA interaction sites of a first RNA-binding Protein (RBP) and a second RBP, in a biological sample, comprising:a) incubating a first RBP-targeting agent comprising a functionalized first DNA barcode, with the first RBP, wherein the first RBP-targeting agent specifically binds the first RBP to form a first complex;b) incubating a second RBP-targeting agent comprising a functionalized second DNA barcode, with the second RBP, wherein the second RBP-targeting agent specifically binds the second RBP to form a second complex;c) incubating the first complex and the second complex with a system comprising:(i) a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a targeting moiety; wherein the targeting moiety binds the first complex;(ii) a second polypeptide construct comprising a second fragment of the reverse transcriptase and a second dimerization domain, wherein the second dimerization domain specifically binds, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form a first barcoded cDNA library and a second barcoded cDNA library;d) amplifying and sequencing the first and the second barcoded cDNA library; and301320428.1 - 202 -e) obtaining one or more interaction site of the first RBP and the second RBP by deconvoluting the sequenced cDNA library based on the first and the second DNA barcode.

98. The method of claim 97, wherein the transcriptase system comprise an RT primer sequence comprising a functional group and biotinylated dNTPs.

99. The method of claim 98, wherein the functional group is an azide functional group.

100. The method of claim 97 or claim 98, wherein the biotinylated dNTPs, and the RT primer sequence comprising the azide functional group, are incorporated into the cDNA to form proximal azide labeled biotinylated cDNAs during reverse transcription in step c.

101. The method of claim 97, wherein the functionalized DNA barcode comprises an alkyne (3'-O-propargyl N 2'-5' linked) functionalized DNA barcode.

102. The method of claim 97, wherein the alkyne functionalized barcodes comprise a nucleic acid sequence as set forth in any one of SEQ ID NO: 65-112, or a sequence at least 80% identical thereto.

103. The method of claim 97, further comprising incorporating the alkyne functionalized first and / or second DNA barcode into the cDNA by reacting the alkyne functionalized first DNA barcode and / or second DNA barcode with the proximal azide labeled biotinylated cDNA of claim 100, using in-situ copper catalyzed azide-alkyne cycloaddition (CuAAC), to obtain a first barcoded biotinylated cDNA library and / or the second biotinylated cDNA library.

104. The method of claim 103, wherein the method further comprises purifying the first and / or the second barcoded biotinylated cDNA library over a streptavidin column prior to step (d).

105. The method of claim 104, further comprising processing the CuAAC using a Klenow Fragment DNA polymerase for second strand synthesis.

106. The method of claim 105, wherein the one or more interaction sites of the first RBP and the second RBP are obtained by deconvoluting the sequenced data based on the first and the second DNA barcodes incorporated into the cDNA.

107. A method of determining spatial distribution of a RNA modification sites on a biological sample bound to a solid surface, comprising:301320428.1 - 203 -a) incubating a modification-targeting agent that specifically binds the modification site on the RNA to form a primary complex;b) incubating the primary complex with a secondary binding agent that specifically bind the primary complex to form a secondary complex;c) incubating the first or the secondary complex with a system comprising a first polypeptide construct comprising a first fragment of a reverse transcriptase, a first dimerization domain and a first targeting moiety; wherein the first targeting moiety binds the first or the secondary complex to form a tertiary complex;d) incubating the tertiary complex with a transcriptase mix and a system comprising a second polypeptide construct comprising a second fragment of the reverse transcriptase, a second dimerization domain and optionally a second targeting moiety, wherein the second dimerization domain binds the first dimerization domain, or can be induced to bind the first dimerization domain, wherein the dimerization of the first and the second polypeptide construct initiates reverse transcription to form cDNA;e) optionally incorporating labelled barcodes into the cDNA;f) sequencing and imaging the biological sample using a single cell genomic imaging technique to determine the one or more modification sites.301320428.1 - 204 -

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