Methods for detection of proteins and nucleic acids

Abstract

This disclosure relates to systems and methods for detection of proteins and nucleic acids in a cell or tissue sample. This disclosure also relates to systems and methods for spatial profiling of the detected proteins and nucleic acids across a cell or tissue sample.

Description

PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCTMETHODS FOR DETECTION OF PROTEINS AND NUCLEIC ACIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Application No. 63 / 697,401 , filed September 20, 2024, which is incorporated by reference herein in its entirety for any purpose.FIELD

[0002] This disclosure relates to methods for detection of an analyte, such as proteins and nucleic acids, in biological samples.INTRODUCTION AND SUMMARY OF INVENTION

[0003] Single-cell and spatial technologies that profile genetic activity across a whole cell or tissue provide invaluable information about the biological processes within cells and tissue, including disease processes or biological processes of pharmacological effect. Conventionally, various attempts to evaluate genetic activity have been focused on genomics, particularly transcriptom ics.

[0004] Proteomics can provide further information about the biological function of cells and organisms. Proteomics includes qualitative and quantitative measurement of gene activity by detecting and quantifying the expression on a protein level rather than the transcriptional level. Proteomics also includes a study of events which are not coded genetically, such as a post-translational modification of proteins and interactions between proteins. There also have been many efforts to measure mRNA (messenger RNA) concentrations as a proxy for protein concentrations. Accordingly, there remains a need for improvement of detection of proteins and nucleic acids, e.g., RNAs such as mRNAs. Thus, in some embodiments, the present disclosure provides for joint spatial profiling of proteins and nucleic acids in a cell or tissue sample.

[0005] Aptamers and antibodies can bind internal epitopes of cells using fixation and permeabilization protocols; however, such binding may be better with the smaller aptamers, having a hydrodynamic diameter of ~2 nm compared to ~15 nm for antibodies. Indeed, many aptamers are readily internalized and staining of intracellular compartments can be more effective than with antibodies. This provides advantages for combining intracellular proteomic and transcriptom ic readouts.

[0006] Accordingly, the present disclosure provides methods for detection of one or more analytes, such as proteins and nucleic acids in a cell or tissue sample. The location of the analyte may be determined by (1) performing nucleic acid amplificationPCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT (e.g., rolling circle amplification) on a padlock probe that hybridizes directly or indirectly to an immobilized analyte, thereby generating a first amplified circularized padlock probe and (2) detecting the amplified circularized padlock probe. This disclosure also relates to systems and methods for spatial profiling of the detected proteins and nucleic acids across the cell or tissue sample.

[0007] In some embodiments, the present disclosure provides for a method of target detection in a biological sample comprising a cell, the method comprising: (a) contacting the biological sample with a polynucleotide comprising an aptamer and a capture probe, wherein the aptamer is capable of binding to a target protein, and wherein the capture probe comprises a first binding sequence and a second binding sequence; (b) hybridizing a first padlock probe to the capture probe of the polynucleotide bound to the target protein; (c) generating a first circularized padlock probe; (d) amplifying the first circularized padlock probe using rolling circle amplification, thereby generating a first amplified circularized padlock probe; and (e) detecting the first amplified circularized padlock probe, thereby identifying the location of the protein in the biological sample.

[0008] In some embodiments, the first padlock probe comprises: (a) a first sequence that is substantially complementary to the first binding sequence of the capture probe of the polynucleotide; (b) a barcode sequence that is unique to the aptamer of the polynucleotide; and (c) a second sequence that is substantially complementary to the second binding sequence of the capture probe of the polynucleotide.

[0009] In some embodiments, generating the circularized padlock probe comprises ligating the first sequence of the padlock probe to the second sequence of the padlock probe.

[0010] In some embodiments, the amplification step comprises: (a) hybridizing one or more amplification primers to the padlock probe; and (b) amplifying the padlock probe with a polymerase to produce an amplified circularized padlock probe.

[0011] In some embodiments, detecting the amplified circularized padlock probes comprises contacting the biological sample with a plurality of labelled probes comprising a sequence complementary to a sequence of the amplified circularized padlock probe.

[0012] In some embodiments, the method further comprises: (a) contacting the biological sample with a padlock probe, wherein the padlock probe is capable of binding to a nucleic acid in the biological sample, and wherein a second padlock probe comprises a first binding sequence complementary to a first sequence of a targetPCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT nucleic acid and a second binding sequence complementary to a second sequence of the target nucleic acid; (b) hybridizing the second padlock probe to the target nucleic acid; (c) generating a second circularized padlock probe; (d) amplifying the second circularized padlock probe using rolling circle amplification, thereby generating a second amplified circularized padlock probe; and (e) detecting the second amplified circularized padlock probe, thereby identifying the location of the target nucleic acid in the biological sample.

[0013] In some embodiments, the method further comprises: (a) contacting the biological sample with a plurality of padlock probes, wherein each padlock probe is capable of binding to a different nucleic acid in the biological sample, and wherein a second padlock probe comprises a first binding sequence complementary to a first sequence of a target nucleic acid and a second binding sequence complementary to a second sequence of the target nucleic acid; (b) hybridizing the second padlock probe to the target nucleic acid; (c) generating a second circularized padlock probe; (d) amplifying the second circularized padlock probe using rolling circle amplification, thereby generating a second amplified circularized padlock probe; and (e) detecting the second amplified circularized padlock probe, thereby identifying the abundance and the location of different nucleic acids in the biological sample. In some embodiments, generating the second circularized padlock probe comprises ligating a first sequence of the second padlock probe to a second sequence of the padlock probe.

[0014] In some embodiments, the amplification step comprises: (a) hybridizing one or more amplification primers to the second padlock probe; and (b) amplifying the second padlock probe with a polymerase to produce an amplified circularized padlock probe. In some embodiments, the one or more amplification primers is complementary to the barcode sequence of the padlock probe.

[0015] In some embodiments, detecting the first or second amplified circularized padlock probes comprises contacting the biological sample with a plurality of labelled probes comprising a sequence complementary to a sequence of the amplified circularized padlock probe. In some embodiments, the labelled probe comprises a sequence that is complementary to at least a portion of the barcode sequence of the amplified circularized padlock probe. In some embodiments, the plurality of labelled probes are fluorescently labelled.

[0016] In some embodiments, the method further comprises removing the labelled probes or the amplified circularized padlock probes. In some embodiments, the method further comprises contacting the labelled probes or the amplified circularized padlockPCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT probe with an enzyme that cleaves nucleic acid sequences. In some embodiments, the enzyme is uracil DNA glycosylase (UDG).

[0017] In some embodiments, the method comprises permeabilizing the cell to allow at least a part of the plurality of polynucleotides to bind to intracellular proteins.

[0018] In some embodiments, the biological sample comprises a formalin-fixed, paraffin-embedded (FFPE) sample. In some embodiments, the biological sample comprises a tissue section. In some embodiments, the biological sample comprises a fresh frozen sample. In some embodiments, the biological sample comprises live cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Fig. 1 is a schematic diagram depicting an illustrative polynucleotide comprising an aptamer and a capture probe in accordance with aspects of the present disclosure. The polynucleotide comprises, from 5’ to 3’, an aptamer capable of binding to a protein, a first binding sequence (P1 ) that can hybridize to a sequence of a padlock probe (P1 ’), and a second binding sequence (P2) that can hybridize a sequence of the padlock probe (P2). Also shown is a padlock probe comprising, from 5’ to 3’, PT, a barcode sequence that is unique to the aptamer of the polynucleotide, and P2’.

[0020] Fig. 2 is a flowchart depicting steps of an illustrative method for detecting a plurality of target proteins and mRNA in a sample, in accordance with aspects of the present disclosure.DETAILED DESCRIPTION

[0021] Various aspects and examples of systems and methods for detection of proteins and nucleic acids are described below and illustrated in the associated drawings. Unless otherwise specified, a system for protein measurement in accordance with the present disclosure, and / or its various components, may contain at least one of the structures, components, functionalities, and / or variations described, illustrated, and / or incorporated herein. Furthermore, unless specifically excluded, the process steps, structures, components, functionalities, and / or variations described, illustrated, and / or incorporated herein in connection with the present disclosure may be included in other similar devices and methods, including being interchangeable between disclosed embodiments. The following description of various examples is merely illustrative in nature and is in no way intended to limit the disclosure, its application, or uses. Additionally, the advantages provided by the examples and embodiments described below are illustrative in nature and not all examples and embodiments provide the same advantages or the same degree of advantages.PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT

[0022] The following definitions are used herein unless otherwise indicated.

[0023] “Comprising,” “including,” and “having” (and conjugations thereof) are used interchangeably to mean including but not necessarily limited to, and are open-ended terms not intended to exclude additional, unrecited elements or method steps.

[0024] Terms such as “first”, “second”, and “third” are used to distinguish or identify various members of a group, or the like, and are not intended to show serial or numerical limitation.

[0025] By substantially complementary, it is meant that the two sequences have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity.

[0026] The term “or” is used in an inclusive sense, i.e. , equivalent to “and / or,” unless the context clearly indicates otherwise.

[0027] In this disclosure, one or more publications, patents, and / or patent applications may be incorporated by reference. However, such material is only incorporated to the extent that no conflict exists between the incorporated material and the statements and drawings set forth herein. In the event of any such conflict, including any conflict in terminology, the present disclosure is controlling.Examples, Components, and Alternatives

[0028] The following sections describe selected aspects of illustrative systems and methods involving polynucleotides including an aptamer for binding a target protein. The examples in these sections are intended for illustration and should not be interpreted as limiting the scope of the present disclosure. Each section may include one or more distinct embodiments or examples, and / or contextual or related information, function, and / or structure.A. Illustrative polynucleotide for binding target proteins

[0029] This section describes an illustrative polynucleotide for binding a protein in a cell. Fig. 1 schematically depicts an illustrative polynucleotide in accordance with aspects of the present disclosure. Unless otherwise specified, the drawings of the present disclosure are schematic and not necessarily to scale with respect to, e.g., polynucleotide length.

[0030] The illustrative polynucleotide comprises, from 5’ to 3’, an aptamer capable of binding to a target protein comprising a first binding sequence (for example, P1 asPCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT shown in Fig. 1 ) and a second binding sequence (for example, P2 as shown in Fig. 1 ). The first and second binding sequences may collectively be referred to as a capture probe.

[0031] In some embodiments, the aptamer (for example, element 1 as shown in FIG. 1 ) comprises 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, or 40-60, 40-50, 50- 100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, or 90-100 nucleotides. In some embodiments, the aptamer comprises 30-90 nucleotides. In some embodiments, the aptamer comprises 32-84 nucleotides. In some embodiments, the aptamer comprises 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,41 , 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 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, or 90 nucleotides.

[0032] In some embodiments, the aptamer comprises one or more chemically modified nucleotides. In some embodiments, the aptamer comprises at least one, at least two, at least three, at least four, or at least five C-5 modified nucleotides. In some embodiments, the aptamer comprises at least one C-5 modified nucleotide. In some embodiments, the aptamer comprises at least two C-5 modified nucleotides. In some embodiments, the aptamer comprises at least three C-5 modified nucleotides. In some embodiments, the aptamer comprises at least four C-5 modified nucleotides. In some embodiments, the aptamer comprises at least five C-5 modified nucleotides. In some embodiments, the modified nucleobase is selected from 5-(N-benzylcarboxamide) (“Bn”)-cytosine, 5-(N-2-phenylethylcarboxamide) (“PE”)-cytosine, 5-(N-3- phenylpropylcarboxamide (“PP”)-cytosine, 5-(N-1 -naphthylmethylcarboxamide) (“Nap”)- cytosine, 5-(N-2-naphthylmethylcarboxamide) (“2Nap”)-cytosine, 5-(N-1 -naphthyl-2- ethylcarboxamide) (“NE”)-cytosine, 5-(N- tyrosylcarboxamide) (”Tyr”)-cytosine, Bn- uracil, Nap-uracil, PE-uracil, 5-(N-isobutylcarboxamide) (“lb”)-uracil, 5-(N-4-fluoro- benzylcarboxamide) (“FBn”)-uracil, 2Nap-uracil, NE-uracil, 5-(N-3,4- methylenedioxybenzylcarboxamide) (“MBn”)-uracil, 5-(N-3- benzofuranylethylcarboxamide) (“BF”)-uracil, 5-(N-3-benzothiophenylethylcarboxamide) (“BT”)-uracil, 5-(N-3-phenylpropylcarboxamide) (“PP”)-uracil, 5-(N- morpholinoethylcarboxamide) (“MOE”)-uracil, Tyr-uracil, 5-(N-3-indole-2- ethylcarboxamide) (“Trp”)-uracil, 5-(n-(R)-2-hydrozypropylcarboxamide) (“Thr”)-uracil, 5-(N-((1 ,1’-biphenyl)-4-yl)ethylcarboxamide) “BPE”-uracil, 5-(N-4- phenylbenzylcarboxamide) (“PBn”)-uracil, 5-(N-4-phenoxybenzylcarboxamide) (“POP”)-PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT uracil, 5-(N-3,3-diphenylpropylcarboxamide) (“DPP”)-uracil, 5-(N-3- phenylbenzylcarboxamide) (“DBM”)-uracil, 5-(N-benzylhydrylcarboxamide) (“BH”)- uracil, BPE-cytosine, PBn-cytosine, POP-cytosine, DPP-cytosine, DBM-cytosine, and BH-cytosine.B. Illustrative Aptamers

[0033] Any nucleic acid-based aptamer that binds a protein target may be used in the present methods.

[0034] One way to detect and quantify the presence of specific proteins in a biological sample is through the use of protein-binding nucleic acid aptamers, such as SOMAmer® (Slow Off-rate Modified Aptamer) reagents. In some embodiments, an aptamer may comprise one or more chemically modified nucleotides that improve the stability and / or binding affinity of the aptamer for the protein target.

[0035] Aptamers are typically single stranded DNA-based protein affinity reagents that may include chemically modified nucleotides. In some embodiments, an aptamer comprises one or more chemically modified nucleotides, expanding the chemical diversity of standard DNA aptamers and enhancing the specificity and affinity of protein- nucleic acid interactions. These modified nucleotides are incorporated into nucleic acid libraries used for iterative selection and amplification of aptamers to a target, using for example, a process called SELEX (Systematic Evolution of Ligands by Exponential enrichment). In some embodiments, the inclusion of certain chemically modified nucleotides allows for the generation of aptamers capable of binding proteins that had been resistant to selection with nucleic acids lacking similar chemical modifications. Aptamers can be selected according to the desirable properties of specificity and slow off-rate, under the assay conditions in which the reagents will be used.

[0036] In some embodiments, an aptamer that binds a desired target is developed using “Systematic Evolution of Ligands by Exponential enrichment,” or SELEX. The SELEX process is a method for the in vitro evolution of nucleic acid molecules for a certain desired activity, such as specific binding to target molecules, such as proteins. In some embodiments, a SELEX-identified nucleic acid capture reagent is a specific ligand of a given target molecule, such as a protein. Molecules of any size or composition can serve as targets.

[0037] The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and stepwise iterations of binding, partitioning and amplification, using the same general selection scheme, toPCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, and then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule. In this manner, aptamers suitable for binding to virtually any target protein can be discovered.

[0038] SOMAmers are protein-binding aptamers that have a rate of dissociation (ti / 2) generally between 5 and 240 minutes, this being the average time it takes for half of the protein-aptamer complexes to dissociate. SOMAmers generally comprise modified nucleosides that improve protein binding, affinity, and / or off rate. In some embodiments, the modifications comprise chemical groups that are attached to the 5-position of the pyrimidine bases. By functionalizing the 5-position with amino acid-like moieties (e.g., benzyl, 2-napthyl, etc.), the chemical diversity of oligonucleotides is expanded, allowing high affinity binding with a wider range of target molecules. Nonlimiting exemplary 5- position modified pyridine nucleobases include 5-(N-benzylcarboxamide) (“Bn”)- cytosine, 5-(N-2-phenylethylcarboxamide) (“PE”)-cytosine, 5-(N-3- phenylpropylcarboxamide (“PP”)-cytosine, 5-(N-1 -naphthylmethylcarboxamide) (“Nap”)- cytosine, 5-(N-2-naphthylmethylcarboxamide) (“2Nap”)-cytosine, 5-(N-1-naphthyl-2- ethylcarboxamide) (“NE”)-cytosine, 5-(N- tyrosylcarboxamide) (”Tyr”)-cytosine, Bn- uracil, Nap-uracil, PE-uracil, 5-(N-isobutylcarboxamide) (“lb”)-uracil, 5-(N-4-fluoro- benzylcarboxamide) (“FBn”)-uracil, 2Nap-uracil, NE-uracil, 5-(N-3,4- methylenedioxybenzylcarboxamide) (“MBn”)-uracil, 5-(N-3- benzofuranylethylcarboxamide) (“BF”)-uracil, 5-(N-3-benzothiophenylethylcarboxamide) (“BT”)-uracil, 5-(N-3-phenylpropylcarboxamide) (“PP”)-uracil, 5-(N- morpholinoethylcarboxamide) (“MOE”)-uracil, Tyr-uracil, 5-(N-3-indole-2- ethylcarboxamide) (“Trp”)-uracil, 5-(n-(R)-2-hydrozypropylcarboxamide) (“Thr”)-uracil, 5-(N-((1 ,1’-biphenyl)-4-yl)ethylcarboxamide) “BPE”-uracil, 5-(N-4- phenylbenzylcarboxamide) (“PBn”)-uracil, 5-(N-4-phenoxybenzylcarboxamide) (“POP”)- uracil, 5-(N-3,3-diphenylpropylcarboxamide) (“DPP”)-uracil, 5-(N-3- phenylbenzylcarboxamide) (“DBM”)-uracil, 5-(N-benzylhydrylcarboxamide) (“BH”)-PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT uracil, BPE-cytosine, PBn-cytosine, POP-cytosine, DPP-cytosine, DBM-cytosine, and BH-cytosine. Nonlimiting exemplary modified nucleosides that may be included in aptamers, such as SOMAmers, include 5-position modified pyridine nucleosides. Exemplary 5-position modified pyridine nucleosides that may be used in aptamers, such as SOMAmers, include, but are not limited to, BndC, 2’-OMe-Bn-C, PEdC, 2’- OMe-PE-C, PPdC, 2’-OMe-PP-C, NapdC, 2’-OMe-Nap-C, 2NapdC, 2’-OMe-2Nap-C, NEdC, 2’-OMe-NE-C, 2NEdC, 2’-OMe-2NE-C, TyrdC, 2’-OMe-Tyr-C, BndU, 2’-0Me- Bn-U, NapdU, 2’-OMe-Nap-U, PEdll, 2’-OMe-PE-U, IbdU, 2’-OMe-lb-U, FBndll, 2’- OMe-FBn-U, 2Napdll, 2’-OMe-2Nap-U, NEdll, 2’-OMe-NE-U, MBndll, 2’-OMe-MBn-U, BFdll, 2’-OMe-BF-U, BTdll, 2’-OMe-BT-U, PPdU, 2’-OMe-PP-U, MOEdll, 2’-0Me- MOE-U, Tyrdll, 2’-OMe-Tyr-U, Trpdll, 2’-OMe-Trp-U, Thrdll, and 2’-OMe-Thr-U, BPEdll, 2’-OMe-BPE-U, 2’-F-BPE-U, PBndll, 2’-OMe-PBn-U, 2’-F-PBn-U, POPdU, 2’- OMe-POP-U, 2’-F-POP-U, DPPdU, 2’-OMe-DPP-U, 2’-F-DPP-U, DBMdll, 2’-0Me- DBM-U, 2’-F-DBM-U, BHdll, 2’-OMe-BH-U, 2’-F-BH-U, BPEdC, 2’-OMe-BPE-C, 2’-F- BPE-C, PBndC, 2’-OMe-PBn-C, 2’-F-PBn-C, POPdC, 2’-OMe-POP-C, 2’-F-POP-C, DPPdC, 2’-OMe-DPP-C, 2’-F-DPP-C, DBMdC, 2’-OMe-DBM-C, 2’-F-DBM-C, BHdC, 2’- OMe-BH-C, and 2’-F-BH-C. See, e.g., PCT Publication Nos. WO 2022 / 0221241 and WO 2018 / 0005974.

[0039] Assays directed to the detection and quantification of physiologically significant molecules in biological samples and other samples are important tools in scientific research and in the health care field. Aptamers are capable of binding to a target molecule in the sample in a highly specific manner and with very high affinity. After appropriate washing and partitioning steps to first remove unbound proteins and then to remove unbound aptamers, aptamers are eluted from the resultant aptamerprotein complexes. The eluted aptamers may then be detected, qualitatively or quantitatively, using the methods provided herein, thereby enabling a determination of the absence, presence, amount, and / or concentration of the target molecules in the sample.B. Illustrative Capture Probes

[0040] In certain embodiments of the present disclosure, the aptamer is linked to a capture probe comprising, from 5’ to 3’, a first binding sequence (shown as P1 in Fig. 1 ) and a second binding sequence (shown as P2 in Fig. 2). In some embodiments, the first binding sequence can hybridize to a first sequence of a padlock probe (shown as PT in Fig. 1 ) and the second binding sequence can hybridize to a second sequence of a padlock probe (shown as P2’ in Fig. 2).PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT

[0041] In general, the capture probe can be any length that accommodates the lengths of its functional components. In one embodiment, the capture probe is between 20 and 100 nucleotides in length. In some embodiments, the capture probe is at least 20, 30, 40, 50, 60, 70, 80, 90 or over 100 nucleotides in length. In other embodiments, the capture probe is 200 to about 400 nucleotides in length. In one embodiment, the capture probe is generally made up of deoxyribonucleic acids (DNA). In one embodiment, the capture probe is a DNA sequence. In other embodiments, the capture probe comprises modified DNA bases. Modifications of DNA bases are known in the art, and can include chemically modified bases including labels. In other embodiments, the capture probe comprises ribonucleic acid (RNA) sequences or modified ribonucleotide bases. Modifications of RNA bases are known in the art, and can include chemically modified bases including labels. In still other embodiments, different portions of the capture probe can comprise DNA and RNA, modified bases, or modified polymer connections (including but not limited to PNAs and LNAs). For a description of modifications to oligonucleotides, see commercial suppliers, e.g., Integrated DNA Technologies, USA website; Custom Oligonucleotide Modifications Guide, Sigma- Aldrich, www.sigmaaldrich.com / technical- documents / articles / biology / custom-dna- oligos-modifications.html, and Modified Oligonucleotides, TriLink, www.trilinkbiotech.com / oligo / modifiedoligos.asp. As described above, in still other embodiments, the polymer construct is composed of polyamides, PNA, etc.

[0042] In some embodiments, the capture probe includes a first binding sequence and a second binding sequence that, together, are unique to the aptamer. In some embodiments, the first and / or second binding sequence has a length of about 5-20 nucleotides. In some embodiments, the first and / or second binding sequence has a length of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the first and / or second binding sequence has a length of 5-20, 5-19, 5- 18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11 , 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6- 17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11 , 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11 , 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8- 13, 8-12, 8-11 , 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11 , 9- 10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11 , 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12- 15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17- 20, 17-19, 17-18, 18-20, 18-19, or 19-20 nucleotides.PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT C. Illustrative Padlock Probes

[0043] This present disclosure provides methods for identifying a location of an analyte (e.g., nucleic acid or protein) in a biological sample using a padlock probe. As used herein, a “padlock probe” or “padlock oligonucleotide” refers to an oligonucleotide that has, at its 5' and 3' ends, sequences (e.g., a first sequence at the 5' end and a second sequence at the 3' end) that are complementary to adjacent or nearby portions (e.g., a first binding sequence P1 and a second binding sequence P2 as shown in FIG. 1 ) of the target nucleic acid or aptamer bound to the target protein (e.g., a SOMAmer). Upon hybridization of the padlock probe to the first and second portions of the analyte or analyte derived molecule, the two ends of the padlock probe are either brought into contact or an end is extended until the two ends are brought into contact, allowing circularization of the padlock probe by ligation (e.g., ligation using any of the methods described herein). The ligated probe resulting from the ligation can be referred to as the “circularized padlock probe.” After circularization of the padlock probe, rolling circle amplification can be used to amplify the circularized padlock probe. In some embodiments, a first sequence of a padlock probe includes a sequence that is substantially complementary to a first binding sequence of the capture probe of the polynucleotide disclosed herein. In some embodiments, the first binding sequence of the target nucleic acid or the capture probe of the polynucleotide is 5' to the second binding sequence of the capture probe of the polynucleotide. In some embodiments, the first sequence is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical) to the first binding sequence.

[0044] In some embodiments, the padlock probe includes an aptamer barcode (see e.g., “barcode” as shown in Fig. 1 ) comprises a sequence for identifying the aptamer. In some embodiments, the aptamer barcode sequence has a length of about 5-20 nucleotides. In some embodiments, the aptamer barcode sequence has a length of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the aptamer barcode sequence has a length of 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11 , 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6- 15, 6-14, 6-13, 6-12, 6-11 , 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11 , 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8- 11 , 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11 , 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11 , 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14,PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14- 16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20 nucleotides. In some embodiments, the aptamer barcode sequence is 3’ of the binding site for a primer for PCR amplification.

[0045] In some embodiments, a backbone sequence of a padlock probe includes a sequence that is substantially complementary to an amplification primer. The amplification primer can be a primer used in a rolling circle amplification reaction (RCA) of the ligated padlock probe hybridized to the capture probe. RCA, or rolling circle amplification, is well known in the art and includes a process by which circularized nucleic acid molecules are amplified with a DNA polymerase with strand displacement capabilities (and other necessary reagents for amplification to occur), thereby creating multiple concatenated copies of the circularized nucleic acid molecules. In some embodiments, the backbone sequence includes a functional sequence. In some embodiments the backbone sequence includes a barcode sequence (e.g., any of the exemplary barcode sequences described herein). In some embodiments, the barcode sequence includes a sequence that is substantially complementary to an amplification primer.

[0046] In some embodiments, a second sequence of a padlock probe includes a sequence that is substantially complementary to a second binding sequence of the capture probe or the target nucleic acid. In some embodiments, the second sequence of the capture probe or the target nucleic acid is 3' to the first binding sequence of the capture probe or the target nucleic acid. In some embodiments, the second sequence is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical) to the second binding sequence.

[0047] In some embodiments, the first sequence is substantially complementary to a first binding sequence of the capture probe or target nucleic acid that is directly adjacent to the second binding sequence of the capture probe or target nucleic acid analyte to which the second sequence is substantially complementary. In such cases, the first sequence is ligated to the second sequence, thereby creating a circularized padlock probe.

[0048] In some embodiments, the first sequence is substantially complementary to a first portion of the capture probe or target nucleic acid analyte that is not directly adjacent to the second portion of the capture probe or target nucleic acid analyte to which the second sequence is substantially complementary. In such cases, a “gap”PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT exists between where the first sequence is hybridized to the first binding sequence and where the second sequence is hybridized to the second binding sequence. In some instances, to generate a padlock probe that includes a first sequence and a second sequence that are close enough to one another to initiate a ligation step, the second sequence is extended enzymatically (e.g., using a polymerase, such as a reverse transcriptase). In some embodiments, the “gap” sequence between the first sequence and the second sequence include 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides. In some embodiments, the “gap” sequence between the first sequence and the second sequence include at least 25 nucleotides, at least 30 nucleotide, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, or at least 50 nucleotides.

[0049] In some embodiments, extending the second sequence of the padlock probe includes a nucleic acid extension reaction (e.g., any of the nucleic acid extension reactions described herein). In some embodiments, extending the second sequence of the padlock probe includes reverse transcribing the capture probe or target nucleic acid analyte. In some embodiments, extending the second sequence of the padlock probe includes using a reverse transcriptase (e.g., any of the reverse transcriptases described herein). In some embodiments, extending the second sequence of the padlock probe includes using a Moloney Murine Leukemia Virus (M-MulV) reverse transcriptase. In some embodiments, the reverse transcriptase includes strand displacement properties. In some embodiments, extending the second sequence of the padlock probe generates a sequence that is complementary to the analyte or the analyte derived molecule. In some embodiments, extending the second sequence of the padlock probe generates an extended second sequence of the padlock probe that is complementary to the capture probe or target nucleic acid analyte. In some embodiments, the second sequence of the padlock probe generates a sequence that is adjacent to the first sequence of the padlock probe.

[0050] Once the first and second sequences in a padlock probe are adjacent, ligation of the two ends can occur. In some embodiments, the ligation step includes ligating the second sequence to the first sequence of the padlock probe using enzymatic or chemical ligation. In some embodiments where the ligation is enzymatic, the ligase is selected from a T4 RNA ligase (Rnl2), a SplintR ligase, a single stranded DNA ligase, or a T4 DNA ligase. In some embodiments, the ligase is a T4 RNA ligase (Rnl2) ligase. In some embodiments, the ligase is a pre-activated T4 DNA ligase as described herein.PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT

[0051] Additionally, this disclosure features a method for identifying which conditions are optimal for releasing an analyte from a biological sample by detection of a signal that corresponds to an amplified circularized padlock probe (which thereby corresponds to a capture probe or target nucleic acid analyte). In some embodiments, the method includes a step of detecting a signal corresponding to the amplified circularized padlock probe on the substrate, thereby identifying whether a reaction condition, such as a permeabilization condition, results in the detection of an analyte in the biological sample.G. Illustrative Methods

[0052] With reference to Fig. 2, this section describes an illustrative method for obtaining qualitative and / or quantitative information about proteins and nucleic acids in a sample. In general, a method for obtaining qualitative and / or quantitative information includes (1 )(a) contacting a biological sample with a polynucleotide comprising an aptamer and a capture probe and (b) hybridizing a padlock probe to the capture probe, (2) generating a circularized padlock probe, (3) amplifying the circularized padlock probe using rolling circle amplification (RCA), and (4) detecting the amplified circularized padlock probe.

[0053] At step (1 )(a), the method includes contacting a biological sample with a plurality of polynucleotides comprising an aptamer and a capture probe. In some embodiments, each aptamer is capable of binding to a different protein. In some embodiments, each capture probe comprises a first binding sequence (P1 ) and a second binding sequence (P2), together, identify the aptamer. Exposing the biological sample to a library of many polynucleotides allows for detecting a large number of target protein species in a single assay. In some embodiments, the polynucleotides comprise aptamers that bind to cell surface epitopes such as proteins expressed on the surface of a cell. In some embodiments, the polynucleotides comprise aptamers that bind to intracellular epitopes such as proteins expressed within the cell. In embodiments in which the polynucleotides comprise aptamers that bind to intracellular epitopes, the cells may be fixed and permeabilized prior to polynucleotide exposure. In some examples, the aptamers are SOMAmers. SOMAmers have slower off-rates than typical aptamers, which in some embodiments allows the SOMAmer reagents to remain bound to the corresponding proteins during execution of additional assay steps such as multiple washes, improving assay performance.PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT

[0054] At step (1 )(b), the method includes hybridizing a padlock probe to the capture probe. In some embodiments, the method includes contacting the biological sample with the padlock probe. In some embodiments, the padlock probe comprises, from 5’ to 3’, a first sequence (P1 ’) that is substantially complementary to the first binding sequence of the capture probe of the polynucleotide (P1 ), a barcode sequence that is unique to the aptamer of the polynucleotide, and a second sequence (P2’) that is substantially complementary to the second binding sequence of the capture probe of the polynucleotide (P2). In some embodiments, the first sequence of the padlock probe (PT) hybridizes to the first binding sequence of the capture probe of the polynucleotide (P1 ). In some embodiments, the second sequence of the padlock probe (P2’) hybridizes to the second sequence of the capture probe of the polynucleotide (P2). In some embodiments, the first sequence of the padlock probe (PT) hybridizes to the first binding sequence of the capture probe of the polynucleotide (P1 ) and the second sequence of the padlock probe (P2’) hybridizes to the second sequence of the capture probe of the polynucleotide (P2).

[0055] In some embodiments, the method of step (1 )(b) may further comprise hybridizing a padlock probe to a nucleic acid. In some embodiments, the method includes contacting the biological sample with the padlock probe. In some embodiments, the padlock probe comprises, from 5’ to 3’, a first binding sequence complementary to a first sequence of the target nucleic acid, a barcode sequence that is unique to the target nucleic acid, and a second binding sequence complementary to a second sequence of the target nucleic acid. In some embodiments, the nucleic acid is an RNA such as a messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), long noncoding RNA (IncRNA), microRNA (miRNA), small interfering RNA (siRNA), short nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), or small nuclear RNA (snRNA). In some embodiments, the nucleic acid is a DNA sequence. In some embodiments, the nucleic acid is a DNA sequence comprising one or more of a single nucleotide polymorphism (SNP), an insertion or deletion (indel), a copy number variant (CNV), or a tandem repeat, a marker of open chromatin, or methylated nucleotide(s).

[0056] At step (2), the method includes generating a circularized padlock probe. In some embodiments, generating the circularized padlock probe comprises ligating the first sequence of the padlock probe to the second sequence of the padlock probe. In some embodiments, the ligation comprises enzymatic ligation or chemical ligation. InPCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT some embodiments, the ligation is performed using a ligase. In some embodiments, the ligase comprises a T4 DNA ligase.

[0057] At step (3), the method includes amplifying the circularized padlock probe using rolling circle amplification (RCA). In some embodiments, amplification comprises the steps of (a) hybridizing one or more amplification primers to the padlock probe and (b) amplifying the padlock probe with a polymerase to produce an amplified circularized padlock probe. In some embodiments, the polymerase has strand displacement activity. In some embodiments, the polymerase is a Phi29 DNA polymerase. In some embodiments, the one or more amplification primer is fully or partially complementary to the barcode sequence of the padlock probe. In some embodiments, the one or more amplification primer is complementary to a sequence of the padlock probe that is not the barcode.

[0058] At step (4), the method includes detecting the amplified circularized padlock probe. In some embodiments, detecting the amplified circularized padlock probe comprises contacting the biological sample with a plurality of labelled probes comprising a sequence complementary to a sequence of the amplified circularized padlock probe. In some embodiments, the labelled probe comprises a sequence that is complementary to at least a portion of the barcode sequence of the amplified circularized padlock probe. In some embodiments, the plurality of labelled probes are fluorescently labelled. In some embodiments, the method further comprises removing the labelled probes or the amplified circularized padlock probes. In some embodiments, the method further comprises contacting the labelled probes or the amplified circularized padlock probe with an enzyme that cleaves nucleic acid sequences. In some embodiments, the enzyme is uracil DNA glycosylase (UDG). In some embodiments, the method comprises permeabilizing the cell to allow at least a part of the plurality of polynucleotides to bind to intracellular proteins.Amplification of Padlock Probes

[0059] In certain embodiments, the methods according to the present disclosure use amplification of the circularized padlock probes. In certain embodiments, the method includes an amplifying step where one or more amplification primers are hybridized to the circularized padlock probe and the circularized padlock probe is amplified using a polymerase. The amplifying step increases the copy number of the capture probe or target nucleic acid analyte for detection. The amplification product(s), i.e. , the increasedPCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT copy number can be detected by detection probes and used to identify the location of the target nucleic acid in the biological sample and thereby determine whether the methods for releasing analytes from a biological sample has been successful. In some embodiments, the amplifying step includes rolling circle amplification (RCA).

[0060] As used herein, rolling circle amplification (RCA) refers to a polymerization reaction carried out using a single-stranded circular DNA (e.g., a circularized padlock probe) as a template and an amplification primer that is substantially complementary to the single-stranded circular DNA (e.g., the circularized padlock probe) to synthesize multiple continuous single-stranded copies of the template DNA (e.g., the circularized padlock probe). In some embodiments, RCA includes hybridizing one or more amplification primers to the circularized padlock probe and amplifying the circularized padlock probe using a DNA polymerase with strand displacement activity, such as Phi29 DNA polymerase, Bst DNA polymerases (e.g., large fragment, 2.0 and 3.0), Klenow fragment, and Vent or DeepVent DNA polymerases. In some embodiments, a first RCA reaction includes a first padlock probe and a first amplification primer (or plurality of first amplification primers). A first RCA reaction can include the first padlock probe hybridizing to a first analyte or first analyte derived molecule.

[0061] In some embodiments, an RCA reaction is carried out using a DNA polymerase and a dNTP mix including uracil, adenine, guanine and cytosine. In such cases, the uracils are incorporated into the amplified padlock probe. In some embodiments, an RCA reaction is carried out using a DNA polymerase and a dNTP mix including uracil, adenine, guanine, cytosine and thymine. In such cases, uracils and thymines are both incorporated into the amplified padlock probe.

[0062] In some embodiments, the method further includes a second RCA reaction. For example, following a first RCA reaction, a second padlock probe and a second amplification primer are added to the substrate. In some embodiments, the second padlock probe hybridizes to a second analyte or second analyte derived molecule and is ligated thereby creating a second circularized padlock probe. The second circularized padlock probe can then be amplified using RCA.

[0063] In some embodiments, the second padlock probe includes a third sequence and a fourth sequence where each are substantially complementary to sequences on a second analyte (e.g., a different analyte then was bound by the first padlock probe in the first RCA reaction).

[0064] In some embodiments, the method includes a second RCA reaction where the second RCA includes digesting the amplified circularized padlock probe generatedPCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT in the first RCA reaction and hybridizing, ligating, amplifying, and subsequently identifying a second padlock probe.

[0065] In some embodiments, an amplification primer includes a sequence that is substantially complementary to one or more of the first sequence, the backbone sequence, or the second sequence of the padlock probe. For example, the amplification primer can be substantially complementary to the backbone sequence. In some embodiments, the amplification primer includes a sequence that is substantially complementary to the padlock probe and an additional portion of the capture probe or target nucleic acid analyte.Detection

[0066] In some embodiments, the detecting step includes contacting the amplified circularized padlock probe with a plurality of detection probes. In some embodiments, a detection probe of the plurality of detection probes includes a sequence that is substantially complementary to a sequence of the padlock probe, circularized padlock probe, or amplified circularized padlock probe and a detectable label. For example, the detection probe of the plurality of detection probes can include a sequence that is substantially complementary to a sequence of amplified circularized padlock probe and a detectable label.

[0067] In some embodiments, the plurality of detection probes include a pool of detection probes where each detection probe includes a sequence different from the other detection probes, thereby enabling detection of signals from two or more different sequences (e.g., two or more different amplified circularized padlock probes).

[0068] In some embodiments, the detectable label is a fluorophore. Non-limiting examples of fluorescent dyes that can bind to single stranded nucleic acids include: TOTO®-1 / TO-PRO®-1 , TOTO®-3 / TO-PRO®-3, TO-PRO®-5, Ethidium bromide, Ethidium homodimer-1 (EthD-1 ), YOYO®-1 / YO-PRO®-1 , YOYO®-3 / YO-PRO®-3, 7- AAD (7-Aminoactinomycin D), and OliGreen®.

[0069] In some embodiments, the detectable label includes a luminescent or chemiluminescent moiety. Common luminescent / chemiluminescent moieties include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and one or more luciferases. These protein moieties can catalyze chemiluminescent or bioluminescent reactions given the appropriate chemical or biological substrates (e.g., an oxidizing reagent plus aPCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT chemiluminescent compound). A number of compound families are known to provide chemiluminescence under a variety of conditions.

[0070] In some embodiments, detecting the signal or signals that correspond to the amplified circularized padlock probe on the substrate includes obtaining an image corresponding to the analyte and / or analyte derived molecule on the substrate.

[0071] In some embodiments, the method includes repeating the detecting step with a second plurality of detection probes. In some embodiments, the method includes removing the bound detection probes from the first detecting step and contacting the amplified circularized padlock probe with a second plurality of detection probes. A detection probe of the second plurality of detection probes includes a sequence that is substantially complementary to a sequence of the padlock probes different from the sequence to which a detection probe from the first plurality of detection probes is substantially complementary.

[0072] In some embodiments, the method further includes obtaining an image of the sample; registering the image data to a spatial location. In some embodiments, the imaging includes a brightfield image. In some embodiments, the method further includes contacting the biological sample with one or more stains. In some embodiments, the one or more stains comprise a histology stain (e.g., any of the histology stains described herein or known in the art). In some embodiments, the one or more stains comprises hematoxylin and eosin. In some embodiments, the one or more stains comprise one or more optical labels (e.g., any of the optical labels described herein).

[0073] Suitable systems for performing spatial analysis may be used. In some embodiments, spatial analysis can be performed using dedicated hardware and / or software well known in the art. See, e.g., Janesick, A., Shelansky, R., Gottscho, A.D. et al. High resolution mapping of the tumor microenvironment using integrated single-cell, spatial and in situ analysis. Nat Commun 14, 8353 (2023) and US11542554B2.Compositions and Kits

[0074] In some embodiments, disclosed herein are compositions, kits, and systems that are used to carry out the methods described herein. In some embodiments, the kit or system includes one or more padlock probes and a ligase (e.g., a T4 DNA ligase (Rnl2), a SplintR ligase, a single stranded DNA ligase, or a T4 DNA ligase). In some embodiments, the kit further includes one or more primers and a polymerase (e.g., a Phi29 DNA polymerase or other strand displacing polymerase).PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT

[0075] In some embodiments, the kit or system further includes a plurality of detection probes, wherein a detection probe from the plurality of detection probes comprises a sequence that is substantially complementary to a sequence of the padlock probe and a detectable label (e.g., any of the exemplary detectable labels described herein (e.g., a fluorophore)).

[0076] In some embodiments, a kit or system used to carry out the methods described herein includes: (a) one or more padlock probe and a ligase; (b) one or more primers and a polymerase; and (c) instructions for performing the method of any one of the preceding claims.

[0077] In some embodiments, a kit or system used to carry out the methods described herein includes: (a) one or more padlock probes and a ligase; (b) one or more primers and a polymerase; (c) one or more fluorescent dyes and / or one or more detection probes; and (d) instructions for performing the method of any one of the preceding claims.

[0078] In some embodiments, a kit used to carry out the methods described herein includes: (a) one or more padlock probes and a ligase; (b) one or more primers and a polymerase; (c) one or more fluorescent dyes and / or one or more detection probes; (d) a uracil-DNA glycosylase enzyme; and (e) instructions for performing the method of any one of the preceding claims.F. Illustrative Analytes

[0079] In some embodiments, the methods of the present disclosure provide for detecting a plurality of proteins and a plurality of nucleic acids.

[0080] In some embodiments, the methods of the present disclosure provide for detecting a plurality of proteins and a plurality of DNA sequences.

[0081] In some embodiments, the methods of the present disclosure provide for detecting a plurality of proteins.

[0082] In some instances, one or more of the proteins in the plurality of proteins is a peptide, a glycoproteins (N-linked or O-linked), a lipoprotein, a phosphoprotein, a specific phosphorylated or acetylated variants of a protein, a amidation variant of a protein, a hydroxylation variants of a protein, a methylation variant of a protein, a ubiquitylation variant of a proteins, a sulfation variant of a protein, a viral coat protein, an extracellular protein, an intracellular protein, an antibody, an antigen binding fragment, or any combination thereof.PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101 -00PCT

[0083] In some embodiments, the method of the present disclosure provides for detecting a plurality of nucleic acids. In some embodiments, the plurality of nucleic acids including DNA. In some embodiments, the plurality of nucleic acids including RNA. In some embodiments, the plurality of nucleic acids including DNA and RNA.

[0084] Examples of nucleic acid analytes include DNA analytes such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids.

[0085] Examples of nucleic acid analytes also include RNA analytes such as various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA. The RNA can be a transcript (e.g., present in a tissue section). The RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi- interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA or single-stranded RNA. The RNA can be circular RNA. The RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).

[0086] Additional examples of analytes include mRNA and cell surface features (e.g., using the labelling agents described herein), mRNA and intracellular proteins (e.g., transcription factors), mRNA and cell methylation status, mRNA and accessible chromatin (e.g., ATAC-seq, DNase-seq, and / or MNase-seq), mRNA and metabolites (e.g., using the labelling agents described herein), a barcoded labelling agent (e.g., the oligonucleotide tagged antibodies described herein) and a V(D)J sequence of an immune cell receptor (e.g., T-cell receptor), mRNA and a perturbation agent (e.g., a CRISPR crRNA / sgRNA, TALEN, zinc finger nuclease, and / or antisense oligonucleotide as described herein). In some embodiments, a perturbation agent can be a small molecule, an antibody, a drug, an aptamer, a miRNA, a physical environmental (e.g., temperature change), or any other known perturbation agents.

[0087] In some embodiments, the method of the present disclosure provides for detecting a plurality of RNAs.

[0088] In some embodiments, the RNA is an endogenous RNA. In some embodiments, the RNA is an exogenous RNA.PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101 -00PCT

[0089] In some embodiments, the RNA is a messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), long noncoding RNA (IncRNA), microRNA (miRNA), small interfering RNA (siRNA), short nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), small nuclear RNA (snRNA).

[0090] In some embodiments, the RNA comprises a poly-A tail. In some embodiments, the RNA does not comprise a poly-A tail.

[0091] In some embodiments, the method of the present disclosure provides for detecting a plurality of DNAs.

[0092] In some embodiments, the target DNA comprises one or more of a single nucleotide polymorphism (SNP), an insertion or deletion (indel), a copy number variant (CNV), or a tandem repeat. In some embodiments, the target DNA comprises a marker of open chromatin. In some embodiments, the target DNA comprises one or more methylated nucleotides.

[0093] In some embodiments, the biological sample comprises a tissue section. In some embodiments, the biological sample comprises a fresh frozen sample. In some embodiments, the biological sample comprises live cells. In some embodiments, the biological sample comprises a FFPE sample.

[0094] The disclosure set forth above may encompass multiple distinct examples with independent utility. Although each of these examples has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the example(s) includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and / or properties disclosed herein.

[0095] Certain combinations and subcombinations regarded as novel and nonobvious are particularly pointed out throughout this disclosure. Other combinations and subcombinations of features, functions, elements, and / or properties may be claimed, with or without variation in scope, in applications claiming priority from this or a related application.

[0096] Explicit reference is hereby made to all examples, embodiments, inventions, labels, terms, descriptions, and illustrative measurements shown in the drawings and / or in any included appendices, whether or not described further herein. To the extent that section headings are used within this disclosure, such headings are for organizational purposes only.

Claims

PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCTWHAT IS CLAIMED IS:1 . A method of target detection in a biological sample comprising a cell, the method comprising: a. contacting the biological sample with a polynucleotide comprising an aptamer and a capture probe, wherein the aptamer is capable of binding to a target protein, and wherein the capture probe comprises a first binding sequence and a second binding sequence; b. hybridizing a first padlock probe to the capture probe of the polynucleotide bound to the target protein; c. generating a first circularized padlock probe; d. amplifying the first circularized padlock probe using rolling circle amplification, thereby generating a first amplified circularized padlock probe; and e. detecting the first amplified circularized padlock probe, thereby identifying the location of the protein in the biological sample.

2. A method of multiplexed target detection in a biological sample comprising a cell, the method comprising: a. contacting the biological sample with a plurality of polynucleotides, wherein each polynucleotide comprises an aptamer and a capture probe, wherein each aptamer is capable of binding to a different protein, and wherein each capture probe comprises a first binding sequence and a second binding sequence; b. hybridizing a first padlock probe to the capture probe of the polynucleotide bound to a target protein; c. generating a first circularized padlock probe; d. amplifying the first circularized padlock probe using rolling circle amplification, thereby generating a first amplified circularized padlock probe; and e. detecting the first amplified circularized padlock probe, thereby identifying the abundance and the location of different proteins in the biological sample.

3. The method of claim 1 or 2, wherein the first padlock probe comprises: a. a first sequence that is substantially complementary to the first binding sequence of the capture probe of the polynucleotide;PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT b. a barcode sequence that is unique to the aptamer of the polynucleotide; and c. a second sequence that is substantially complementary to the second binding sequence of the capture probe of the polynucleotide;4. The method of any one of claims 1-3, wherein generating the circularized padlock probe comprises ligating the first sequence of the padlock probe to the second sequence of the padlock probe.

5. The method of claim 4, wherein the ligating comprises enzymatic ligation or chemical ligation.

6. The method of claim 5, wherein the enzymatic ligation utilizes a ligase.

7. The method of claim 6, wherein the ligase comprises a T4 DNA ligase.

8. The method of any one of claims 1-7, wherein the amplification step comprises: a. hybridizing one or more amplification primers to the padlock probe; and b. amplifying the padlock probe with a polymerase to produce an amplified circularized padlock probe.

9. The method of claim 8, wherein the polymerase has strand displacement activity.

10. The method of claim 8 or 9, wherein the polymerase is a Phi29 DNA polymerase.11 . The method of any one of claims 8-10, wherein the one or more amplification primers is complementary to the barcode sequence of the padlock probe.

12. The method of any one of claims 1-11 , wherein detecting the amplified circularized padlock probes comprises contacting the biological sample with a plurality of labelled probes comprising a sequence complementary to a sequence of the amplified circularized padlock probe.

13. The method of claim 12, wherein the labelled probe comprises a sequence that is complementary to at least a portion of the barcode sequence of the amplified circularized padlock probe.

14. The method of claim 12, wherein the plurality of labelled probes are fluorescently labelled.

15. The method of any one of claims 1 -14, wherein the method further comprises: a. contacting the biological sample with a padlock probe, wherein the padlock probe is capable of binding to a nucleic acid in the biological sample, and wherein a second padlock probe comprises a first binding sequence complementary to a first sequence of a target nucleic acid andPCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT a second binding sequence complementary to a second sequence of the target nucleic acid; b. hybridizing the second padlock probe to the target nucleic acid; c. generating a second circularized padlock probe; d. amplifying the second circularized padlock probe using rolling circle amplification, thereby generating a second amplified circularized padlock probe; and e. detecting the second amplified circularized padlock probe, thereby identifying the location of the target nucleic acid in the biological sample.

16. The method of any one of claims 1 -14, wherein the method further comprises: a. contacting the biological sample with a plurality of padlock probes, wherein each padlock probe is capable of binding to a different nucleic acid in the biological sample, and wherein a second padlock probe comprises a first binding sequence complementary to a first sequence of a target nucleic acid and a second binding sequence complementary to a second sequence of the target nucleic acid; b. hybridizing the second padlock probe to the target nucleic acid; c. generating a second circularized padlock probe; d. amplifying the second circularized padlock probe using rolling circle amplification, thereby generating a second amplified circularized padlock probe; and e. detecting the second amplified circularized padlock probe, thereby identifying the abundance and the location of different nucleic acids in the biological sample.

17. The method of claim 15 or 16, wherein generating the second circularized padlock probe comprises ligating a first sequence of the second padlock probe to a second sequence of the padlock probe.

18. The method of claim 17, wherein the ligating comprises enzymatic ligation or chemical ligation.

19. The method of claim 18, wherein the enzymatic ligation utilizes a ligase.

20. The method of claim 19, wherein the ligase comprises a T4 DNA ligase.21 . The method of any one of claims 15-20, wherein the amplification comprises: a. hybridizing one or more amplification primers to the second padlock probe; andPCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT b. amplifying the second padlock probe with a polymerase to produce an amplified circularized padlock probe.

22. The method of claim 21 , wherein the polymerase has strand displacement activity.

23. The method of claim 21 or claim 22, wherein the polymerase is a Phi29 DNA polymerase.

24. The method of any one of claims 21 -23, wherein the one or more amplification primers is complementary to the barcode sequence of the padlock probe.

25. The method of any one of claims 1 -24, wherein detecting the first or second amplified circularized padlock probes comprises contacting the biological sample with a plurality of labelled probes comprising a sequence complementary to a sequence of the amplified circularized padlock probe.

26. The method of claim 25, wherein the labelled probe comprises a sequence that is complementary to at least a portion of the barcode sequence of the amplified circularized padlock probe.

27. The method of claim 25 or 26, wherein the plurality of labelled probes are fluorescently labelled.

28. The method of any one of the preceding claims, further comprising removing the labelled probes or the amplified circularized padlock probes.

29. The method of claim 28, comprising contacting the labelled probes or the amplified circularized padlock probe with an enzyme that cleaves nucleic acid sequences.

30. The method of claim 29, wherein the enzyme is uracil DNA glycosylase (UDG).31 . The method of any one of claims 1 -30, wherein the method comprises permeabilizing the cell to allow at least a part of the plurality of polynucleotides to bind to intracellular proteins.

32. The method of any one of claims 1 -31 , wherein the protein is a peptide, a protein, a glycoprotein (N-linked or O-linked), a lipoprotein, a phosphoprotein, a specific phosphorylated or acetylated variant of a protein, an amidation variant of a protein, a hydroxylation variant of a protein, a methylation variant of a protein, a ubiquitylation variant of a protein, a sulfation variant of a protein, a viral coat protein, an extracellular protein, an intracellular protein, an antibody, an antigen binding fragment, or any combination thereof.

33. The method of any one of claims 1 -32, wherein the nucleic acid is an RNA.PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT34. The method of any one of claims 1-33, wherein the nucleic acid is a messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), long noncoding RNA (IncRNA), microRNA (miRNA), small interfering RNA (siRNA), short nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), or small nuclear RNA (snRNA).

35. The method of any one of claims 1-34, wherein the nucleic acid is a DNA sequence.

36. The method of any one of claims 1-35, wherein the nucleic acid is a DNA sequence comprising one or more of a single nucleotide polymorphism (SNP), an insertion or deletion (indel), a copy number variant (CNV), or a tandem repeat.

37. The method of any one of claims 1-36, wherein the nucleic acid is a DNA sequence comprising one or more of a marker of open chromatin.

38. The method of any one of claims 1-37, wherein the nucleic acid is a DNA sequence comprising one or more methylated nucleotides.

39. The method of any one of claims 1-38, wherein the biological sample comprises a formalin-fixed, paraffin-embedded (FFPE) sample.

40. The method of any one of claims 1-39, wherein the biological sample comprises a tissue section.41 . The method of any one of claims 1 -40, wherein the biological sample comprises a fresh frozen sample.

42. The method of any one of claims 1-41 , wherein the biological sample comprises live cells.

43. A kit, comprising: a. a plurality of polynucleotides, wherein each polynucleotide comprises an aptamer and a capture probe, wherein each aptamer is capable of binding to a different protein, and wherein each capture probe comprises a first binding sequence and a second binding sequence; b. one or more padlock probes and a ligase; c. one or more primers and a polymerase; and d. instructions for performing the method of any one of the preceding claims.

44. The kit of claim 43, further comprising a plurality of labelled probes, each labelled probe comprising a sequence complementary to a sequence of the padlock probe and a detectable label.PCT / US25 / 46511 16 September 2025 (16.09.2025)Attorney Docket No. 01137-0101-00PCT45. The method or kit of any one of claims 1-44, wherein each of the aptamers in the plurality of polynucleotides independently comprises 20-100, 20-90, 20-80, 20- 70, 20-60, 20-50, 30-100, 30-90, 30-80, 30-70, 30-60, 40-100, 40-90, 40-80, 40- 70, or 40-60 nucleotides.

46. The method or kit of any one of claims 1-45, wherein each of the aptamers in the plurality of polynucleotides independently comprises 30-90 nucleotides.

47. The composition of any one of claims 1-46, wherein each of the aptamers in the plurality of polynucleotides independently comprise at least one, at least two, at least three, at least four, or at least five C-5 modified nucleobases.

48. The composition of claim 47, wherein the modified nucleobase is selected from 5-(N-benzylcarboxamide) (“Bn”)-cytosine, 5-(N-2-phenylethylcarboxamide) (“PE”)-cytosine, 5-(N-3-phenylpropylcarboxamide (“PP”)-cytosine, 5-(N-1- naphthylmethylcarboxamide) (“Nap”)-cytosine, 5-(N-2- naphthylmethylcarboxamide) (“2Nap”)-cytosine, 5-(N-1 -naphthyl-2- ethylcarboxamide) (“NE”)-cytosine, 5-(N- tyrosylcarboxamide) (”Tyr”)-cytosine, Bn-uracil, Nap-uracil, PE-uracil, 5-(N-isobutylcarboxamide) (“lb”)-uracil, 5-(N-4- fluoro-benzylcarboxamide) (“FBn”)-uracil, 2Nap-uracil, NE-uracil, 5-(N-3,4- methylenedioxybenzylcarboxamide) (“MBn”)-uracil, 5-(N-3- benzofuranylethylcarboxamide) (“BF”)-uracil, 5-(N-3- benzothiophenylethylcarboxamide) (“BT”)-uracil, 5-(N-3- phenylpropylcarboxamide) (“PP”)-uracil, 5-(N-morpholinoethylcarboxamide) (“MOE”)-uracil, Tyr-uracil, 5-(N-3-indole-2-ethylcarboxamide) (“Trp”)-uracil, 5-(n- (R)-2-hydrozypropylcarboxamide) (“Thr”)-uracil, 5-(N-((1 ,1’-biphenyl)-4- yl)ethylcarboxamide) “BPE”-uracil, 5-(N-4-phenylbenzylcarboxamide) (“PBn”)- uracil, 5-(N-4-phenoxybenzylcarboxamide) (“POP”)-uracil, 5-(N-3,3- diphenylpropylcarboxamide) (“DPP”)-uracil, 5-(N-3-phenylbenzylcarboxamide) (“DBM”)-uracil, 5-(N-benzylhydrylcarboxamide) (“BH”)-uracil, BPE-cytosine, PBn- cytosine, POP-cytosine, DPP-cytosine, DBM-cytosine, and BH-cytosine.

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