Dnazyme detection of metal IONS and gene expression in tissues

Abstract

Nucleic acid enzymes such as DNAzymes are provided, including nucleic acid enzymes that can be used for detection or quantification of a cofactor, such as a metal ion, in a cell. Methods for concurrent detection of mRNA from cells are also provided and can benefit from modifications to the nucleic acid enzyme such as, e.g., inclusion of a polyadenine (poly-A) tail and / or nonnative chemical groups. The methods may utilize SNAIL probes for spatial transcriptomics, combining SNAIL probes with DNAzymes for spatial metallomics, e.g., to image metal ions spatially.

Description

DESCRIPTIONDNAZYME DETECTION OF METAL IONS AND GENE EXPRESSION IN TISSUESBACKGROUND

[0001] This application claims the benefit of United States Provisional Patent Applications No. 63 / 643,836, filed May 7, 2024, and 63 / 729,788, filed December 9, 2024, the entirety of which are incorporated herein by reference.

[0002] This invention was made with government support under Grant no. R35 GM141931 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on April 21, 2025, is named UTFBP1363WO.xml and is 46,704 bytes in size.1. Field

[0004] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, it concerns DNAzymes and related methods of use.2. Description of Related Art

[0005] Messenger RNAs (mRNAs) are nucleic acids that are transcribed into proteins, coordinating structure, function, and regulation of the cell, tissue, and whole organisms (Goss and Theil, 2011). mRNAs have long been known to have specific 3D regulatory structures naturally, in which endogenous ribonucleotides, such as ribozymes and riboswitches, are small molecule sensing mRNAs that regulate gene expression (Batey, 2015; Breaker, 2018; Kauppinen et al. , 2005). For example, the binding of labile Fe2+ to a ~30 nucleotide RNA iron responsive element in mRNA initiates the binding of eukaryotic initiation factors and starts translation. Changing the distribution of iron metabolic mRNAs controls the rate of protein synthesis. However, the identification of other metal ion responsive mRNAs has lagged due to limited methods to identify different metal abundances in different cell types and how to identify related genes.

[0006] DNAzymes are sequences of single turn-over catalytic nucleic acids that cleave upon binding a target metal ion (Breaker and Joyce, 1994; Hwang et al., 2019; Lake et al.,2019). They can have up to 10,000X selectivity for a target metal ion of interest compared to chemically similar off-target metal ions. DNAzymes as sensors are highly generalizable, with sensors published for cellular important metals like Mg2+, Li+, Fe2+, Fe3+, Na-i- and others (Hong et al., 2020; McGhee et al., 2021; Torabi et al., 2015; Zhang et al. , 2017; Yigit et al., 2008; Xiang et al., 2011).

[0007] Nonetheless, current methods for measuring metal ions in cells have limitations. Methods to probe how labile metal ions influence cellular processes are inductively coupled plasma-mass spectrometry (ICP-MS), fluorescent sensors, radiolabeling, and genetically encoded protein-based sensors. ICP-MS provides no spatial information and destroys the biological sample of interest, fluorescent sensors often have low selectivity between similar targets (MagFura2 senses Ca2+ over Mg2+), radiolabels can be toxic and / or cost prohibited, and genetically encoded sensors may potentially alter native cellular conditions and face expression level differences in different cell types and locations (Raju etal., 1989; Livingston, 1995; Abbasi 1989; Jepsen et al., 2018). For how other metals influence gene expression, metals are typically added to modulate native metal pools; however, adding metals on the scale of 100-10000-fold higher than native levels may have side effects on gene expression that are not legitimately linked to the metal’s influence on mRNA expression or result in protein mismetallation. Moreover, the spatial distributions of mRNAs and metals are not uniform through different cell types, requiring methods to identify single cell level differences from complex hetereogenous samples, but these methods only work in traditional monocellular cultures. Ultimately, while there are decades of precedent showing that labile metal ions directly influence mRNA, there is still no large-scale, direct, and generalizable technique that can study the native metal pool simultaneously with native gene expression. However, recent advancements in the field of RNA sequencing have allowed for unprecedented insights into gene expression on the individual cell level in complex heterogenous tissues, demonstrating the importance of studying intact tissues, as bulk cell culture models do not accurately represent complex biological processes (Wu et al., 2023; De Bie etal., 2007; Song et al., 2008). Clearly, there is a need for a generalizable, high throughput method to simultaneously read out how metal ion flux changes with mRNA expression and validate these findings to discover new metal ion-related genes on the single cell level.SUMMARY

[0009] The present disclosure overcomes limitations in the prior art by providing modified nucleic acid enzymes (e.g., DNAzymes) that can be used to detect a cofactor such as a metal ion in methods that are optimized for the concurrent analysis of mRNA. In some preferred embodiments, the nucleic acid enzyme or DNAzyme is modified to include an affinity tag, preferably a poly-A tail. The DNAzyme may result in a cleavage of the DNAzyme in the presence of the cofactor. By purifying the nucleic acid enzyme or DNAzyme based on the presence of the poly-A tail, one or more mRNA can be simultaneously purified and analyzed. The nucleic acid enzyme may preferably be photocaged and / or contain a modification (e.g., a 10 serinol dithiol modification is present at the 3' position on the nucleic acid enzyme) to promote entry into and distribution within cells. These approaches can be used with next-generation sequencing (NGS) and / or multiplex PCR to detect or measure expression across one or more mRNA, optionally using single cell imaging. These approaches are illustrated in FIGS. 1A-C. In some aspects, DNAzymes are provided that self-hybridizes upstream and downstream from the catalytic loop within the DNAzyme, wherein the presence of a cofactor (e.g., a metal ion) can cause the DNAzyme to self-cleave and release either the upstream or downstream self-hybridized portion of the DNAzyme, revealing a single stranded portion of the DNAzyme that can be detected, for example using SNAIL probes and / or Padlock probes comprising a barcode amplified using rolling circle amplification (RCA). When using SNAIL probes, the DNAzyme may detect a point mutation that is outside of the catalytic loop of the DNAzyme. Combining SNAIL probes with DNAzymes can be utilized for spatial metallomics, e.g., to image metal ions spatially. In some aspects, the methods can be used to correlate spatial metallomics e.g., detection or quantification of a metal ion) with spatial transcriptomics (e.g. , detection or quantification of mRNA expression). By detecting selfcleaved and uncleaved DNAzyme, one can detect or quantify the presence of a cofactor, and these approaches can be used with spatial transcriptomics such as Spatially resolved Transcript Amplicon Readout Mapping (STARmap). An illustration of this methodology is provided in FIGS. 7A-C.

[0010] As shown in the below examples, metal ion- selective DNAzymes were adapted for read outs through single cell transcriptomics. The adapted DNAzymes undergo a cleavage reaction in response to intracellular metal ions; the ratio of cleaved to uncleaved DNAzyme sensor can thus be used as a measure of endogenous metal ion concentrations and readsimultaneously with mRNA expression from single cells, e.g. , as illustrated in FIG. 1A. This can allow for the identification of metal ion abundances in individual cells from complex cell mixtures and for the discovery of metal ion-related genes. As a proof-of-concept, the inventors demonstrated this platform with a photocaged Na+-selective DNAzyme in monocultured liver cells and in periphereal blood mononuclear cells (PBMCs). The results can be validated with ICP-MS, Northern blot assays, and confocal imaging. This technique is generalizable, allowing for its extension to other DNAzymes developed for essential cellular metal ions, combination with methods for cell surface protein expression profiling and CRISPR-guideRNA sequencing, and expandable to spatial transcriptomics.

[0011] More specifically, the nucleic acid enzyme comprising a DNAzyme was generated in single-stranded form. The DNAzyme was modified in several ways to make it compatible with NGS. First, the DNAzyme was poly- adenylated by adding 20 adenine bases to the 3’ position, in order to make the DNAzyme chemically similar to mRNA’s poly-A tail and allow for its extraction from cells using dT-oligo beads. The poly-A tail at this position allowed for both the full length and cleaved DNAzyme to be extracted. Second, the DNAzyme cleavage site was photocaged. As extracellular sodium levels are higher than intracellular levels, photoprotection inhibited DNAzyme cleavage during the delivery process. It was found that photoprotection prevented DNAzyme activity in buffer, while the photoprotecting group readily removed with light (as shown in FIG. 5). Third, 10 serinol dithiol modifications were added to the 3' position. The dithiol modifications were to allow for DNAzyme delivery into primary cell types. Without wishing to be bound by any theory, the dithiol modifications may interact with membrane bound cysteines and promote internalizing the DNAzyme through translocation. These allowed for uniform distribution through the cell (FIG. 6). As a control strand, a point mutation sequence was generated, in which an essential base for activity was mutated to abolish any sodium-specific response. This control in cells accounted for signal due to DNA degradation and could be background subtracted from the active DNAzyme cleavage ratios. After cellular delivery and intracellular cleavage, the DNAzyme and mRNA were extracted with dT-oligo bead purification. Upon binding to dT-oligo beads, the DNAzyme and mRNA were eluted with first strand synthesis buffer and random primer mix. Then the elution mixture was phosphorylated with T4 PNK to convert the free 5 ’OH on the DNAzyme to a phosphoryl group for primer recognition. The first strand cDNA synthesis of both the mRNA and the DNAzyme was carried out using dT as a primer. The resulting cDNA contained a universal sequence to anneal to a template switching oligo. Second strand cDNA synthesiswas then carried out to produce double- stranded cDNA from the first- strand reaction without the need for intermediate organic extraction of ethanol precipitation steps. Then sequencing adaptors were added, with subsequent PCR enrichment of adaptor ligated DNA and addition of i5 / i7 primers for compatibility with Illumina instruments. The general concept of this approach and results are shown in FIGS. 1A-C. Data was obtained for these methods using NGS (FIGS. 2A-B, FIGS. 3A-E) and measurements of intracellular metal ions and correlating genes were detected and measured using these methods in PBMC cells (FIGS. 4A-G). An example of a Na+sensing DNAzyme comprising photocaging with DEACM is shown in FIGS. 23A-E. Since labile metal ions can control a wide range of cellular processes through their interactions with RNAs and proteins, including cell-to-cell communication, protein degradation, post-translational modifications, and general gene expression, it is anticipated that the methods provided herein can be used to analyze a wide variety of cellular functions and / or disease states. These methods can be used, e.g. , to measure or semiquantitatively identify metal ion concentrations in single cells and directly correlate metal ion flux with single cell transcriptomics.

[0012] An aspect of the present disclosure relates to an in vitro method of detecting or measuring a cofactor in a cell comprising: (a) contacting the cell with a nucleic acid enzyme comprising: a cofactor binding site, optionally an effector binding site, and a nucleic acid affinity tag; wherein the nucleic acid enzyme can undergo a cleavage reaction in the presence of the cofactor; and (b) quantitatively amplifying and detecting portions of the nucleic acid enzyme using a first primer and a second primer; wherein the first primer that can selectively hybridize to the nucleic acid enzyme upstream of the cleavage site, and wherein the second primer can selectively hybridize to the nucleic acid enzyme downstream of the cleavage site. The method may comprise purifying the nucleic acid enzyme by binding the nucleic acid affinity tag. The affinity tag may be a polyadenine (poly-A). The poly-A may be from 5 to 250 nucleotides in length, preferably from 10-100 nucleotides in length, even more preferably about 15-35 or 20; or wherein the poly-A has the formula: -Ax-, wherein X is from 5 to 250, preferably X is from 10 to 100, even more preferably X is from 15 to 35 or 20. The nucleic acid affinity tag can preferably be located at the 3’ terminal of the nucleic acid enzyme. In some aspects, the nucleic acid affinity tag can be located at the 5’ terminal of the nucleic acid enzyme. The nucleic acid enzyme may comprise a barcode nucleic acid. The method may further comprise purifying and detecting one or more mRNA, wherein the purifying is by binding polyadenine (poly-A) in the mRNA. The nucleic acid enzyme may comprise a single-stranded DNAzyme, , an RNAzyme, or an aptazyme (e.g., comprising SEQ ID NO: 8, optionally wherein the aptazyme comprises a spacer or linker, such as a PEG (polyethylene glycol) linker or a cl8 spacer). The aptazyme may comprise a barcode or nucleic acid sequence that can be targeted by a SNAIL probe. The aptazyme may optionally comprise a fluorophore and quencher at opposite terminal ends of the nucleic acid (e.g., a fluorophore such as ce6 at the N-terminal end, and a BHQ2 quencher at the C-terminal end). The nucleic acid enzyme may be an RNAzyme (ribozyme). The cleavage reaction may occurs in a solution comprising a sodium concentration of from about 4 mM to about 135 mM, or about 4-20 mM sodium. The single-stranded DNAzyme comprises any one of SEQ ID NOs:L4, or a sequence having at least 95% sequence identity thereto. The quantitatively amplifying may comprise quantitative PCR (qPCR). The quantitatively amplifying and detecting can preferably comprise nextgeneration sequencing (NGS). The NGS may comprise sequencing by synthesis (SBS) or sequencing by ligation (SBL). The method may further comprise detecting an mRNA in the cell. The NGS may comprise single cell RNA sequencing. The method may further comprise detecting multiple mRNA in the cell via multiplex PCR. In some preferred aspects, the mRNA and the nucleic acid enzyme are both substantially purified, preferably in a single reaction step or purification step. In some preferred aspects, the affinity tag is a polyadenine (poly- A), and wherein the mRNA and the nucleic acid enzyme are substantially purified by binding polyadenines in the mRNA and the nucleic acid enzyme. The mRNA and the nucleic acid enzyme may be substantially purified using dT-oligo magnetic beads or dT-oligo oil-based beads. In some aspects, the nucleic acid enzyme comprises a DNAzyme, and after the purification the nucleic acid enzyme is phosphorylated to convert the free 5 ’OH on the DNAzyme to a phosphoryl group. In some aspects, after the purification the nucleic acid enzyme is phosphorylated with T4 PNK to convert the free 5 ’OH on the DNAzyme to a phosphoryl group. The DNAzyme may comprise a universal sequence for template-switching. The mRNA may be detected using quantitative PCR (qPCR). The quantitatively amplifying and detecting may comprise next-generation sequencing (NGS). The NGS may comprise sequencing by synthesis (SBS) or sequencing by ligation (SBL). In some aspects, the mRNA and the nucleic acid enzyme are sequenced substantially simultaneously. The sequencing may be performed using quantitative PCR (qPCR) and next-generation sequencing (NGS). The sequencing may comprise amplifying with a first primer and a second primer, for example wherein the first primer is SEQ ID NO:5 or SEQ ID NO:6, and the second primer is SEQ ID NO:7. The nucleic acid enzyme may comprise a DNAzyme, and the DNAzymes may for example be diluted to a concentration of about 1-1000 pg / pL before the quantitativelyamplifying and detecting. The cofactor may be a nitrogen fertilizer, pesticides, dioxin, phenols, 2,4-dichlorophenoxyacetic acid, a metal ion, glucose, insulin, hCG-hormone, HIV, HIV proteins, anthrax, small pox, nerve gases, TNT, DNT, cocaine, or an antibiotic. Preferably, the cofactor is a metal ion. The metal ion may be an ion of K, Pb, Hg, As, UO2, Fe, Zn, Ca, Cu, Co, Cd, Cr, Li, or Mn. The metal ion may be Mg(II), K(l+), Fe(II), Ag(I), Pb(II), Hg(II), As(III), Fe(III), Zn(II), Cd(II), Cu(II), Sr(II), Ba(II), Ni(II), Co(II), As(V), U(VI), or Cr(VI). The metal ion has a +2 formal oxidation state or a +3 formal oxidation state. In some aspects, Fe2+ and Fe3+ are simultaneously measured. The nucleic acid enzyme may comprise a DNAzyme (e.g., a 8-17 DNAzyme, EtNa DNAzyme, Li+-DNAzyme, 11-5 DNAzyme, or 10- 23 DNAzyme). The DNAzyme may comprise any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In some aspects, the ratio of the amplification products of the first primer and second primer indicate presence of the cofactor. The cofactor may be a metal ion. The nucleic acid enzyme may comprise a non- natural nucleotide (e.g. , a phosphorothioate (PS), locked nucleic acid (LNA), or wherein the non-natural nucleotide comprises a 2'-0-Me (2'-O- Me) modification, 2'-O-(N-(aminoethyl)carbamoyl) methyl, 2'-deoxyadenosine analogue, 2'deoxyuridine derivative containing a guanidinium group, phosphorodiamidate morpholino oligonucleotides (PMO), 2'-fluoroarabino nucleic acid (FANA) backbone, a-l-threofuranosyl nucleic acid backbone (XNA) backbone, or an amino acid-like nucleotide modification such as a histidine or a carboxylate). The nucleic acid enzyme may preferably comprise a photocaged moiety. The photocaged moiety may be photocaged nitrobenzyl phosphoramidte or diethylaminocoumarin (DEACM). In some aspects, the nucleic acid enzyme comprises a DNAzyme, wherein the DNAzyme cleavage site is photocaged. The nucleic acid enzyme may contain serinol dithiol modifications, or phosphorothioate backbone modifications to improve cell delivery. In some aspects, a series of 5-20, more preferably 10-20, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 serinol dithiol modification are present at the 3' position on the nucleic acid enzyme. In some aspects, a first primer upstream of the cleavage site in the nucleic acid enzyme and a second primer downstream of the cleavage site in the nucleic acid enzyme are used to amplify reaction products and measure a rate of the cleavage reaction. The method may further comprise exposing the cell to a control DNA sequence to detect DNA degradation. The rate of DNA degradation may be subtracted from the rate of the cleavage reaction. The nucleic acid enzyme may be comprised in a lipid nanoparticle, polymer, metal nanoparticle, biological microvesicle, polyplex, nanoconjugate, micelle, nanocapsule, dendrimer, or a virus- associated vector.

[0013] Another aspect of the present disclosure relates to an in vitro method of detecting or measuring a cofactor in a cell comprising: (a) contacting the cell with a nucleic acid enzyme comprising: a cofactor binding site, wherein the nucleic acid enzyme comprises a catalytic loop, and wherein the nucleic acid enzyme can self-hybridize upstream from the catalytic loop and downstream from the catalytic loop; wherein the nucleic acid enzyme can undergo a cleavage reaction in the presence of the cofactor; wherein the cleavage reaction releases a nucleic acid strand that was self-hybridized upstream or self-hybridized downstream to the nucleic acid enzyme, thereby providing a single-stranded primer site in the nucleic acid enzyme and (b) detecting the cleavage reaction by contacting a first primer that selectively binds the single- stranded primer site in the nucleic acid enzyme and amplifying a nucleic acid using a polymerase chain reaction (PCR) or rolling circle amplification (RCA). The nucleic acid enzyme may comprise a single- stranded DNAzyme, an RNAzyme, or an aptazyme (e.g., comprising SEQ ID NO: 8, optionally wherein the aptazyme comprises a linker cush as a PEG spacer or a cl 8 spacer). The aptazyme may comprise a barcode or nucleic acid sequence that can be targeted by a SNAIL probe. The aptazyme may optionally comprise a fluorophore and quencher at opposite terminal ends of the nucleic acid (e.g. , a fluorophore such as ce6 at the N- terminal end, and a BHQ2 quencher at the C-terminal end). The first primer may comprise a barcode. The first primer may be a primer for rolling circle amplification (RCA). The first primer may be a SNAIL probe, a FISSEQ probe, a PLAYR probe, or a Padlock probe. The SNAIL probe or padlock probe may comprise a barcode, wherein the said amplifying comprises using rolling circle amplification (RCA) to generate a rolling circle amplification product, and wherein the rolling circle amplification product is detected with one or more detection probes that selectively bind the barcode. The detection probe may comprise a fluorescent label. The RCA may comprise amplification with a DNA polymerase, preferably a Bst polymerase, a Bsm polymerase, Vent® polymerase, a polymerase without 5’-3’ exonuclease activity, phi29, or a T7 RNA polymerase. The method may comprise spatial transcriptomics detection and analysis of the amplification product in a plurality of cells or in a tissue. The spatial transcriptomics may comprise a Spatially resolved Transcript Amplicon Readout Mapping (STARmap) , merFISH, seqFISH, FISSEQ, non-gapfilling padlock probe, PLAYR, or gapfilling padlock / barSeq methodology. The method may further comprise detecting or measuring total nucleic acid enzyme levels by: (i) hybriding a second primer to the nucleic acid enzyme, wherein the second primer selectively binds a single-stranded portion of the nucleic acid enzyme that is not within the self-hybridized region upstream and is not within self-hybridized region downstream from the catalytic loop; and (ii) amplifying nucleicacid from the second primer using polymerase chain reaction (PCR) or rolling cycle amplification (RCA). The second primer may be a SNAIL primer or a padlock probe. The cofactor may be a nitrogen fertilizer, pesticides, dioxin, phenols, 2,4-dichlorophenoxyacetic acid, a metal ion, glucose, insulin, hCG-hormone, HIV, HIV proteins, anthrax, small pox, nerve gases, TNT, DNT, cocaine, or an antibiotic. The cofactor may be a metal ion, such as an ion of K, Pb, Hg, As, UO2, Fe, Zn, Ca, Cu, Co, Cd, Cr, Li, or Mn, or Mg(II), K(l+), Fe(II), Ag(I), Pb(II), Hg(II), As(III), Fe(III), Zn(II), Cd(II), Cu(II), Sr(II), Ba(II), Ni(II), Co(II), As(V), U(VI), or Cr(VI). The metal ion may have a +2 formal oxidation state or a +3 formal oxidation state. In some aspects the Fe2+ and Fe3+ are simultaneously measured. The nucleic acid enzyme may comprise or consist of a DNAzyme. The DNAzyme is may be an 8-17 DNAzyme, EtNa DNAzyme, Li+-DNAzyme, 11-5 DNAzyme, or 10-23 DNAzyme. The DNAzyme comprises any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The nucleic acid enzyme may comprise a non-natural nucleotide. The non-natural nucleotide may be a phosphorothioate (PS), locked nucleic acid (LNA), or wherein the non-natural nucleotide comprises a 2'-O-Me (2'-0-Me) modification, 2'-O-(N-(aminoethyl)carbamoyl) methyl, 2'- deoxyadenosine analogue, 2'deoxyuridine derivative containing a guanidinium group, phosphorodiamidate morpholino oligonucleotides (PMO), 2'-fluoroarabino nucleic acid (FANA) backbone, a-l-threofuranosyl nucleic acid backbone (XNA) backbone, or an amino acid-like nucleotide modification such as a histidine or a carboxylate. The nucleic acid enzyme may comprise a photocaged moiety such as, e.g., nitrobenzyl phosphoramidte or diethylaminocoumarin (DEACM). The nucleic acid enzyme may comprise a DNAzyme, wherein the DNAzyme cleavage site is photocaged. The nucleic acid enzyme may contain a serinol dithiol modification, or a phosphorothioate backbone modification. In some aspects, a series of 5-20, more preferably 10-20, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 serinol dithiol modification are present at the 3' position on the nucleic acid enzyme. The nucleic acid enzyme may be comprised in a lipid nanoparticle, polymer, metal nanoparticle, or biological microvesicle.

[0014] Yet another aspect of the present disclosure relates to a composition comprising a nucleic acid enzyme, wherein the nucleic acid comprises: a cofactor binding site, optionally an effector binding site, and a nucleic acid affinity tag; and wherein the nucleic affinity tag is a polyadeninde (poly-A). The poly-A is from 5 to 250 nucleotides in length, preferably from 10-100 nucleotides in length (e.g., 10-30, 10-20, 20-30, or 20 nucleotides in length). The nucleic acid affinity tag may preferably be located at the 3 ’ terminal of the nucleic acid enzyme.The nucleic acid affinity tag may be located at the 5’ terminal of the nucleic acid enzyme. The nucleic acid enzyme may comprise a DNAzyme. The DNAzyme may comprise any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The nucleic acid enzyme may comprise a single- stranded DNAzyme, an RNAzyme, or an aptazyme. The apatazyme may comprise SEQ ID NO:8, and the aptazyme may optionally comprise a PEG linker or a c!8 (hexaethylene glycol) spacer. The aptazyme may compsise a barcode or nucleic acid sequence that can be targeted by a SNAIL probe. The aptazyme may optionally comprise a fluorophore and quencher at opposite terminal ends of the nucleic acid (e.g., a fluorophore such as ce6 at the N-terminal end, and a BHQ2 quencher at the C-terminal end). The nucleic acid enzyme may comprise a non-natural nucleotide. The non-natural nucleotide may be a phosphorothioate, locked nucleic acids, or other modified base to increase DNAzyme selectivity, efficiency, or sensitivity (e.g., a histidine or carboxylate). The nucleic acid enzyme preferably comprises a photocaged moiety. The photocaged moiety may be photocaged nitrobenzyl phosphoramidte or diethylaminocoumarin (DE ACM). The nucleic acid enzyme may comprise a barcode nucleic acid.

[0015] Another aspect of the present disclosure relates to a composition comprising a nucleic acid enzyme, wherein the nucleic comprises a catalytic loop, and wherein the nucleic acid enzyme comprises a self-hybridized regions both upstream and downstream from the catalytic loop; wherein the nucleic acid enzyme can self-cleave in the presence of a cofactor, the nucleic acid enzyme can self-cleave in the presence of a cofactor, and wherein the selfcleavage can release a nucleic acid from either the upstream self-hybridized region or the downstream self-hybridized region of the nucleic acid enzyme, thereby forming a singlestranded structure in the nucleic acid enzyme. The nucleic acid enzyme may comprise a non- natural nucleotide. The non-natural nucleotide may be a phosphorothioate, locked nucleic acid, or other modified base to increase DNAzyme selectivity, efficiency, or sensitivity (e.g., a histidine or carboxylate). The nucleic acid enzyme may comprise a photocaged moiety such as, e.g., photocaged nitrobenzyl phosphoramidte or diethylaminocoumarin (DE ACM). DEACM is a photoremovable protecting group for 2'-deoxyguanosine in oligonucleotides (Menge et al., 2011). An example of a Na+sensing DNAzyme comprising photocaging with DEACM is shown in FIGS. 23 A-E. The nucleic acid enzyme may comprise a barcode nucleic acid. The barcode nucleic acid may be comprised in the upstream self-hybridized region or the downstream self-hybridized region of the nucleic acid enzyme. The cofactor may be a metal ion such as, e.g., an ion of K, Pb, Hg, As, UO2, Fe, Zn, Ca, Cu, Co, Cd, Cr, Li, or Mn.

[0016] Yet another aspect of the present disclosure relates to a kit comprising the composition described above or herein in a container means.

[0017] Another aspect of the present disclosure relates to a nucleic acid encoding the nucleic acid enzyme described above or herein. The nucleic acid may encode any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The nucleic acid may be comprised in a vector (e.g., a viral vector or a non- viral vector). The vector may be comprised in liposomes, nanoparticles, disulfides, or a nucleic acid delivery vehicle. The nucleic acid delivery vehicle may be a polyplex, nanoconjugate, micelle, nanocapsule, dendrimer, or a virus-associated vector.

[0018] Yet another aspect of the present disclosure relates to a cell comprising the nucleic acid described above or herein. The cell may be comprised in a tissue sample.

[0019] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0020] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0021] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.

[0022] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0023] As used in this specification and claim(s), 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.

[0024] The term “sample” or “test sample” is defined as a composition that will be subjected to analysis that is suspected of containing the analyte of interest. Typically, a sample for analysis is in a liquid form, and preferably the sample is an aqueous mixture. A sample may be from any source, such as an industrial sample from a waste-stream or a biological sample, such as blood, urine, or saliva. A sample may be a derivative of an industrial or biological sample, such as an extract, a dilution, a filtrate, or a reconstituted precipitate.

[0025] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0027] FIGS. 1A-C. DNAzyme-Seq concept and sensor incorporation. (A) The single- stranded DNAzyme sensor has several key modifications. First, the sensor is polyA- tailed to allow for extraction with mRNA in bulk cell culture models in from single cells, Second, the sensor is modified with multiple dithiols to allow for delivery into hard to transfect cells through thiol-mediated uptake. Third, the second is photocaged to prevent premature sensor activation during the delivery process. In cells, the modified sensor and the point mutation control are delivered through thiol-mediated uptake. Once internalized, the sensor is photoactivated and cleaves in response to different metal ion levels inside different cell types. Both the cleaved and full length sequences are extracted simultaneously with mRNA from different cell types. Due to the long sequence length, the strands undergo library preparation alongside endogenous mRNA (i.e. purification, barcoding, fragmentation). As NGS describes differential gene expression, it can also determine the cleavage ratio of the full length to cleaved DNAzyme based upon the number of reads for the distinct sequences. This ratio is indicative of metal ion amount. Because the sensor output is ratio-based, different delivery efficiencies to different cell types does not bias results. Barcoding the sensor alongside the mRNA from single cells also allows for correlation with differential expressed genes, allowing for the identification of new metal ion related genes. (B) As a proof-of-concept, the Na+-DNAzyme was used to validate this idea. Due to the multiple modifications, the sensor activity compared to its point mutation control was ensured to remain. After incubating the point mutation control and the active sensor with a range of physiologically relevant intracellular sodium concentrations for 1 hour at 37°C, denaturing PAGE gel analysis showed no detectable cleavage for the control (left), while the active sensor displayed proportional cleavage with increasing sodium levels (right). (C) To test if the sensor cleavage ratio extended even after cDNA formation and exponential amplification (such as in NGS), qRT-PCR was performed with DNAzyme specific primers. After incubating the DNAzyme with varying levels of sodium for 1 hour at 37°C, post qRT-PCR analysis of the active sensor showed proportional changes in Cqvalues with increasing sodium levels, indicating more of the cleaved sensor detected.Meanwhile, the point mutation control showed no significant change in Cqvalue, indicating no non-specific cleavage even during the exponential amplification.

[0028] FIGS. 2A-B. Quantifying potential artifacts that may influence the modified DNAzyme. (A) While sensors may be specific for their target of interest when their target is the only analyte of interest present, real life systems, such as cellular environments, contain several different chemically similar species. The cell’s high ionic strength environment, with species such as 150 mM potassium and 2 mM magnesium, may cause non-specific Na+- DNAzyme cleavage. To test the Na+-DNAzyme’s activity in a high ionic strength, cellular environments, the Na+-DNAzyme was incubated in an intracellular mimicking buffer for 1 hour at 37 °C. Dosing was done in increasing levels of intracellular sodium levels. No additional cleavage was found in response to the intracellular buffer and could still observe increasing cleavage yields with increasing sodium concentrations in the high ionic strength buffer (right). Meanwhile, the point mutation control under these conditions displayed no detectable cleavage (left). (B) To test if these results extended after cDNA formation and exponential amplification, qRT-PCR was performed on the DNAzymes after incubation in these high ionic strength conditions with increasing levels of sodium. qRT-PCR analysis of the active sensor showed proportional changes in Cqvalues with increasing sodium levels, indicating more of the cleaved sensor detected. Similar degree changes in Cqvalues were observed compared to those measured under low ionic strength conditions with just the presence of sodium (FIG. 1C).

[0029] FIGS. 3A-E. Measuring intracellular Na -DNAzyme response and identification of correlating genes in bulk cell culture models through NGS. (A) To test if the Na+-DNAzyme could reflect different cell type’s intracellular sodium levels, HepG2 cells were stimulated to internalize more sodium compared to their baseline levels. Increased intracellular sodium levels were verified with Sodium Green™, a commercially available sodium sensor, through confocal microscopy. (B) After cell delivery, sensor photoactivation, sensor intracellular cleavage, sensor extraction with mRNA, and library preparation, the full length and cleaved DNAzymes were counted through NGS. The point mutation control was found to have consistent cleavage yields. After normalizing the active sensor response by subtracting out the background cleavage from the point mutation control, proportional increases in DNAzyme cleavage ratio corresponding to amounts of modulated intracellular sodium were found. This indicated the DNAzyme was sensitive enough to report on single cell levels of sodium and the cleavage ratio was an accurate indicator of intracellular sodium levels.(C) To test gene expression level distributions in each sample type, the NGS results were visualized as fragments per kilobase million (FPKM). These metrics normalized for sequencing depth and gene length. These found no significant effects of the DNAzyme on gene expression reads in each sample. (D) To identify general similarities and specific outliers in gene expressions between samples, scatter plots were visualized between each sample. (E) To further delineate significance of differentially expressed genes between each sample, volcano plots were generated comparing all groups. Several significantly differentially expressed genes were identified in response to modulate sodium levels, indicating the effect of different sodium levels on gene expression.

[0030] FIGS. 4A-G. Measuring intracellular Na+-DNAzyme response and identification of correlating genes in PBMCs. (A) Clustered t-SNEplot of PBMCs to identify specific cell types. (B) Normalized unique molecular identifier counts for the total (full length and cleaved) active DNAzyme. (C) Normalized unique molecular identifier counts for the total (full length and cleaved) point mutation control DNAzyme). (D) Normalized unique molecular identifier counts for the uncleaved region of both the active and inactive DNAzyme sequences. (E) Ratio of active DNAzyme cleavage efficiency grouped into no cleavage (0%), low cleavage (1-49%), high cleavage (50-99%), and 100% cleavage. Cleavage ratios are overlayed over total PBMC t-SNE. (F) Protein interaction network analysis of significantly enriched genes in the 100% cleavage group. The nodes show protein name abbreviations, with edges representing network interactions between specific expressed proteins. (G) Heat map of significantly enriched genes in each DNAzyme ratio group correlations of major groups of differentially expressed genes with sodium levels.

[0031] FIG. 5. Photoprotection of sensor. To test the activity of the sensor when photocaged and after photocage removal, the point mutation control and the active Na+- DNAzyme were incubated in 135 mM sodium at 37°C for 1 hour. It was found that the photocage prevented DNAzyme cleavage. After removal of the photoprotecting group, the DNAzyme activity was restored. Under all conditions tested, the point mutation control did not cleave.

[0032] FIG. 6. Dithiol modifications for PBMC delivery. A range of fluorescein- labeled DNAzyme was incubated for 1 hour at 37°C with purified PBMCs. After cell washing, the nuclei were stained with Hoechst 33342 (blue) as white blood cells contain nuclei, while red blood cell contaminants do not contain nuclei. High DNA delivery (green) was found tooverlap with the nuclei containing cells. As the DNA concentration increased, increasing fluorescence intensity was found internalized in the PBMCS.

[0033] FIGS. 7A-C. Schematic of SNAIL probes used to a) detect mRNA targets b) detect DNAzyme cleavage c) to simultaneously detect metal ions pools and unique mRNAs.

[0034] FIGS. 8A-C. Barcoded DNAzyme A) activity characterization and B) selectivity verification and C) in vitro sodium- specific SNAIL probe ligation and amplification.

[0035] FIG. 9. Cellular imaging of actin encoding Mrna and active total (blue) and cleaved (pink) DNAzyme showing metal ion pools.

[0036] FIG. 10. in vitro ligation using only PLP with Barcoded DNAzymes for barcoded detection and DNAzyme cleavage.

[0037] FIG. 11. Thiol-mediated uptake of ssDNA and disulfide-modified DNA into plant roots

[0038] FIG. 12. Fluorescence-based activity of the disulfide-modified Na-Specific DNAzyme active and inactive sensor

[0039] FIG. 13. Delivery and imaging of the Na-specific DNAzyme into plant roots.

[0040] FIG. 14. Imaging of gene-expression in Arabidopsis roots.

[0041] FIG. 15. Design of barcode and synthesis of DNAzyme. Strand A, B and C hybridization test using a native gel to see potential secondary structure formations that might lead to side product formation. There is a very strong interaction that is happening between the B strand and the splint, this might be causing strand B to self-ligate and produce a large side product decreasing the yield of the BC DNAzyme. (SEQ ID NOS:9-46)

[0042] FIG. 16. Using SNAIL probes, it was attempted to read the DNAzyme’ s point mutation on the catalytic loop. The results of these experiments did not distinguish between active and inactive DNAzyme. Additionally, the conformational change upon metal addition also inhibits the binding of SNAIL probes trying to read total DNAzyme.

[0043] FIG. 17. System cannot distinguish between inactive and active DNAzymes and is conformational dependent not cleaved dependent.

[0044] FIG. 18. In this experiment, there was improvement in the removal of over ligation bands by using less DNA in ligation reaction and less T4 ligase, however the point PLP cannot distinguish between point and active DNA because there is ligation in both bands.

[0045] FIG. 19. In this experiment, there was again improvement in the removal of over ligation bands by using less DNA in ligation reaction and less T4 ligase and the active PLP seems that it is able to distinguish between point and active.

[0046] FIG. 20. T4 DNA Ligase Concentration Titration with SNAIL probes.

[0047] FIGS. 21A-D. Mock DNAzyme sequence ligation with SNAIL probes. FIG. 21A: 1H ligation at room temperature FIG. 21B: 1H ligation at 28C. FIG. 21C: 16 hour ligation at 16C. FIG. 21D: 16 hour ligation at 28C.

[0048] FIGS. 22A-E. DEACM caging of Na DNAzyme. FIG. 22A: dG-DEACM was integrated to the G50 position of the Substrate strand of the Na DNAzyme. The DEACM group can be removed by shinning 4051ight for 1 minute and the Na DNAzyme activity can be recovered. FIG. 22B: Version three: before cleavage. FIG. 22C: Version three: After cleavage. FIG. 22D: Before cleavage secondary structures. FIG. 22E: After cleavage secondary structure. (SEQ ID NOS:47-48)

[0049] FIGS. 23A-B. In silico hybridization studies. (SEQ ID NOS:49-50)

[0050] FIG. 24: In vitro ligation studies.DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0052] Methods for detecting or measuring a cofactor (e.g., a metal ion) due to a cleavage reaction by a nucleic acid enzyme (e.g. , a DNAzyme) are provided herein. Detection of the cleavage event can be performed using a polymerase reaction or a rolling cycle amplification (e.g., using SNAIL probes and / or Padlock probes). The methods may be used to quantify levels of the cofactor. In some aspects, detection of the cofactor can be performed in cells or a tissue using spatial transcriptomics.

[0053] In some aspects, nucleic acid enzymes such as DNAzymes are provided herein that can be more effectively utilized with sequencing such as NGS, and such approaches may utilize DNAzyme-based sequencing (DNAzyme-Seq) as described herein. As DNAzymes cleave proportionally in response to their target metal ions, it was hypothesized that the ratio of cleaved DNAzyme to full length DNAzyme can be correlated with metal ion concentration. Using DNAzymes in Next Generation Sequencing has several advantages. From the sequencing perspective, the sequencing read out allows for single base resolution, reducing false positives that are common in fluorescence outputs due to DNA degradation (Lu et al., 1989). Moreover, read depth on the several hundred million reads can allow for a lower limit of detection compared to more traditional microscopy detection methods. The unique sequence of each DNAzyme allows for the possibility of multiplexing multiple DNAzymes in one experiment, leading to a more detailed and comprehensive understanding of how many metal ions interact with mRNAs at once. From the biological perspective, DNAzymes can sense endogenous levels of metal ions, compared to other sensors which rely on the addition of metal, which may perturb native gene expression. While different cell types may have different DNAzyme uptake efficiencies, the sensor output in this application is ratio-based, so sensor delivery efficiencies do not bias readout results.

[0054] Designs are provided herein that utilize strategies to exploit the potential of nucleic acid tools in NGS. First, the DNAzyme was polyadenylated to allow it to be extracted simultaneously with mRNA using dT-oligo magnetic beads, which may compatible with methodologies for CITE-seq or REAP-seq, in which a polyadenylated-oligo is used to barcode antibodies for cell surface protein expression profiling (Peterson et al., 2017; Stoeckius et al., 2017). Using this approach, exogenous polyA-oligo tag can be purified out with native mRNA from live cells. Another improvement provided herein is compatibility with Perturb-seq, in which multiple guideRNAs in CRISPR screens can be sequenced simultaneously (Datlinger etal., 2017; Adamson er al., 2016; Dixit et al., 2016). This allows for unambiguous explanations for target gene knockdown and allows for multiple gene perturbations to occur at once. Lastly, incorporating the selection of new DNAzyme candidates with NGS has been achieved (Sednev et al., 2022). This method allows for the large-scale screening of thousands of potential DNAzyme candidates based upon their cleavage activity in a single sequencing experiment. These technologies can be used with DNAzyme-based sensing and correlation to gene expression through RNA-sequencing.

[0055] As described in the below examples, a photocaged-poly-A tailed unimolecular Na+DNAzyme was into HepG2 cells to monitor sodium flux with gene expression in the classic cell model of the body’s metabolic hub and in human peripheral mononuclear blood cells to monitor sodium flux with immune cells. To test that the modifications did not hinder the DNAzyme’ s activity, the inventors verified it retained sodium-initiated cleavage abilities and high selectivity, even in cellular ionic strength conditions. After verifying the Na+- DNAzyme had proportional cleavage yields in response to increasing sodium levels, the inventors measured if its response could still be retained after multiple rounds of amplification, which occurs in NGS. It was found that the DNAzyme cleavage ratio was retained even after exponential amplification, and that these cleavage ratios still correlated to the sodium levels used to induce cleavage. To test this in live cells, intracellular levels of sodium were modulated and the DNAzyme was delivered into live cells to allow for it to respond to the endogenous and modulated levels of sodium. After DNAzyme extraction with mRNA and sequencing library preparation, the DNAzyme ratio was read out through NGS, with its ratio corresponding to relative sodium modulation levels. To determine individual cell sodium levels and correlations with gene expression, PBMCs were chosen as proof-of-concept. Previous reports found that increased sodium chloride in mice diets resulted in the induction of mouse TH17 cells. High salt conditions activated the critical p38 / MAPK pathway, Na+-sensing kinases, and pro-inflammatory cytokines, like TNF-alpha (Kleinewietfeld et al., 2013; Wu et al., 2013). These studies can be used for measuring, detecting, and correlating the effect of sodium on gene expression and disease states including autoimmune disease; however, the sodium modulation may have resulted in artifact changes in gene expression and different cell type contributions to the differential gene expression were not elucidated. To overcome these limitations, the Na+-DNAzyme provides significant advantages, since it can cleave itself in response to the intracellular range of sodium and can be barcoded in standard library preparation techniques, alongside mRNAs from single cells.I. Definitions

[0056] A “co-factor” is an ion or molecule involved in the catalytic process of nucleic acid enzyme-catalyzed reactions and is required for catalytic activity.

[0057] An “effector” is a molecule that, when bound to an enzyme having an effector binding site, can enhance or inhibit enzyme catalysis. An “effector binding site” may be “specific,” that is, binding only one effector molecule in the presence of other effector molecules. An example of effector binding site specificity is when only an adenosine molecule binds in the presence of many other similar molecules, such as cytidine, gaunosine and uridine. Alternatively, an effector binding site may be “partially” specific (binding only a class of molecules), or “non-specific” (having molecular promiscuity). Examples of effectors include environmental pollutants, such as nitrogen fertilizers, pesticides, dioxin, phenols, or 2,4- dichlorophenoxyacetic acid; heavy metal ions, such as Pb(II), Hg(II), As(III), UO2(II), Fe(III), Zn(II), Cu(II), or Co(II); biological molecules, such as glucose, insulin, hCG-hormone, HIV or HIV proteins; chemical and biological terrorism agents, such as anthrax, small pox, or nerve gases; explosives, such as TNT or DNT; drugs, such as cocaine or antibiotics.

[0058] A “nucleic acid enzyme” is an enzyme that principally contains nucleic acids, such as ribozymes (RNAzymes), deoxyribozymes (DNAzymes), and aptazymes. Nucleic acids may be natural, unnatural or modified nucleic acids. Peptide nucleic acids (PNAs) are also included. A nucleic acid enzyme requires a “co-factor” for efficient substrate cleavage and / or specific effector binding. Common co-factors include Mg(II), Ca(II), Zn(II), Mn(II), Co(II) and Pb(II). DNA enzymes, DNAzymes are also referred to as catalytic DNA and preferably can perform a specific chemical reaction via catalytic function. DNAzymes may be used as amplifying labels for the development of optical or electronic sensors, or for detecting the presence of an analyte (e.g., Kahn et al. , 2021).

[0059] “Polynucleotide” refers to a nucleic acid sequence having at least two or more nucleotides. Polynucleotides may contain naturally occurring nucleotides and synthetic nucleotides. PNA molecules are also embraced by this term.

[0060] “Sensitivity” refers to the smallest increase of a cofactor or effector concentration that can be detected by the sensor.

[0061] “Detection limit” refers to the limits of detection of an analytical device. In the context of the DNAzyme- and aptazyme-based sensors of the present invention, detection limit refers to the lowest concentration of a cofactor or effector that the sensor can differentiate from the background.

[0062] “Base-pairing” refers to the ability of a polynucleotide to form at least one hydrogen bond with a nucleotide under low stringency conditions. The nucleotide may be part of a second polynucleotide or to a nucleotide found within the first polynucleotide. A polynucleotide is partially complementary to a second polynucleotide when the first polynucleotide is capable of forming at least one hydrogen bond with the second polynucleotide. To be partially complementary, a polynucleotide may have regions wherein base pairs may not form surrounded by those regions that do, forming loops, stem-loops, and other secondary structures.

[0063] The complementarity relationships described herein are not necessarily 100% complementarity. In some embodiments, the complementarity relationships described herein are >95%, >90%, >85%, or >80% complementarity. In other words, if a first sequence is defined as being complementary to a second sequence, then the reverse complement of the first sequence may be 100%, >95%, >90%, >85%, or >80% identical to the second sequence. By way of example, if one sequence is defined as being at least 80% complementary to another sequence, then that sequence is at least 80% identical to the reverse complement of the other sequence.

[0064] In some embodiments, two nucleic acid molecules are complementary if they can hybridize with each other under stringent conditions. As used herein “stringent conditions” are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but precludes hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. By way of example, stringent conditions may comprise low salt and / or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of solvent(s) in ahybridization mixture. It is also understood that these ranges, compositions and conditions for hybridization are mentioned by way of non-limiting examples only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to one or more positive or negative controls. In some embodiments, two nucleic acid molecules are complementary if they can hybridize with each other under low stringency conditions. Non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20°C to about 50°C. Of course, it is within the skill of one in the art to further modify the low or high stringency conditions to suit a particular application. In some embodiments, two nucleic acid molecules are non- complementary if they are unable to hybridize with each other under low stringency conditions.

[0065] “Aptamer” refers a polynucleotide which contains an effector binding cite. An “effector binding site” may be “specific,” that is, binding only one effector molecule in the presence of other effector molecules. An example of effector binding site specificity is when only an adenosine molecule binds in the presence of many other similar molecules, such as cytidine, gaunosine and uridine. Alternatively, an effector binding site may be “partially” specific (binding only a class of molecules), or “non-specific” (having molecular promiscuity).

[0066] “Aptazyme” refers to a nucleic acid enzyme that includes an aptamer region which binds an effector. The binding of the effector can enhance or inhibit catalysis.

[0067] The term “analyte” is defined as one or more substance potentially present in the sample. The analysis process determines the presence, quantity, or concentration of the analyte present in the sample.

[0068] The term “sensitivity” refers to the lower concentration limit at which a sensor system can detect an analyte. Thus, the more sensitive a sensor system is to an analyte, the better the system is at detecting lower concentrations of the analyte.

[0069] The term “vector” refers to small carrier nucleic acid molecule, a plasmid, virus (e.g., AAV vector, retroviral vector, lend viral vector), or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid. Vectors, such as viral vectors, can be used to introduce / transfer nucleic acid sequences into cells, such that the nucleic acid sequence therein is transcribed and, if encoding a protein, subsequently translated by the cells.IL Nucleic Acid Enzymes

[0070] A variety of nucleic acid enzymes have discovered or developed that can be used with methods provided herein, including those described in US2009 / 0011402, US2006 / 0094026, and US20040175693, which are incorporated herein by reference. The catalytic activity of the nucleic acid enzymes may depend on or require one or more co-factors, such as a metal ion. In vitro selection may be used to “enhance” selectivity and sensitivity for a particular ion. In some preferred embodiments, the nucleic acid enzymes catalyzes a molecular association (ligation, phosphorylation, and amide bond formation) or dissociation (cleavage or transfer), such that the nucleic acid product(s) can be detected (e.g., via amplification, PCR, quantitative PCR (qPCR), and / or Next Gen sequencing). In some preferred embodiments, the nucleic acid enzyme results in self-cleavage of the nucleic acid enzyme e.g., DNAzyme) such that the reaction products can be detected to quantify the presence of the co-factor (e.g. , metal ion) that may be present in a biological sample (e.g. , tissue sample) or environmental sample (e.g., water sample from nature or industrial setting). The nucleic acid enzyme may preferably contain an affinity tag (e.g. , a poly- A tail) that may allow for improved purification and sequencing of the reaction products generated by exposure to the co-factor (e.g., metal ion). In some preferred embodiments, the nucleic acid enzyme (e.g., DNAzyme) is photocaged, and such metal-dependent DNAzymes may be used for the quantitative detection of metal ions, e.g., in living cells (Hwang et al., 2019). If desired, the nucleic acid enzyme (e.g., preferably a DNAzyme) can be immobilized to a substrate, for example for use as a biosensor, and a variety of immobilization strategies can be used (e.g., Khan et al., 2021). DNAzyme-based biosensors can be used, e.g., for the detection of pathogens, metal ions, and clinical biomarkers.

[0071] A nucleic acid enzyme that catalyzes the cleavage of a nucleic acid in the presence of an effector is preferably used in methods provided herein. The nucleic acid enzyme may be RNA (ribozyme), DNA (deoxyribozyme), a DNA / RNA hybrid enzyme, or a peptide nucleic acid (PNA) enzyme. PNAs are the DNA / RNA analogues in which sugar-phosphate backbone is replaced by N-2-aminoethylglycine repeating units (e.g., Gupta et al., 2017). Ribozymes that may be used include group I and group II introns, the RNA component of the bacterial ribonuclease P. hammerhead, hairpin, hepatitis delta virus and Neurospora VS ribozymes. The ribozyme may be selected using an in vitro method (e.g., Tang and Breaker 2000). Because ribozymes are typically be less stable than deoxyribozymes, DNAzymes canbe preferably used. The DNAzyme may be modified or include an extended chemical functionality, e.g., as described in Santoro et al. (2000).

[0072] Methods of producing ribozymes and deoxyribozymes include chemical oligonucleotide synthesis, polymerase chain reaction (PCR), DNA cloning and replication. Preferably the nucleic acid enzymes are DNA / RNA hybrids and PNAs. Nucleotides containing modified bases, phosphates, or sugars may also be used; in some instances, these modified nucleotides may be advantageous for stability or confer effector specificity. Examples of modified bases include inosine, nebularine, 2-aminopurine riboside, N7-deazaadenosine, and O6-methylguanosine (Earnshaw and Gait 1998). Modified sugars and phosphates include 2'- deoxynucleoside, abasic, propyl, phosphorothioate, and 2'-O-allyl nucleoside (Earnshaw and Gait 1998). DNAzymes can be used to detect a variety of cofactors for different purposes. DNAzymes that bind metal ions that influence transcriptomic activity, including heavy metal ions e.g., lead, mercury, cadmium, chromium) as well as ions of iron, copper, zinc, can be used in the methods provided herein.A. Biological response to heavy metal contamination

[0073] DNAzymes can be used to detect heavy metal contaminants in tissues or the environment. There are many DNAzymes for environmental heavy metal contaminants, including lead (Li and Lu, 2000), mercury (Li and Lu, 2007), cadmium (Huang and Liu, 2015), and chromium (Zhou et al., 2016). The presence of these metals in the body can lead to bioaccumulation and significant diseases, such as kidney dysfunction, nervous system disorders, immune system dysfunction, lung damage, birth defects, and tumors (Jaishankar et al., 2014). Studying the detailed biological effects of heavy metals in different model organs with single cell resolution can identify specific roles of these metals in causing severe diseases.B. Redox active metals in biology

[0074] In some preferred aspects, a DNAzyme that can catalyzes a chemical reaction such as a nucleic acid cleavage is used in methods provided herein to detect or quantify a redox active metal, such as metal ions of copper or iron. Redox active metals exist in biology and may accumulate in different cell types and catalyze different processes depending on their oxidation state. DNAzymes for the detection of different oxidation states of copper (Liu and Lu, 2007b; Carmi et al., 1998) and iron (Wu et al., 2023) have been developed, and can be used in methods provided herein. These metals can switch between two different functionalstates under native cellular environments, resulting in specific effects, such as differential enzyme activities and gene expression activation. These metals can be especially advantageous for detection and analysis with the methods provided herein that utilize a DNAzyme and subsequent purification and amplification (“DNAzyme-Seq”). While DNAzymes can successfully differentiate between copper and iron oxidation states, other sensor classes often fail to distinguish between oxidation states (Wu et al. , 2023). Different levels of redox active metals have been linked to diseases. For example, the trace metal copper is linked to well- known copper-deficient or overload diseases, such as Menkes disease or Wilson disease (De Bie et al., 2007). Studies in yeast and mammalian cells have identified transcription factors that can act as endogenous copper sensors and regulate copper transporter expression (Song et al. , 2008). Fe2+can influence gene expression, and Fe2+has well-characterized iron responsive elements in mRNA. Iron binding to these mRNA structures can result in gene expression activation (Piccinelli and Samuelsson, 2007). Iron redox cycling is important or required for a range of biological processes, such as nucleotide synthesis, oxygen transport, and respiration. However, diseases with reactive oxygen species formation, such as cancers and neurodegenerative diseases, often lead to toxic levels of unregulated iron species (Aron et al., 2017).

[0075] The nucleic acid enzyme (e.g. , DNAzyme) may contain a variety of modifications to reduce degradation and / or alter catalytic activity, stability, or selectivity. These modifications include phosphorothioate (PS), locked nucleic acid (LNA), or wherein the non-natural nucleotide comprises a 2'-0-Me (2'-0-Me) modification, 2'-O-(N- (aminoethyl)carbamoyl) methyl, 2'-deoxyadenosine analogue, 2'deoxyuridine derivative containing a guanidinium group, phosphorodiamidate morpholino oligonucleotides (PMO), 2'- fluoroarabino nucleic acid (FANA) backbone, a-l-threofuranosyl nucleic acid backbone (XNA) backbone. Additional modifications that may be included in the nucleic acid enzyme are described, e.g., in Larcher et al. (2023).C. Redox inactive metals on biological processes.

[0076] In some preferred aspects, the DNAzyme can bind a redox inactive metal such as zinc. Redox inactive metals with multiple modes of action can regulate a range of biological processes. Zinc influences biological processes through a range of catalytic, structural, and regulatory effects (Cousins, 1998). Catalytic effects in metalloenzymes can require or be affected by zinc bound to an active site of the metalloenzyme. Whether a zinc-containingmetalloenzyme is metalated or not may determine its activity and structure, such as in metallo beta-lactamases, which leads to antibiotic resistance (Gonzalez et al., 2023). Structurally, zinc can bind to zinc binding domains in zinc finger proteins, which results in protein stabilization. This has many downstream effects, such as increased transcription, DNA recognition, RNA packing, and lipid binding (Berg, 1989). Zinc can affect cell regulation functions. For example, zinc directly binds to metallothionein, resulting in cell specific direct regulation of gene transcription rate (Moilane et al., 1999). In systems without lead, the 8-17 DNAzyme can be used as a zinc sensor1.

[0077] The DNAzyme may use calcium as a cofactor. Other metals, such as calcium, can also serve as trace and fleeting messengers. Increased levels of intracellular calcium modify transcription factors that interact with DNA regulatory elements in promoters. This effect is mainly known and studied in the brain, in which calcium serves as the source for electrical activity-dependent transcriptional factors (Johnson et al., 1997). Identifying calcium levels can indicate activated neurons in response to a stimulus of interest, such as physical activity, stressors, rewards, etc. The EtNa DNAzyme can be used as a calcium sensor (Yu et al., 2018).D. Therapeutic metals

[0078] The DNAzyme may bind as a cofactor a metal that is also used in some contexts as a therapeutic (e.g., lithium). Lithium in the body is typically absorbed from food sources in trace amounts, but has no known natural biological functions. Lithium is internalized in cells through transporters for other monovalent ions (Jakobsson et al., 2017). The effects of unneeded lithium hijacking transporters for essential metals, like sodium and potassium, could be elucidated with DNAzyme-seq. While lithium naturally is an unessential cellular metal, it has been used to treat bipolar disorder. The therapeutic dosage window for this metal is narrow. At low mM doses, lithium displays no observable effects. Above >1 mM, lithium results in side effects including diarrhea, tremors, and / or muscle weakness. Studies into its mechanism of action support the idea that lithium can modulate essential neurotransmitter abundance, but detailed studies on how individual brain cell contributions respond to lithium are largely unknown (Jakobsson et al., 2017). While there are no known lithium responsive genes in mammalian systems, bacteria express lithium riboswitches that are activated in high lithium environments to prevent lithium induced toxicity (White et al. , 2022). Excess lithium when used as a bipolar disorder therapeutic is also toxic. Without being bound by any theory, it is anticipated that structures may also exist in mammalian systems as well, and bacteria presentin mammals (e.g. , in digestive systems) may express lithium riboswitches. The Li+-DNAzyme (McGhee et al. , 2021) may be used in methods provided herein to detect or quantify lithium.E. Metals with unknown roles and in the host-pathogen interface

[0079] The DNAzyme may bind a metal ion that has either no known role in biology or that can affect or sense infection by a pathogen, such as manganese. While intracellular levels of manganese are some of the lowest biological metal concentrations, manganese levels vary up 1,000-fold depending on the organ, with excess levels accumulating in the brain (Balachandran et al. , 2020; Johnston et al., 2006). Exposure to excess manganese results in brain accumulation, a biomarker for attention-deficient hyperactivity disorder and Parkinson’s disease. Manganese is mainly used as a sole cofactor for only a few enzymatic reactions. There are several basic questions still unanswered about manganese biology, such as cellular distributions and biological impacts of varying manganese levels. Manganese biology is difficult to study because many reactions catalyzed by manganese can also be catalyzed by magnesium. Due to magnesium’s higher abundance, magnesium is often preferred to catalyze general reactions over manganese.

[0080] Apart from serving as a biomarker, Mn2+acts as a signal for virulence at the host-pathogen interface. By modulating manganese uptake from bacteria residing in human environments, both organisms can limit metal toxicity. Pathogens residing in human hosts express riboswitches that respond to manganese. Under high manganese conditions, manganese binds to the riboswitch, resulting in the expression of manganese exporter proteins (Murdoch and Skaar, 2022). Questions to potentially explore could be if similar riboswitches are expressed in other species and what are other roles of manganese in biology. The 11-5 DNAzyme can be used to study manganese (Fan et al., 2023).F. Metals for biological structures

[0081] The DNAzyme may bind magnesium as a cofactor. Magnesium is one of the most abundant biological metal ions. Magnesium can coordinate nucleotide and protein structures and can facilitate ideal folding and function. Magnesium can also affect gene expression. For example, magnesium may bind to a ubiquitous open reading frame for genes that encode phosphatases. Cellular concentrations of magnesium can correlate with protein expression, with increased phosphatase expression resulting from or associated with lower levels of magnesium. This process may cause rewiring of cellular metabolism, and Mg can beused as a biomarker for many cancers (Hardy et al., 2019). The 10-23 DNAzyme can be used to sense magnesium (Breaker and Joyce, 1994; Xiong et al., Angew. 2020).G. Modifications to increase stability

[0082] A variety of modifications can be made to increase the half-life or stability of a nucleic acid enzyme such as a DNAzyme. Since DNAzymes typically contain at least some DNA, they can be degraded by endonucleases and exonucleases. Since select methods provided herein utilize reading the full length and cleaved sequences corresponding to the nucleic acid enzyme activity, degraded DNAzyme would be unread. To increase the sensor stability in cells, modifications unrecognized by the endonucleases can be incorporated into the nucleic acid enzyme (e.g., DNAzyme), such as including phosphorothioates in the backbone and / or locked nucleic acids in the nucleotide sugars.H. Modifications to increase delivery efficiency

[0083] The nucleic acid enzyme (e.g., DNAzyme) may contain one or more modifications to improve absorption or uptake into cells. 10 serinol dithiol modification may preferably be included at the 3’ end of the nucleic acid enzyme, preferably a DNAzyme, to promote delivery into cells. Such dithiol modifications may interact with membrane bound cysteines and promote internalization of the DNAzyme through translocation. These approaches can be used to allow for uniform distribution throughout a cell, as shown for example in FIG. 6.

[0084] The nucleic acid enzyme may contain one or more modifications to increase absorption or transport into cells. Primary cells and complex tissues typically have advanced developed cell membranes that may prevent the absorption or update of these DNA-sensors through traditional delivery methods such as commercial lipid nanoparticles and electroporation. While this first method incorporated dithiol modifications for enhanced delivery through thiol-mediated uptake with cell surface cysteines, other modifications and delivery vehicles can enhance the uptake of the nucleic acid enzyme. Optimized synthetic lipid nanoparticles, polymers, metal nanoparticles, protein-based expression, disulfide modifications, and biological micro vesicles can be used to increase transport of the nucleic acid enzyme into cells.III. Methods of quantifying cofactor concentrations

[0085] Concentrations of cofactor may preferably be quantified by amplifying cleaved and uncleaved polynucleotide as shown in FIG. 1, and this method may use PCR or quantitative PCR (qPCR). The polynucleotides may be purified based on the presence of an affinity tag present on the nucleic acid enzyme and the reaction products, e.g., using magnetic beads as described in Green et al. (2019). Differences in the size of the cleaved and uncleaved polynucleotides can be used to determine the concentration of the cofactor (e.g., metal ion) in the sample (e.g., environmental sample, tissue sample, etc.). This amplification can also be used to simultaneously amplify and / or quantify one or more mRNA species. The mRNA and nucleic acid enzyme may be purified in a single step by targeting the same affinity tag (e.g., poly-A tail) that is present in both the nucleic acid enzyme and the mRNA. In some embodiments, the DNAzyme self-hybridizes upstream and downstream from the catalytic loop of the DNAzyme, wherein the presence of a cofactor can cause the DNAzyme to self-cleave and release either the upstream or downstream self-hybridized portion of the DNAzyme, revealing a single stranded portion of the DNAzyme that can be detected, for example using SNAIL probes and RCA. An illustration of this methodology is provided in FIGS. 7A-C.

[0086] The methods provided herein can be used to measure or quantify concentrations of a cofactor (e.g., metal ion) in both human and nonhuman tissue samples. While this proof- of-concept was demonstrated in human samples in the below examples, methods provided herein can be used with tissue samples or in cells from other mammalian species with complex cell compositions and tissue architecture, including mammals (e.g., mice, rats, rodents, horses, cows, cats, dogs, domesticated animals or livestock), non-mammal animals such as birds, reptiles, amphibians, fish, and plants.

[0087] In some embodiments, multiple nucleic acid enzymes (e.g., multiple DNAzymes) can be used to simultaneously detect and / or quantify multiple cofactors (e.g., two or more metal ions). Multiplexing may thus be achieved by using multiple nucleic acid enzyme sensors to detect different metal ions. Multiple sensors can be delivered at the same time, and these approaches can allow for analysis of various biological processes (e.g., how different metals influence gene expression in the same cell). Such approaches can be particularly beneficial for analysis of redox active species, including iron in combination with other cofactors or metal ions.

[0088] The methods provided herein can be used in combination with multiomics sequencing platforms for other biomolecules. The methods of detecting a cofactor provided herein can be used to correlate metal ion concentrations or location and with another cellular characteristic (e.g. , gene expression or cell surface protein expression). The methods provided herein can also be used in combination with CRISPR screening (e.g., Dixit et al., 2016). These approaches may improve biological understanding of a wide variety of biological processes.

[0089] The methods provided herein can also be used to detect and quantify a wide variety of RNA. For example methods using a DNAzyme (“DNAzyme-Seq”) to quantify both a metal ion in combination with one or more mRNA are provided in the below examples. In addition to detecting and quantifying mRNA expression, other RNA species can also be detected and quantified using the methods provided herein. For example, noncoding RNA species, such as transfer and ribosomal RNAs, can be targeted by the nucleic acid enzyme (e.g., DNAzyme). While transfer RNAs and ribosomal RNAs have well-understood roles of assisting protein synthesis, the vast majority of other noncoding RNAs still have unknown roles.

[0090] The methods provided herein can be used in combination with Spatial transcriptomics methodologies. The DNAzyme cleavage ratio can also be read out through imaging methods that evaluate gene expression. By keeping tissue samples intact, the spatial heterogeneities and relationships between different cell types can be analyzed.

[0091] The DNAzyme or nucleic acid enzyme may be encoded by a vector or nucleic acid under the control of a promoter in the cell. These approaches can be utilized to observe expression in select tissues. For example, the promoter may be inducible by a compound or drug (e.g., tetracycline-on promoter, tetracycline-off promoter, etc.) or the promoter may be active in select tissue types. To achieve tissue sensing without delivery, the DNA-based sensors may be expressed under the activation of a promoter, similar to RNA and protein expression.

[0092] A variety of amplification methods can be used with the methods provided herein including PCR, qPCR, rolling circle amplification, and hybridization chain reaction. To account for low delivery, the sensors can be designed to allow for signal amplification of the DNAzymes. For example, methods such as rolling circle amplification and hybridization chainreaction, can enhance the DNAzyme readout and allow for incorporation with spatial transcriptomics in cells and in tissue.

[0093] The nucleic acid enzyme may be photocaged using a variety of photocaging approaches. To account for potentially toxic effects of sensor light activation, the photoprotecting group may be modified to be lower energy red-shifted light. Likewise, other caging groups on the sensors may be incorporated, such as “pro-drug”-like modifications for enzyme activation. Exemplary photocaging approaches that can be used include those described in (Xu et al., 2021).

[0094] In some aspects the DNAzyme comprises a barcode. A variety of techniques can be used to detect the barcode and / or cleavage event of the DNAzyme (e.g., cleavage of the DNAzyme in response to the presence of a metal ion or cofactor). Simultaneous measurement of mRNA expression in cells can also be performed using a variety of techniques including those listed in Table 1. In this way, the cleavage event of the DNAzyme and measurement of a cofactor can be correlated with a wide variety of mRNA expression in cells. Table 1: Nucleic Acid Detection MethodsA. RNA Detection and Imaging with Rolling Circle Amplification (RCA)

[0095] A clevage event by a DNAzyme may be detected using a rolling cycle amplifcation (RCA) such as, e.g., SNAIL. If desired, the RCA reaction may utilize additional probes to measure expression of one or more mRNA of interest. RNA detection imaging can be performed with rolling circle amplification. Gene expression at the single-cell level with improved spatial and sequence resolution can be performed with RCA or SNAIL. Direct detection of mRNA by rolling circle amplification may enable imaging of mRNA with singlenucleotide and near-single molecule resolution in single cells (Deng, R., Zhang, K., Sun, Y.,Ren, X., & Li, J. (2017). Highly specific imaging of mRNA in single cells by target RNA- initiated rolling circle amplification. Chemical Science, 8(5), 3668-3675). An RCA reaction typically comprises a single- stranded circular nucleic acid template, at least one primer annealing to the circular nucleic acid template, and a polymerase with strand displacement activity. Polymerases with strand displacement activity can provide the denaturation of doublestranded nucleic acids during polymerization. Rolling circle amplification typically use circular template nucleic acids. Select uses of detection of a cleavage event by a DNAzyme and mRNA sequencing and detection using SNAIL probes are shown in FIGS. 7A-C. The circulized SNAIL probe may comprise a barcode for transcription into a Rolling Circle amplificaiton product that can be detected with a detection probel (e.g., a nucleic acid comprising a detectable label).

[0096] During an RCA reaction, after the primer anneals to a circular nucleic acid template, the synthesis of its complementary strand may begin, typically continuing until the polymerase reaches the 5 ’-end of the primer. At this point, the polymerase may displace the strand that interferes with it and continue synthesis along the circle, creating a long singlestranded RCA product. RCA can be performed with circular DNA templates or RNA templates. In the case of RNA templates, the reaction may be called rolling circle transcription (RCT). An RCT reaction may utilize an RNA-dependent polymerase, and initiation of transcription typically requires the presence of promoter sequences.

[0097] Any of the following polymerases may be used in an RCA reaction: DNA polymerases Bst, Bsm, and Vent without 5’-3’ exonuclease activity, phi29, T7 RNA polymerases, among others. Phage T7 RNA polymerase may be used for RCA, but may require the preence of promoter sequences or a template with increased content of pyrimidine bases. Reverse transcriptases may also be used for RCA.

[0098] RCA can be used with various reporter systems to detect amplification. Exemplary reporter systems that may be used for detection include: fluorescent (e.g. intercalating dyes, fluorescent hybridization probes, fluoregenic substrates, fluorescent nucleotides, metal nanoparticles and metal complexes), colorimetric (e.g. chromogenic substrates, colorimetric hybridization probes), and electrochemical (e.g. hybridization probes with electroactive or a conductive label, nucleic acid binding of electroactive particles) (Garafutdinov, R. R., Sakhabutdinova, A. R., Gilvanov, A. R., & Chemeris, A. V. (2021).Rolling Circle Amplification as a Universal Method for the Analysis of a Wide Range of Biological Targets. Russian Journal of Bioorganic Chemistry, 47(6), 1172-1189).B. Padlock Probes

[0099] A clevage event by a DNAzyme may be detected using one or more padlock probes. Circularizing oligonucleotide probes, also known as padlock probes, can be used for in in situ analyses, genotyping and measurement of gene expression. Padlock probes are oligonucleotides that may become circularized by DNA ligation in the presence of an appropriate target DNA or RNA sequence. Padlock probes typically use two target- complementary segments, one at each end of the probe, hybridize to a target sequence for circularization to occur. Rolling circle amplification can then be used to amplify the circularized probe. Padlock probes may be 70-100 nucleotides in length (C. Larsson, I. Grundberg, O. Soderberg, M. Nilsson, In situ detection and genotyping of individual mRNA molecules. Nat. Methods 7, 395-397 (2010). doi:10.1038 / nmeth,1448 Medline; Antson, D.-O. (2000). PCR- generated padlock probes detect single nucleotide variation in genomic DNA. Nucleic Acids Research, 28(12), 58e- 558).C. Specific Nucleic Acid detection via Intramolecular Ligation (SNAIL) Probes

[0100] A clevage event by a DNAzyme may be detected using Specific Nucleic Acid detection via Intramolecular Ligation (SNAIL) and Rolling Circle Amplification (RCA). SNAIL-RCA employs a structural adaptation of the padlock / proximity ligation / rolling circle amplification system.

[0101] In SNAIL-RCA, two primers may be used, one of which may bind to RNA at a 3’ position and contain the sequence cognate to a common ligation junction. The second oligonucleotide may contain sequence proximal, which may bind slightly upstream on the RNA and anneal to the ligation junction. When both primers hybridize to the target RNA, the padlock probe can be circularized, such that rolling-circle amplification can amplify the target.D. Spatial Transcriptomics

[0102] Spatial transcriptomics methods can be used, e.g., to allow multiplexed analysis of cellular transcripts. With spatially resolved transcriptomic methods, transcriptomic data canbe determined with positional context of those cells in a tissue (e.g., Marx, V. Method of the Year: spatially resolved transcriptomics. Nat Methods 18, 9-14 (2021)). High-degree multiplexing can be achieved with serial hybridizations or barcoding systems. Gene expression can be detected using fluorescent in situ hybridization (FISH), which may utilize combinatorial barcoding or spatial barcoding. In spatial barcoding, fluorophores targeted to different segments along an RNA may be resolved by microscopy. FISH may also rely on spectral barcoding. In spectral barcoding, the targets may be labeled with a specific combination of fluorophores. FISH may also rely on temporal barcoding. In temporal barcoding, multiple cycles of smFISH hybridization and stripping may repeatedly label the same RNA molecules in a predefined color sequence.

[0103] In some aspects, RNA can be sequenced directly. In situ sequencing may use target-specific padlock probes for sequencing in situ. FISSEQ may also be employed to sequence amplified cellular RNA content directly.

[0104] An alternative to in situ sequencing may be to use patterned microarrays, carrying barcoded oligo-dT primers, to capture mRNA frm a tissue section. During cDNA synthesis on the array, barcodes may indicate the location of each spot, and such barcodes may be incorporated into the cDNA which may be pooled and sequenced. This may allow for mapping to the specific spatial coordinates (E. Lein, L. E. Borm, S. Linnarsson, The promise of spatial transcriptomics for neuroscience in the era of molecular cell typing. Science 358, 64- 69 (2017)).E. Fluorescent in situ sequencing (FISSEQ)

[0105] Fluorescent in situ sequencing (FISSEQ) begins with fixing cells on a glass slide and performing reverse transcription (RT) in siu. After RT, any residual RNA may be degraded to prevent inhibition of the next ligation step. Next, cDNA fragments can be circularized. To prevent cDNA fragments from diffusing away, primary amines may be incorporated, which may be subsequently crosslinked using BS(PEG)9. Each cDNA circle may then be linearly amplified using rolling circle amplification. The RCA product can then be crosslinked to form a highly porous 3D nucleic acid matrix in the cell. Next, sequencing primers are hybridized to multiple copies of the adaptor sequence in RCA amplicons, followed by ligation of dinculeo tide- specific fluorescent oligonucleotides. After imaging, the fluorophores may be cleaved and ligation of fluorescent oligonucleotides may be repeated to interrogate dinucleotidepairs. To fill in the gaps between dinucleotide pairs, the whole ligation complex may be stripped off, and additional sequencing primers with a single base offset may be used to repeat dinucleotide interrogation, which may generate 3D image stacks representing dinucleotide compositions at all base positions over time (J. H. Lee, E. R. Daugharthy, J. Scheiman, R. Kalhor, T. C. Ferrante, R. Terry, B. M. Turczyk, J. L. Yang, H. S. Lee, J. Aach, K. Zhang, G. M. Church, Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat. Protoc. 10, 442-458 (2015). doi: 10.1038 / nprot.2014.191 Medline).F. Spatially resolved Transcript Amplicon Readout Mapping (STARmap)

[0106] Spatially resolved Transcript Amplicon Readout Mapping (STARmap) may be used to detect a cleavage event by a DNAzyme. The process can begin with labelling of cellular RNAs by pairs of DNA probes followed by enzymatic amplification so as to produce a DNA nanoball (amplicon). Rolling-circle amplification can be performed after circularization of the probe by means such as, for example, SNAIL or padlock probes. The tissue may then be transformed into a 3D hydrogel DNA chip by anchoring DNA amplicons via an in situ- synthesized polymer network and removing proteins and lipids. This hydrogel-tissue chemistry may replot amplicons onto an optically transparent hydrogel coordinate system. Then, the identity of each species can be encoded by a five-base barcode and detected via in situ sequencing. STARmap can be used to simultaneously detect more than 1000 genes over six imaging cycles (Wang, X., Allen, W. E., Wright, M. A., Sylwestrak, E. L., Samusik, N., Vesuna, S., Evans, K., Liu, C., Ramakrishnan, C., Liu, J., Nolan, G. P., Bava, F.-A., & Deisseroth, K. (2018). Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science, 361(6400), eaat5691. ; R. Ke, M. Mignardi, A. Pacureanu, J. Svedlund, J. Botling, C. Wahlby, M. Nilsson, In situ sequencing for RNA analysis in preserved tissue and cells. Nat. Methods 10, 857-860 (2013)).G. Single-Cell RNA Sequencing (scRNA-seq)

[0107] Single-cell RNA sequencing (scRNA-seq) can be used to detect a clevage event by a DNAzyme as provided herin in single cells. In scRNA-seq, single cells are first isolated. This may be accomplished with limiting dilutin, micromanipulation, flow-activated cell sorting, or laser capture microdissection. scRNA-seq libraries may be generated by cell lysis, reverse transcription into first-strand cDNA, second-strand synthesis, and cDNA amplification. For cDNA preparation, an engineered version of the Moloney murine leukaemia virus reversetranscriptase with low RNase H activity can be used. Second strands may be generated using poly(A) tailing or template-switching. Conventional or other PCR methods may then be used for cDNA amplification. Sequencing may then be performed on, for examples, Illumina’s HiSeq4000, NextSeq500, or MiSeq (Hwang, B., Lee, J. H., & Bang, D. (2018). Single-cell RNA sequencing technologies and bioinformatics pipelines. Experimental & Molecular Medicine, 50(8), 1-14.).IV. Processing of Nucleic AcidsA. Amplification of Nucleic Acids

[0108] “Amplification,” as used herein, refers to any in vitro process for increasing the number of copies of a nucleotide sequence or sequences. Nucleic acid amplification results in the incorporation of nucleotides into DNA or RNA. As used herein, one amplification reaction may consist of many rounds of DNA replication. For example, one PCR reaction may consist of 30-100 “cycles” of denaturation and replication.

[0109] “Polymerase chain reaction,” or “PCR,” means a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors well-known to those of ordinary skill in the art, e.g., exemplified by the references: McPherson et al., editors, PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively).

[0110] “Primer” means an oligonucleotide, either natural or synthetic that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Usually primers are extended by a DNA polymerase. Primers are generally of a length compatible with its use in synthesis ofprimer extension products, and are usually are in the range of between 8 to 100 nucleotides in length, such as 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, and so on, more typically in the range of between 18-40, 20-35, 21-30 nucleotides long, and any length between the stated ranges. Typical primers can be in the range of between 10-50 nucleotides long, such as 15-45, 18-40, 20-30, 21-25 and so on, and any length between the stated ranges. Primers may be no more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.

[0111] The term “PCR” encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, assembly PCR and the like. Reaction volumes range from a few hundred nanoliters, e.g., 200 nL, to a few hundred microliters, e.g., 200 pL. “Reverse transcription PCR,” or “RT-PCR,” means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, e.g., Tecott et al., U.S. Pat. No. 5,168,038. “Real-time PCR” means a PCR for which the amount of reaction product, i.e., amplicon, is monitored as the reaction proceeds. There are many forms of real-time PCR that differ mainly in the detection chemistries used for monitoring the reaction product, e.g., Gelfand et al., U.S. Pat. No. 5,210,015 (“Taqman”); Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecular beacons). Detection chemistries for real-time PCR are reviewed in Mackay et al., Nucleic Acids Research, 30: 1292- 1305 (2002). “Nested PCR” means a two-stage PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon. As used herein, “initial primers” in reference to a nested amplification reaction mean the primers used to generate a first amplicon, and “secondary primers” mean the one or more primers used to generate a second, or nested, amplicon. “Multiplexed PCR” means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g. Bernard etal. (1999) Anal. Biochem., 273:221-228 (two-color realtime PCR). Usually, distinct sets of primers are employed for each sequence being amplified. “Quantitative PCR” means a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen. Techniques for quantitative PCR are well-known to those of ordinary skill in the art, as exemplified in the following references: Freeman et al., Biotechniques, 26:112-126 (1999); Becker- Andre et al., Nucleic Acids Research, 17:9437- 9447 (1989); Zimmerman et al., Biotechniques, 21:268-279 (1996); Diviacco etal., Gene, 122:3013-3020 (1992); Becker- Andre et al., Nucleic Acids Research, 17:9437-9446 (1989); and the like.

[0112] As used herein, “RNA-seq” (RNA Sequencing), is a technology that uses the capabilities of next-generation sequencing to reveal a snapshot of RNA presence and quantity from a genome at a given moment in time. Single-cell mRNA sequencing (scRNA-Seq) can be used for unbiased transcriptional profiling of hundreds to thousands of individual cells from a single-cell suspension (scRNA-Seq). The term “scRNA-Seq,” as used herein, generally refers to a single-cell RNA sequencing method to obtain expression profiles of individual cells. For example, single-cell libraries can be prepared from single-cell suspensions. Such single-cell libraries can be sequenced. Sequencing reads may be processed, for example, by alignment, filtration, de-duplication, and / or conversion into a digital count matrix.

[0113] cDNA synthesis can be performed where first strand synthesis is primed with an (anchored) oligo dT primer (or potentially with a randomer or a combination of the two) which is optionally appended with a sample barcode (BC), an amplification primer binding site, and a template switch (TS) primer sequence. The optional barcode can be preceded or followed by a molecular barcode (unique molecular identifier, or “UMI”) that would allow for the detection of PCR duplicates. When the reverse transcriptase reaches the 5' end of the RNA template, the enzyme’s terminal transferase activity adds a few additional non-template nucleotides to the 3' end of the cDNA. A template- switch oligo, designed to base-pair with this non-template nucleotide stretch, anneals and creates an extended template to enable the RT to continue polymerizing to the end of the template switching oligonucleotide. This incorporates the complement of the template switching oligonucleotide into the first strand cDNA. The first strand cDNA can subsequently be rendered double stranded, and optionally amplified in a PCR reaction, with the template switching primer.

[0114] Varied choices of polymerases exist with different properties, such as temperature, strand displacement, and proof-reading. Amplification can be isothermal, such as multiple displacement amplification (MDA) described by Dean et al., Comprehensive human genome amplification using multiple displacement amplification, Proc. Natl. Acad. Sci. U.S.A., vol. 99, p. 5261-5266. 2002; also Dean et al., Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification, Genome Res., vol. 11, p. 1095-1099. 2001; also Aviel-Ronen et al., Large fragment Bst DNA polymerase for whole genome amplification of DNA formalin-fixed paraffin-embeddedtissues, BMC Genomics, vol. 7, p. 312. 2006. Amplification can also cycle through different temperature regiments, such as the traditional polymerase chain reaction (PCR) popularized by Mullis et al., Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol., vole 51, p. 263-273. 1986. Other methods include Polony PCR described by Mitra and Church, In situ localized amplification and contact replication of many individual DNA molecules, Nuc. Acid. Res., vole 27, pages e34. 1999; emulsion PCR (ePCR) described by Shendure et al., Accurate multiplex polony sequencing of an evolved bacterial genome, Science, vol. 309, p. 1728-32. 2005; and Williams et al., Amplification of complex gene libraries by emulsion PCR, Nat. Methods, vol. 3, p. 545-550. 2006. Any amplification method can be combined with a reverse transcription step, a priori, to allow amplification of RNA. According to certain aspects, amplification is not absolutely required since probes, reporters and detection systems with sufficient sensitivity can be used to allow detection of a single molecule using template non-hybridizing nucleic acid structures described. Ways to adapt sensitivity in a system include choices of excitation sources (e.g. illumination) and detection (e.g. photodetector, photomultipliers). Ways to adapt signal level include probes allowing stacking of reporters, and high intensity reporters (e.g. quantum dots) can also be used.

[0115] Exemplary methods for amplifying nucleic acids include the polymerase chain reaction (PCR) (see, e.g., Mullis et al. (1986) Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1:263 and Cleary et al. (2004) Nature Methods 1:241; and U.S. Pat. Nos. 4,683,195 and 4,683,202), anchor PCR, RACE PCR, ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. U.S. A. 91:360-364), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:1874), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:1173), Q-Beta Replicase (Lizardi et al. (1988) BioTechnology 6: 1197), recursive PCR (Jaffe et al. (2000) 7. Biol. Chem. 275:2619; and Williams et al. (2002) 7. Biol. Chem. 277:7790), the amplification methods described in U.S. Pat. Nos. 6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and 5,612,199, isothermal amplification (e.g., rolling circle amplification (RCA), hyperbranched rolling circle amplification (HRCA), strand displacement amplification (SDA), helicase-dependent amplification (HD A), PWGA) or any other nucleic acid amplification method using techniques well known to those of skill in the art.

[0116] A barcode, such as a sample barcode, may be added to the target nucleic acid molecules during amplification. One method involves annealing a primer (e.g., a truth marker DNA oligonucleotide) to the nucleic acid molecule, the primer including a first portion complementary to the nucleic acid molecule and a second portion including a barcode; and extending the annealed primer to form a barcoded nucleic acid molecule. Thus, the primer may include a 3' portion and a 5' portion, where the 3' portion may anneal to a portion of the nucleic acid molecule and the 5' portion comprises the barcode.B. Sequencing of Nucleic Acids

[0117] Methods are also provided for the sequencing of the library of nucleic acid molecules. Any technique for sequencing nucleic acids known to those skilled in the art can be used in the methods of the present disclosure. DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, sequencing-by- synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing-by-synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, and SOLiD sequencing.

[0118] The nucleic acid library may be generated with an approach compatible with Illumina sequencing such as a Nextera™ DNA sample prep kit, and additional approaches for generating Illumina next-generation sequencing library preparation are described, e.g., in Oyola et al. (2012). In other embodiments, a nucleic acid library is generated with a method compatible with a SOLiD™ or Ion Torrent sequencing method (e.g. , a SOLiD® Fragment Library Construction Kit, a SOLiD® Mate-Paired Library Construction Kit, SOLiD® ChlP- Seq Kit, a SOLiD® Total RNA-Seq Kit, a SOLiD® SAGE™ Kit, a Ambion® RNA-Seq Library Construction Kit, etc.). Additional methods for next- generation sequencing methods, including various methods for library construction that may be used with embodiments of the present disclosure are described, e.g., in Pareek (2011) and Thudi (2012).

[0119] In particular aspects, the sequencing technologies used in the methods of the present disclosure include the HiSeq™ system (e.g., HiSeq™ 2000 and HiSeq™ 1000) and the MiSeq™ system from Illumina, Inc. The HiSeq™ system is based on massively parallel sequencing of millions of fragments using attachment of randomly fragmented genomic DNAto a planar, optically transparent surface and solid phase amplification to create a high density sequencing flow cell with millions of clusters, each containing about 1,000 copies of template per sq. cm. These templates are sequenced using four-color DNA sequencing-by-synthesis technology. The MiSeq™ system uses TruSeq™, Illumina’s reversible terminator-based sequencing-by-synthesis.

[0120] Another example of a DNA sequencing platform is the QIAGEN GeneReader platform — a next generation sequencing (NGS) platform utilizing proprietary modified nucleotides whose 3' OH groups are reversely terminated by a small moiety to perform sequencing-by-synthesis (SBS) in a massively parallel manner. Briefly, the sequencing templates are first clonally amplified on a solid surface (such as beads) to generate hundreds of thousands of identical copies for each individual sequencing template, denaturized to generate single-stranded sequencing templates, hybridized with sequencing primer, and then immobilized on the flow cell. The immobilized sequencing templates are then subjected to a nucleotide incorporation reaction in a reaction mix that includes modified nucleotides with a cleavable 3' blocking group that enables the incorporation and detection of only one specific nucleotide onto each sequencing template in each cycle. See U.S. Pat. Nos. 6,664,079; 8,612,161; and 8,623,598, each of which is incorporated by reference herein.

[0121] Another example of a DNA sequencing platform is the Ion Torrent PGM™ sequencer (Thermo Fisher) and the Ion Torrent Proton™ Sequencer (Thermo Fisher), which are ion-based sequencing systems that sequence nucleic acid templates by detecting ions produced as a byproduct of nucleotide incorporation. Typically, hydrogen ions are released as byproducts of nucleotide incorporations occurring during template-dependent nucleic acid synthesis by a polymerase. The Ion Torrent PGM™ sequencer and Ion Proton™ Sequencer detect the nucleotide incorporations by detecting the hydrogen ion byproducts of the nucleotide incorporations. The Ion Torrent PGM™ sequencer and Ion Torrent Proton™ sequencer include a plurality of nucleic acid templates to be sequenced, each template disposed within a respective sequencing reaction well in an array. The wells of the array are each coupled to at least one ion sensor that can detect the release of H+ ions or changes in solution pH produced as a byproduct of nucleotide incorporation. The ion sensor comprises a field effect transistor (FET) coupled to an ion-sensitive detection layer that can sense the presence of H+ ions or changes in solution pH. The ion sensor provides output signals indicative of nucleotide incorporation, which can be represented as voltage changes whose magnitude correlates withthe H+ ion concentration in a respective well or reaction chamber. Different nucleotide types are flowed serially into the reaction chamber, and are incorporated by the polymerase into an extending primer (or polymerization site) in an order determined by the sequence of the template. Each nucleotide incorporation is accompanied by the release of H+ ions in the reaction well, along with a concomitant change in the localized pH. The release of H+ ions is registered by the FET of the sensor, which produces signals indicating the occurrence of the nucleotide incorporation. Nucleotides that are not incorporated during a particular nucleotide flow will not produce signals. The amplitude of the signals from the FET may also be correlated with the number of nucleotides of a particular type incorporated into the extending nucleic acid molecule thereby permitting homopolymer regions to be resolved. Thus, during a run of the sequencer multiple nucleotide flows into the reaction chamber along with incorporation monitoring across a multiplicity of wells or reaction chambers permit the instrument to resolve the sequence of many nucleic acid templates simultaneously. Further details regarding the compositions, design and operation of the Ion Torrent PGM™ sequencer can be found, for example, in U.S. Pat. Publn. Nos. 2009 / 0026082; 2010 / 0137143; and 2010 / 0282617, all of which are incorporated by reference herein in their entireties.

[0122] Another example of a DNA sequencing technique that can be used in the methods of the present disclosure is 454 sequencing (Roche) (Margulies et al., 2005). 454 sequencing involves two steps. In the first step, DNA is sheared into fragments of approximately 300-800 base pairs, and the fragments are blunt ended. Oligonucleotide adaptors are then ligated to the ends of the fragments. The adaptors serve as primers for amplification and sequencing of the fragments. The fragments can be attached to DNA capture beads, e.g., streptavidin-coated beads using, e.g., Adaptor B, which contains 5'-biotin tag. The fragments attached to the beads are PCR amplified within droplets of an oil- water emulsion. The result is multiple copies of clonally amplified DNA fragments on each bead. In the second step, the beads are captured in wells (pico-liter sized). Pyrosequencing is performed on each DNA fragment in parallel. Addition of one or more nucleotides generates a light signal that is recorded by a CCD camera in a sequencing instrument. The signal strength is proportional to the number of nucleotides incorporated.

[0123] Another example of a DNA sequencing technique that can be used in the methods of the present disclosure is SOLiD technology (Life Technologies, Inc.). In SOLiD sequencing, genomic DNA is sheared into fragments, and adaptors are attached to the 5' and 3'ends of the fragments to generate a fragment library. Alternatively, internal adaptors can be introduced by ligating adaptors to the 5' and 3' ends of the fragments, circularizing the fragments, digesting the circularized fragment to generate an internal adaptor, and attaching adaptors to the 5' and 3' ends of the resulting fragments to generate a mate-paired library. Next, clonal bead populations are prepared in microreactors containing beads, primers, template, and PCR components. Following PCR, the templates are denatured and beads are enriched to separate the beads with extended templates. Templates on the selected beads are subjected to a 3' modification that permits bonding to a glass slide.

[0124] Another example of a DNA sequencing technique that can be used in the methods of the present disclosure is the lonTorrent system (Life Technologies, Inc.). Ion Torrent uses a high-density array of micro-machined wells to perform this biochemical process in a massively parallel way. Each well holds a different DNA template. Beneath the wells is an ion- sensitive layer and beneath that a proprietary Ion sensor. If a nucleotide, for example a C, is added to a DNA template and is then incorporated into a strand of DNA, a hydrogen ion will be released. The charge from that ion will change the pH of the solution, which can be detected by the proprietary ion sensor. The sequencer will call the base, going directly from chemical information to digital information. The Ion Personal Genome Machine (PGM™) sequencer then sequentially floods the chip with one nucleotide after another. If the next nucleotide that floods the chip is not a match, no voltage change will be recorded and no base will be called. If there are two identical bases on the DNA strand, the voltage will be double, and the chip will record two identical bases called. Because this is direct detection — no scanning, no cameras, no light — each nucleotide incorporation is recorded in seconds.

[0125] Another example of a sequencing technology that can be used in the methods of the present disclosure includes the single molecule, real-time (SMRT™) technology of Pacific Biosciences. In SMRT™, each of the four DNA bases is attached to one of four different fluorescent dyes. These dyes are phospholinked. A single DNA polymerase is immobilized with a single molecule of template single stranded DNA at the bottom of a zeromode waveguide (ZMW). A ZMW is a confinement structure which enables observation of incorporation of a single nucleotide by DNA polymerase against the background of fluorescent nucleotides that rapidly diffuse in and out of the ZMW (in microseconds). It takes several milliseconds to incorporate a nucleotide into a growing strand. During this time, the fluorescent label is excited and produces a fluorescent signal, and the fluorescent tag is cleaved off.Detection of the corresponding fluorescence of the dye indicates which base was incorporated. The process is repeated.

[0126] A further sequencing platform includes the CGA Platform (Complete Genomics). The CGA technology is based on preparation of circular DNA libraries and rolling circle amplification (RCA) to generate DNA nanoballs that are arrayed on a solid support (Drmanac el al. 2010). Complete genomics’ CGA Platform uses a novel strategy called combinatorial probe anchor ligation (ePAL) for sequencing. The process begins by hybridization between an anchor molecule and one of the unique adapters. Four degenerate 9- mer oligonucleotides are labeled with specific fluorophores that correspond to a specific nucleotide (A, C, G, or T) in the first position of the probe. Sequence determination occurs in a reaction where the correct matching probe is hybridized to a template and ligated to the anchor using T4 DNA ligase. After imaging of the ligated products, the ligated anchor-probe molecules are denatured. The process of hybridization, ligation, imaging, and denaturing is repeated five times using new sets of fluorescently labeled 9-mer probes that contain known bases at the n + 1, n + 2, n + 3, and n + 4 positions.

[0127] A further sequencing platform includes nanopore sequencing (Oxford Nanopore). Nanopore detection arrays are described in US2011 / 0177498; US2011 / 0229877; US2012 / 0133354; WO2012 / 042226; WO2012 / 107778, and have been used for nucleic acid sequencing as described in US2012 / 0058468; US2012 / 0064599; US2012 / 0322679 and WO2012 / 164270, all of which are hereby incorporated by reference. A single molecule of DNA can be sequenced directly using a nanopore, without the need for an intervening PCR amplification step or a chemical labelling step or the need for optical instrumentation to identify the chemical label. Commercially available nanopore nucleic acid sequencing units are developed by Oxford Nanopore (Oxford, United Kingdom). The GridlON™ system and miniaturised MinlON™ device are designed to provide novel qualities in molecular sensing such as real-time data streaming, improved simplicity, efficiency and scalability of workflows and direct analysis of the molecule of interest. Using the Oxford Nanopore nanopore sequencing platform, an ionic current is passed through the nanopore by setting a voltage across this membrane. If an analyte passes through the pore or near its aperture, this event creates a characteristic disruption in current. Measurement of that current makes it possible to identify the molecule in question. For example, this system can be used to distinguish between the four standard DNA bases G, A, T and C, and also modified bases. It can be used to identify targetproteins, small molecules, or to gain rich molecular information, for example to distinguish between the enantiomers of ibuprofen or study molecular binding dynamics. These nanopore arrays are useful for scientific applications specific for each analyte type; for example when sequencing DNA, the technology may be used for resequencing, de novo sequencing, and epigenetics.V. Kits

[0128] A kit comprising a nucleic acid enzyme comprising a nucleic acid affinity tag (e.g., DNAzyme comprising a poly- A tail) and primers for amplification is contemplated. The kit may further comprise materials for purification of the nucleic acid enzyme, such as beads e.g., magnetic beads) or a column for separation of compounds comprising the nucleic acid affinity tag e.g. , poly-A tail). When a kit is supplied, the different components of the composition may be packaged in separate containers and admixed immediately before use. Packaging of the components preferably permits long-term storage of the active components. The nucleic acid enzyme(s) and primers may be supplied in separate containers.

[0129] The reagents included in the kits can be supplied in containers of any sort such that the life of the different components are preserved and are not adsorbed or altered by the materials of the container. For example, sealed glass ampules may contain one of more of the reagents, or buffers that have been packaged under a neutral, non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc.; ceramic, metal or any other material typically employed to hold similar reagents. Other examples of suitable containers include simple bottles that may be fabricated from similar substances as ampules; and envelopes that may comprise foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to be mixed. Removable membranes may be glass, plastic, rubber, etc.

[0130] The kits may also contain other reagents and items useful for detecting the targeted cofactor or effector and / or for amplifying one or more specific mRNA may be provided in the kit. The reagents may include standard solutions containing known quantities of the cofactor or effector, dilution and other buffers, pretreatment reagents, primers, PCR reagents, etc.

[0131] Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and / or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.IV. Examples

[0132] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1 - Design of DNAzymes for DNAzyme-seq

[0133] During DNAzyme generation, DNAzymes are initially selected in singlestranded form. Usually, they are converted into double- stranded sequences by removing unnecessary bases for activity and selectivity in order to reduce synthesis cost. To remove a variable of two strands for cleavage and allow for read lengths more similar to mRNA lengths, the selected single- stranded Na+-DNAzyme was used. The Na+-DNAzyme was modified in several ways to make it compatible with NGS. First, the DNAzyme was poly-adenylated by adding 20 adenine bases to the 3’ position. This was to make the DNAzyme chemically similar to mRNA’s poly-A tail and allow for its extraction from cells using dT-oligo beads. The poly- A tail at this position allowed for both the full length and cleaved DNAzyme to be extracted. Second, the DNAzyme cleavage site was photocaged. As extracellular sodium levels are higher than intracellular levels, photoprotection inhibited DNAzyme cleavage during the delivery process. It was found that photoprotection prevented DNAzyme activity in buffer, while the photoprotecting group readily removed with light (FIG. 5). Third, 10 serinol dithiol modifications were added to the 3' position. The dithiol modifications were to allow for DNAzyme delivery into primary cell types, that are known to not be transfected by standardcommercially available lipid transfection agents (Gomes et al., 2023). The dithiol modifications interact with membrane bound cysteines and internalized the DNAzyme through translocation. These allowed for uniform distribution through the cell (FIG. 6). As a control strand, a point mutation sequence was generated, in which an essential base for activity was mutated to abolish any sodium-specific response. This control in cells accounted for signal due to DNA degradation and could be background subtracted from the active DNAzyme cleavage ratios.

[0134] After cellular delivery and intracellular cleavage, the DNAzyme and mRNA were extracted with dT-oligo bead purification. Upon binding to dT-oligo beads, the DNAzyme and mRNA were eluted with first strand synthesis buffer and random primer mix. Then the elution mixture was phosphorylated with T4 PNK to convert the free 5 ’OH on the DNAzyme to a phosphoryl group for primer recognition. The first strand cDNA synthesis of both the mRNA and the DNAzyme was carried out using dT as a primer. The resulting cDNA contained a universal sequence to anneal to a template switching oligo. Then the second strand cDNA synthesis was carried out to produce double-stranded cDNA from the first-strand reaction without the need for intermediate organic extraction of ethanol precipitation steps. Then then added sequencing adaptors, with subsequent PCR enrichment of adaptor ligated DNA and addition of i5 / i7 primers for compatibility with Illumina instruments.Example 2 - Materials and Methods

[0135] DNA sequences*NB: nitrobenzyl photoprotecting group; SS: di thiol serinol modifications

[0136] Reagents. Primer sequences were purchased from Integrated DNA Technologies and further gel purified by 20% urea denaturing PAGE gel electrophoresis. All DNAzyme sequences were synthesized at UT Austin using standard phosphoramidite chemistry on an Applied Biosystems 392 DNA / RNA synthesizer. HepG2 lines were purchased from American Type Culture Collection. Fresh human PBMCs were purchased from StemCell Technologies. Water was MilliQ water for buffer preparation. Buffer reagents and chemicals were purchased from Sigma- Aldrich at the highest purity available. qPCR was performed with a BioRad instrument and SYBR Green 2X MasterMix. HepG2 libraries were prepared using the NEBNext Ultra II Directional cDNA synthesis kit. PBMC libraries were prepared using the 10X Genomics Chromium Single Cell 3’ Gene Expression kit and Chromium system. All sequencing was performed at UT Austin’s Next Generation Sequencing facility on a NovaSeq 6000 SP, paired end 150, with prior sample quality checks through Bioanalyzer for RNA purification and library preparation.

[0137] Cell Lines. HepG2 cells were cultured in DMEM (Corning) with 10% fetal bovine serum (Corning) with 100 U / mL penicillin-streptomycin (Gibco). All cells wereincubated at 37°C in 5% CO2. PBMCs were thawed in RPMI (Coming) with 10% FBS (Coming). PBMCs were freshly thawed and directly used in each experiment.

[0138] Light irradiation. Light irradiation was performed with an Asahi Xe lamp with band filters of 490 nm (band width 10 nm) from Thorlabs. Sample lids were removed prior to light exposure.

[0139] DNA synthesis. All DNAzymes were synthesized in house. Synthesis carried out using standard solid phase phosphoramidite chemistry on an Applied Biosciences 392 DNA / RNA synthesizer on a 1 pM scale, with Pac-dA-CEP, Pac-dG-CEP, dT-CEP, dC-CEP, dithiol serinol phosphoramidite, 0.40 M tetrazole in acetonitrile, 5% phenoxyacetic anhydride in THF, 16% 1 -methylimidazole in THF, 3% dichloroacetic acid in dichloromethane, and 0.02 M iodine in THF / pyridine / water from Glen Research. The photocaged nitrobenzyl phosphoramidte was synthesized as previously reported2. Photocaged monomers were reacted for 15 minute coupling times, while all standard monomers used 25 second coupling times. DNA was purified through reverse-phase HPLC (XBridge Prep Cl 8, 19 X 250 pm column with Agilent 1260 Infinity II). A gradient of 5% acetonitrile in 95% triethylammonium acetate, pH 7.0 to 60% acetonitrile in 40% triethylammonium acetate over 20 minutes was used for DMT-on purification. Samples were concentrated in butanol. DMT was deprotected with neat acetic acid for 30 minutes at room temperature. DNA was purified through ethanol precipitation, with masses verified by MALDI-TOF.

[0140] qRT-PCR information and protocol. qRT-PCR was initially used to screen if different DNAzyme cleavage amounts with different metal ion concentrations could be distinguished post amplification with DNAzyme-specific primers. The cleavage reaction was carried out by incubating 500 ng of the ssDNAzyme containing rA with 20 pl of reaction buffer containing 1.5 mM MgCh, 50 mM KC1, 10 mM sodium citrate, 105 mM NaCl, 50 mM BisTris (pH 7.0) over 1 h. 5 nmol was then incubated with 10X PNK buffer A (5 pL, 500 mM Tris-Hcl, 10 mM MgCh, 50 mM DTT), ATP (5 pL, 10 mM), and T4 PNK (5 uL, 10 U / pL) at 37°C for 30 min and then heat inactivated for 30 minutes at 65 °C. cDNA was made with the iScript kit (Bio-Rad), in which a 2X master mix was used with 2 pL dilution from the phosphorylation reaction. The master mix included dT primers, random primers, dNTPs, and reverse transcriptase. Post cDNA construction, 2 pL from this reaction was added to a 96 well plate with 10 pM forward primer (either corresponding to full length uncleaved DNAzyme or the cleaved DNAzyme), 10 pM reverse primer (dT oligo), nuclease free water, and 2X SYBRGreen master mix (Thermo Fisher). The plate was sealed, centrifuged down, and then thermal cycled at 95°C for 10 min, 95°C for 15 seconds, 49°C for 1 minute, 72°C for 30 seconds, with this process repeated for a total of 40 cycles. Controls of no template and point mutated DNAzyme were used, and experiments were performed in triplicate. Post amplification, 20 pL of each well (the whole well sample) was run on a 20% denaturing PAGE gel compared to 2 pL of the cDNA pre-amplification.

[0141] Monocultured cell delivery. HepG2 cells were plated in Corning 6 well plates the day before delivery to prevent large clump formation. The DNA has essentially no delivery to deep intracellular regions within these large clumps. All plates used were pre-TC treated, individually wrapped, and sterilized by the manufacturer. 400 nM of functional-DNA was transfected into cells in serum-free DMEMCells were incubated with the probe for 1 hour in the cell culture incubator. Before light activation, the media was replaced with fresh DMEM (10% FBS, 100 U / mL penstrep). Light activation was performed for 10 minutes. The cells were then incubated for an additional 1 hour in the incubator before downstream sample preparation.

[0142] RNA and DNA extraction protocol. The manufacturer’s protocol for TRIzol RNA and DNA purification was followed. Briefly, 0.4 mL of TRIzol was directly added to the cell culture dish per well. The sample was pipetted up 10 times to homogenize the lysate, transferred to 1.5 mL RNase free Eppendorf tubes, and incubated at room temperature for 5 minutes. 80 p L of chloroform was added to each tube and incubated for 3 minutes. The samples were centrifuged for 15 minutes at 12000 g at 4°C. RNA in the aqueous phase was pipetted out and 200 pL isopropanol was added. The sample was centrifuged for 10 minutes at 12,000g at 4°C and washed with freshly prepared 75% ethanol. The resulting RNA pellet was then air dried for 10 minutes and dissolved in 20 pL RNase free water and incubated at 55°C for 10 minutes. For DNA extraction, 120 p L of freshly open 100% ethanol was added to the interphase and the sample was incubated for 3 minutes. The tube was centrifuged for 5 minutes at 2000 at 4°C. The resulting pellet was resuspended in 400 pL of 0.1 M sodium citrate in 10% ethanol, pH 8.5 and incubated at room temperature for 30 minutes. The sample was centrifuged for 5 minutes at 2000g at 4°C, with discarding of the supernatant. The pellet resuspension was repeated. The pellet was then resuspended in 800 pL of freshly prepared 75% ethanol and incubated at room temperature for 20 minutes. The sample was centrifuged for 5 minutes at 2000g at 4°C. The resulting pellet air dried for 10 minutes and resuspended in 300 pL nuclease free water. The sample was centrifuged for 10 minutes at 12,000g at 4°C and the supernatantwas transferred to a new 1.5 mL Eppendorf tube. The RNA and DNA samples were then combined. RNA / DNA amounts were independently checked with Qubit ssDNA and RNA broad sensitivity assays (Thermo Fisher).

[0143] mRNA and DNAzyme purification. To purify poly-A nucleic acids, 50 pL of dT-oligo magnetic beads (NEB) were resuspended and washed with 200 pL of lysis / binding buffer for 2 minutes (NEB). 10 ug of DNA / RNA was added to each bead tube and rotated at room temperature for 10 minutes. 250 pL of wash buffer 1 was added, the samples were vortexed vigorously, and rotated for 1 minute. Supernatant was removed and this was repeated one more time. 250 pL of wash buffer 2 was added, the samples were vortexed vigorously, and rotated for 1 minute. Supernatant was removed and this was repeated one more time. 250 pL of low salt buffer was added, the samples were vortexed vigorously, and rotated for 1 minute, and the supernatant was removed. 50 pL of the elution buffer was added and beads were vortexed vigorously. Samples were mixed in a Thermomixer (Fisher) at 50°C for 2 minutes at 500 rpm. Supernatant was kept in a RNase free Eppendorf tube. Purified RNA / DNA amounts were independently checked with Qubit ssDNA and RNA broad sensitivity assays.

[0144] Library preparation for monocultured experiments. NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) was used with 10 ng input RNA per sample post poly A enrichment. All manufacturer’s instructions were followed.

[0145] PBMC delivery and library preparation. Remove cryovials from storage and immediately thaw in the water bath at 37 °C until a tiny ice crystal remains without fully submerging the entire vial in the water bath. Using a wide-bore pipette tip, added 1 mL warm Complete Growth Media (RPMI with 10% FBS) to the cells in cryovial. The cells were resuspended with warm media to melt all remaining ice. The resuspended cells were then transferred to a 15 mL conical tube with 9 mL warm complete growth media. The cryovial was then rinsed with 1 mL warmed complete growth media and transferred the remaining cells to the 15 mL conical tube. The tube was centrifuged at 300 ref for 5 minutes at room temperature and resuspended the cells with 500 pL RPMI with a regular bore pipette to break up any clumps. The cells were counted and split the sample into 3 1.5 mL Eppendorf tubes at a concentration of 1000 cells / pL. Chilled PBS + 0.04% BSA was then added for a total of 1 mL per tube and centrifuged the tubes at 300 ref for 5 minutes at 4°C. The supernatant was removed and the cell pellet was resuspended in 50 pL RPMI. For the no DNA control, the total volume was brought up to 100 pL in RPMI. For the point DNAzyme control and active DNAzymegroup, the DNA was added to a final concentration of 400 nM from 1-2 pM stocks. All final volumes were brought to 100 pL using RPMI. The mixtures were gently pipetted 10X with the pipette set to 90 pL. The tubes were incubated in a cell culture incubator for 1 hour at 37 °C to allow the DNA to be internalized. The cells were then washed by transferring the cells to 15 mL conical tubes and adding 3.5 mL chilled PBS + 0.04 % BSA. The sample were centrifuged at 4°C for 5 min, 400 ref and then the cell pellets were resuspended in 100 pl room temperature PBS. After transferring the cells to a new 15-mL tube, they incubated for 5 min at room temperature. The light activation protocol was then followed. The tubes were incubated in a cell culture incubator for 1 hour at 37°C to allow the DNAzyme to cleave. The cells were then washed by transferring the cells to 15 mL conical tubes and adding 3.5 mL chilled PBS + 0.04 % BSA. The samples were centrifuged at 4°C for 5 min, 400 ref and then the cell pellets were resuspended in 100 pl room temperature PBS. For the second wash, the cells were resuspended in 3.5 ml chilled PBS + 1% BSA and centrifuged at 4°C for 5 min, 400 ref. For the third and fourth wash, the second wash was repeated twice more. The cells were then diluted in PBS + 1% BSA (starting point 100 pL) until concentrations of 1000 cells / pL were reached without any cell clumps, as visualized through microscopy. Cells were required to be above 90% viable before then following the manufacturer’s instructions for 10X Chromium Single Cell 3’ Gene Expression library preparation (performed by UT Austin’s Genomic Sequencing and Analysis Facility).

[0146] Data processing and clean up through bioinformatics. The quality of data was assessed by FastQC, with adaptor clean up, mapped genes, differentially expressed genes normalized and identified, DNAzyme read counts, and final standard data visualizations (expression heatmaps, Volcano plots, etc) were prepared. All scripts to generate data and raw data are deposited on GitHub.

[0147] Verification of metal ion amounts with ICP-MS. For ICP-MS analyses, 10 ml culture was pelleted and digested in 100 pL nitric acid (69%, ARISTAR grade, VWR International) spiked with 345 p.p.b. indium (VWR International) at room temperature (~20 °C) for ~3 h. Samples were then diluted to a final concentration of 2% v / v nitric acid and 10 p.p.b. indium before analysis. Serial dilutions of ICP-Multi-element solution IV (Merck, Certipur) were used for calibration of all the metals analysed and to check for instrument drift. A standard reference material, SRM1643e (National Institute of Standards and Technology), was analyzed to validate the calibration. Indium was used to correct for dilution errorsintroduced during handling. ICP-MS data reported are based on three replicate flasks, each sampled every time point n = 3). Results presented were verified in a replicate experiment, and outliers were excluded if they were >2 s.d. from the mean.Example 3 - Validations qRT-PCR validation to test amplification of cleaved and noncleaved DNAzyme

[0148] To perform in vitro validation of the modified Na+-DNAzyme activity, its response was measured with different concentrations of biologically relevant sodium, and adaptability with sequencing preparation. After incubating the point mutation control sequence and the active DNAzyme in a range of intracellular sodium concentrations, it was found through urea denaturing PAGE gel analysis that the active DNAzyme cleavage yields proportionally responded in amounts of cellular sodium levels, while the point mutation did not. Then qRT-PCR of these reactions was performed to measure the ability of the cleaved DNAzymes to amplify under NGS like conditions. It was found that the limit of quantification (Cq) allowed for the identification of the cleavage ratios of the DNAzyme even after amplification using two DNAzyme-specific forward primer and generalized dT reverse primer. Briefly, the samples were split into two separate forward primer reactions. One forward primer read the full length DNAzyme, while the second forward primer read the cleaved region. The cleaved length primer amplified both the full length and cleaved DNAzyme, while the full length primer only amplified unreacted DNAzyme. This corresponding Cqvalues proportionally decreased with increasing sodium levels, indicating the cleavage ratio trend remained intact even after amplification. Meanwhile, the point mutation control did not display any changes in Cqvalues. Controls of no template and primer dimer did not show significant amplification. This indicated the modified DNAzyme could also report relative metal levels through NGS. Results are shown in FIGS. 1A-C.DNAzyme sensor validation in response to potential signal artifacts

[0149] To confirm the DNAzyme was sodium-specific and not reacting with other metal ions in the sample preparation buffer or under high ionic salt intracellular conditions, the DNAzyme cleavage was tested in an intracellular mimicking high ionic strength buffer containing 15 mM sodium chloride, 150 mM potassium chloride, 2 mM magnesium chloride, and 0.1 mM calcium chloride. No additional cleavage was found for either the point mutation control or active sensor in a denaturing gel (FIG. 2A). To test if these positive results extendedeven after cDNA formation and DNAzyme exponential amplification, qRT-PCR was performed using DNAzyme specific primers. After amplifying the DNAzymes, Cqvalues did not change from low ionic strength buffer (FIG. 2B).Measuring intracellular Na+-DNAzyme response through NGS

[0150] Because extracellular sodium is ~10X higher than intracellular sodium, the DNAzymes were photocaged with a nitrobenzyl protecting group to prevent premature cleavage. It was verified that the photoprotecting group prevented DNAzyme activity and optimized activity restoration after decaging (FIG. 5). Cell studies were done next to evaluate if the DNAzyme cleavage ratio could respond to salt modulation in monocultured cells. HepG2 cells were stimulated with 10 mM sodium chloride and 20 mM sodium chloride over 2 days. No modulation was used as a control corresponding to 0 mM extra sodium added. To test if intracellular sodium levels increased in response to additional sodium in the cell media, Sodium Green™ (a commercially available sodium sensor) was used to image the intracellular sodium levels. A clear increase was found in Sodium Green™’ s fluorescence intensity with increased sodium in the cell media (FIG. 3A). With verification the stimulated sodium resulted in increased intracellular sodium, the photocaged sensors were delivered into the stimulated cells. After cell internalization and washing to remove any DNAzyme remaining in the buffer, the DNAzymes were uncaged and allowed to cleave in response to intracellular sodium. The mRNA and DNAzyme was then extracted with dT-oligo magnetic beads, and prepared sequencing libraries with the polyA-enriched samples. To understand the effects of intracellular conditions to buffer, control groups were used of the point and active sequences in ~20 mM sodium in buffer and prepared these samples for NGS sequencing as well. It was found that through the number of reads for the full length versus cleaved DNAzyme, a 92% cleavage ratio for the 20 mM sodium sample, 87% cleavage ratio for the 10 mM sodium sample, and 84% cleavage for the no sodium added sample could be relatively read (FIG. 3B). Additionally, the point mutation remained consistent in all samples at background cleavage values of 75-78% (FIG. 3B). When normalizing the background cleavage from each groups’ active sensor cleavage, proportional increases in DNAzyme cleavage yields were found in response to modulated intracellular sodium levels (FIG. 3B). This indicated the DNAzyme could accurately report on different cell types intracellular sodium levels in future single cell experiments. After gene expression normalization for sequencing depth and gene length in different samples (FIG. 3C), each sample was visualized for differentially expressed genes(FIG. 3D, FIG. 3E). Several differentially expressed genes were identified in response to sodium modulation, indicating that different intracellular sodium levels have effects on gene expression. These positive results of sensitive DNAzyme cleavage ratio read outs through NGS and the effect of sodium on gene expression confirmed that DNAzyme-seq was suitable to advance to single cell experiments.

[0151] With the evidence that the DNAzyme cleavage ratio read out through NGS accurately represented intracellular sodium levels, the platform was next applied in single cell RNA sequencing. For the single cell experiments, the effects of different endogenous sodium levels was quantified in PBMCs because high sodium diets in mice have been found to initiate helper T cell induction, but these experiments did not report on effects in different PBMC cell types. These reports also served as literature background to compare the accuracy of the findings to previous literature examples. For the DNAzyme delivery into the mixed cell types of the PBMCs, dithiol modifications were added to the 3’ region of the DNAzyme. Through thiol-mediated uptake, the DNAzymes were delivered into all cell types containing cell membrane cysteines. After delivery, the DNAzyme were decaged and allowed to react for 1 hour in the PBMC samples. The point and active sequences were delivered into separate samples to prevent cytotoxity due to excess DNA in one sample. For the single cell barcoding, equal numbers of cells from both samples were mixed together. Library preparation then immediately commenced.

[0152] To identify gene expression correlation with DNAzyme activity, similar single cells were first visualized in a t-SNE plot, based upon their gene expression profiles (FIG. 4A). To identify the delivery efficiencies for both the active DNAzyme and the point mutation control, the library was searched with barcodes covering the point mutation site. These sequences were called “total” because they are present in both full length unreacted DNAzyme and cleaved DNAzyme. Visualization of the log2 normalized number of unique molecular identifiers for active (FIG. 4B) and point mutation (FIG. 4C) showed similar delivery efficiencies throughout the heterogenous samples. Importantly, cells with the point mutation sequence did not overlap with cells with the active DNAzyme sequence, indicating successful single cell and base resolution. Verification that these sequences were not present in the PBMC control library demonstrated they were accurate reads of the artificial DNAzyme. To identify the unreacted DNAzymes, the library was searched for the “uncleaved” DNAzyme, encompassing the 5’ starting point of the DNAzyme to the cleavage site. This was the samesequence for both the point mutation and the active DNAzyme. Mapping of the log2 normalized unique molecular identifier counts for the uncleaved DNAzymes showed stronger overlap of the full length, unreacted sequence with the point mutation compared to the active sensor (FIG. 4D). To account for different delivery efficiencies to different cells, the uncleaved reads were subtracted from the total active reads and divided the sum by the total active reads per cell. For the active DNAzyme, 0% cleavage was grouped into no cleavage, 1-49% cleavage was grouped into low cleavage, 50-99% cleavage was grouped into high cleavage, and 100% was grouped into 100% cleavage. By overlaying the cleavage ratios onto the total t-SNE plot, relative sodium levels were visualized in the heterogenous samples (FIG. 4E). Importantly, higher levels of 100% cleaved DNAzyme was noted in T helper cells and monocytes. Significantly enriched differentially expressed genes correlated was compared with each cleavage cluster and a mixture of known and putative sodium-related genes were identified, with varying degrees of known interactions between each other (FIG. 4F). When plotting these significantly enriched differentially expressed genes in a heat map based upon each cluster, it was found major gene clusters that correlated with DNAzyme cleavage ratio, potentially indicating correlation with sodium levels as well.

[0153] To validate the relationship of sodium with unknown sodium-related genes, a series of key experiments will be performed. First, sodium levels will be modulated in THP1 monocyte cells and evaluate several identified gene expression levels through qRT-PCR. Second, the effects of differential sodium levels on protein expression will be evaluated through immunofluorescence and Western blots. Third, sodium’s effect on interacting proteins will be evaluated to determine if differential sodium levels affect interacting cell networks. If these are successful, differential sodium levels will be verified through ICP-MS and experiments on sodium’s relationships to gene and protein production in isolated primary cells will be repeated.

[0154] Two limitations with using synthetic nucleic acids in cells are the cellular immune response and sensor degradation. The cytosolic accumulation of DNA results in innate immune response. To account for this, a no DNA control was used and changes in gene expression profiles were compared to those sequenced in the point mutation control sample and the active DNAzyme sample. DNA modifications, such as with locked nucleic acid bases and phosphorothioate backbones, do not interfere with sensor activity and limit DNA degradation by decreasing recognition from DNases in the cell (Kauppinen et al. , 2005; Crooke et al., 2020). However, it should be noted that DNA faces less degradation than RNA, and theuse of guideRNAs in sequencing has been widely reported already with discussion on this limitation (Zhu et al., 2018; Wang et al., 2020; Allen et al. , 2021; Anderegg et al., 1998). Because the sequencing output has single base resolution, true signal could be determined compared to false background by the sequencing read of the correct full length versus cleaved sensor.Example 4 - DNAzyme-Seq: Direct Correlation of Metal Ions with Transcriptomics in Single Cells using DNAzyme-based SequencingSummary of methods for optimization:

[0155] While generating this method, the DNAzyme design was redesigned to ensure applicability toward intracellular metal concentrations, reagents for amplification, and methods for amplification. For the initial DNAzyme design, the single- stranded DNAzyme was first developed by joining together the double stranded enzyme strand and substrate strand with flexible polyT linkers. A terminal poly- A tail was also added for extraction with mRNA, so the DNAzyme had two repetitive sequences (polyT and poly A). This approach was chosen initially because double stranded DNAzymes have been optimized to eliminate bases nonessential for activity, which ultimately increases synthesis yield. It was found that the polyT linkers did not alter the DNAzyme cleavage abilities because their flexibilities still allowed the DNAzyme to form its optimal structure and cleave in response to the metal of interest. However, the polyT linkers inhibited successful amplification of the intact and cleaved DNAzymes. During the methods for amplification and read-out, the single stranded DNAzyme must first be converted to its complementary strand. This is to integrate the DNAzyme with RNA-sequencing, in which mRNA is converted to cDNA during sample preparation, as cDNA is more stable than RNA and conversion to cDNA eliminates unwanted portions of RNA sequences. During DNAzyme cDNA formation, two additional repetitive sequences were generated: a poly-A linker and a polyT tail.

[0156] When first attempting to amplify the DNAzyme, no amplification occurred (amplified 8-17 point zinc with cleaved length primer ). This was because high primer annealing temperatures were used for enhanced primer binding specificity to the DNAzyme. However, at these temperatures, the primers could not selectively identify between the similar sequences within the DNAzyme. After decreasing the primer annealing temperature, multiple nonspecific amplification bands were found. While traditionally, these multiple unspecific bands can result from off-target amplification, these unspecific amplifications were distinct anddue to the four repetitive polyT and poly-A sequences. These repetitive sequences interfered with primer recognition and base addition during amplification.

[0157] To eliminate two of the repetitive sequences, the original selected singlestranded form of the DNAzyme was chosen and only extended the 3’ end of the DNAzyme with a poly-A tail. While these original sequences do contain nonessential bases, using these original strands eliminated having to completely redesign the sensor (FIG. 17). When trying the amplification with DNAzyme specific primers for the forward reaction and a polyT primer for the reverse reaction, less off-target amplification was observed, but still failed to achieve significant amplification corresponding to the exact full length and cleaved DNAzymes and still observed nonspecific amplification (FIG. 18).

[0158] To address the presence of nonspecific amplification, one additional DNAzyme specific base was added to the reverse polyT primer. With all primers, annealing temperature was optimized, ultimately reaching 49°C as the optimal annealing temperature for both primers. While this temperature allowed for some amplification, the amplification did not reach the ideal exponential levels of amplification. (FIGS. 19-20)

[0159] With the optimized DNAzyme sequence, primer design, and primer conditions, specific exponential amplification was not observed. The inventors next worked to address this failure. It was hypothesized the nmol levels of DNAzyme being utilized were too unsimilar to the pg levels of mRNA concentrations found normally. To determine if excess DNAzyme was inhibiting the amplification reagents and enzymes, the cDNAzymes were diluted to 1 pg (1 pg for 10 uL total reaction) before amplification. The combination of the enhanced DNAzyme sequence, primer sequence, and amplification methods allowed for successful exponential amplification. When extracting full length and cleaved DNAzyme detection limits from the amplification data, it was found they corresponded to the sodium amounts used for cleavage (e.g., qPCR was performed and displayed varying sodium cleavage rates). To determine that these cleavage ratios were not artifacts of incorrect DNAzyme amplification, the sequences were checked for amplification specificity. Only one product was found corresponding to the full length sequence and one product corresponding to the cleaved sequence, both of which were at the correct molecular weight (e.g., qPCR was performed and displayed varying sodium cleavage rates). These results indicate that specific DNAzyme amplification has occurred.Example 5 - Spatial DNAzyme-Seq: spatially correlating endogenous metal ion abundance with the transcriptome by reading DNAzyme cleavage

[0160] Metal ions are critical in cellular function and health as they play a significant role in basal cellular processes such as enzymatic catalysis, structural stabilization, and gene regulation. More specifically, it is well-studied that metal ions can influence mRNA populations through stability and translation. For example, iron can regulate mRNA translation via iron-responsive elements and iron-responsive proteins. Additionally, magnesium ions play a critical role in RNA splicing, a critical step in mRNA maturation, through structural stabilization. Additionally, metal ions are known to be distributed heterogeneously throughout a cell. Areas containing high metal ion content are known as metal ion pools . These pools can change depending on the health state of the cells. For example, in cancer cell models, metals are significantly increased to meet the metabolic demands of rapidly growing cells; in disease models such as multiple sclerosis, sodium accumulates, which can be correlated to high sodium diets. Even though there are strong validations that metal ions can influence gene expression, there is no one method to spatially correlate mRNA and endogenous metal ions in cells or tissue samples. Methods investigating the influence of metal ions on gene expression, such as Microarray analysis, compare the gene expression profiles of healthy samples to samples supplemented with metals. Although these methods have yielded indispensable insight into how metals can modulate gene expression, adding exogenous metal can affect the natural biological processes inside the cell and thus is not an accurate depiction of the metal functions.

[0161] To overcome these limitations, spatial DNAzyme-Seq has been developed as described herein that combines DNAzymes and standard spatial transcriptomic methods to quantify metal ion abundances in a three-dimensional manner. DNAzymes are single-turnover catalytic nucleotides that can perform a phosphodiester cleavage reaction in the presence of specific metal ions. DNAzymes are highly generalizable and selective as they are obtained through in vitro selection. For example, K+, Mg2+, Li+, Fe2+, Fe3+, and Na-i- specific DNAzymes have already been selected and characterized. Since DNAzymes cleave proportionally to metal ion concentrations, metal abundances were semi-quantified by calculating a ratio between cleaved and total DNAzyme. As such, a high ratio between cleaved and total DNAzyme signifies high metal ion abundance and a low ratio signifies low metal abundances.

[0162] Spatial DNAzyme-Seq builds upon DNAzyme metal ion cleavage and in situ hybridization and sequencing technologies to identify cleaved and total DNAzyme and spatially resolve metal ion abundances in cells. Spatial DNAzyme-Seq, in combination with mRNA in situ hybridization and sequencing technologies, can allow one to identify and quantify endogenous metal ion pools and correlate it with the transcriptome, simultaneously removing any biases that might arise from a two-method correlation. Other methods to identify and quantify metal ions, such as ICP-MS, radiolabeling, and genetically encoded sensors, cannot be readily combined with spatial transcriptomic methods. Additionally, since DNAzymes can react with endogenous levels of metal ions, any influence can be removed that might arise from adding exogenous metal ions, giving a more direct readout of how metals influence gene expression. To perform spatial DNAzyme-seq, a sodium- specific cis-DNAzyme was modified with a unique barcode at the three prime end that can be targeted by specific amplification of nucleic acids via intramolecular ligation (SNAIL) probes containing a sequence identifier (FIG. 7A). SNAIL probes are commonly used to spatially identify and sequence mRNA transcripts in mammalian cells and brain tissue (FIG. 7A). To obtain a ratio between total and cleaved DNAzyme, a pair of SNAIL probes was designed to target 1) the additional barcode of the DNAzyme (to read total) and 2) the bases released post DNAzyme cleavage reaction (to read cleaved)(FIG. 7B). SNAIL probes designed to read the bases released post-cleavage reaction cannot hybridize with uncleaved DNAzymes (FIG. 7B). Once bound to its DNAzyme target, the SNAIL probes, with sequence identifiers, can be circularized and amplified in situ (FIG. 7B). The amplification of the unique sequence identifiers provides a high signal-to-noise ratio, which can expand the usage of this method in tissue imaging for cell-specific metal ion mapping. The location of the total and cleaved DNAzyme is recorded using sequence by hybridization chemistry (SHC) that can target the unique sequence identifiers on the DNA amplicons (FIG. 7B). Unique mRNA targets can be simultaneously identified by striping DNAzyme- specific probes after identifying the metal ion pool and adding mRNA-specific probes. Additionally, through sequential rounds of probing, imaging, and stripping, a three-dimensional map of both metal ion abundance and the transcriptome can be obtained (FIG. 7C).Results and Discussion

[0163] Design and characterization of barcoded DNAzymes. To target DNAzymes with SNAIL probes, a 40-base barcode was integrated into the three prime ends of the sodiumspecific DNAzyme. As a negative control, an inactive sequence containing a point mutationwas also included that obliterates the cleavage reaction of the DNAzyme. This negative control, called inactive DNAzyme (iE), is critical as it accounts for any background ligation that might arise from the DNAzyme's degradation inside the cell. Gel-based activity assays were done to verify that the activity of the sodium-specific DNAzyme was not compromised by the addition of the 40-base barcode to the 3 prime ends. The results show that the DNAzyme cleavage is sodium-dependent as more sodium yields a higher cleavage product (lower band in FIG. 8A). In addition, the cleavage happens only in the presence of sodium, and no other biologically abundant metals, such as potassium and magnesium, can promote this reaction (FIG. 8B). Additionally, when a point mutation is added, the sodium DNAzyme is inactivated and cannot perform the cleavage reaction.

[0164] Sodium-specific in vitro ligation and amplification of SNAIL probes. To test the compatibility of DNAzymes with SNAIL probes and the capability of SNAIL probes to ligate only in the presence of sodium in vitro ligation assay was run. The cellular environment was mimicked by performing the DNAzyme cleavage reaction. Then the SNAIL probes were hybridized and added. Optimizations were conducted to allow high target specificity and minimize background ligation. Once hybridized, T4 DNA ligase enzyme was added to the DNAzyme and SNAIL probes mixture. The ligation reaction was monitored through denaturing PAGE as ligation yields a circularized product characterized by a mobility shift towards a heavier weight (shift up.) From the ligation assay, it was observed that 1) SNAIL probes identify the total barcoded DNAzyme, and its ligation is not affected by the presence of sodium ions, and 2) SNAIL probes reading the bases released upon cleavage can only ligate in the presence of sodium (FIG. 7C). Although some background ligation is still seen without the addition of sodium, it was removed by subtracting the background seen in the inactive DNAzyme control. This background ligation could be attributed to DNA degradation or a strand displacement reaction displacing the uncleaved product with the SNAIL probes. To counteract this phenomenon, the SNAIL probe's binding region was optimized to the bases released upon cleavage.

[0165] An amplification reaction was conducted to further verify sodium-specific ligation by taking the ligated samples and incubating them with Phi29 DNA polymerase. The formation of an RCA amplicon can be seen through a 1.2% agarose gel. When an amplicon is present, its size will be too large to traverse through the gel matrix and be seen stuck inside the gel wells. This amplification reaction verified the ligation only in the presence of sodium whenthe SNAIL probes read the bases released upon DNAzyme cleavage (FIG. 8C). Both in vitro ligation and amplification reactions demonstrate the capability of SNAIL probes to read DNAzyme cleavage and the adaptability of DNAzymes to spatial transcriptomic methods.

[0166] Cellular imaging of metal ion pools using spatial DNAzyme -seq. Yielding promising results in the in vitro studies, cellular studies were conducted, where a sodiumspecific photocaged barcoded DNAzyme was delivered into live SH-SY5Y cells using commercially available transfection agents. Following the delivery, the cells were fixed and permeabilized according to previous protocols. Afterwards, the DNAzyme specific SNAIL probes were hybridized, ligated, and amplified in situ. Following that, bridge probes and detection probes containing fluorophores were incubated with samples to detect the amplicons generated from detecting the DNAzyme inside cells. Some of the concern going into cells was signal saturation. However, based on the imaging, clear dotted signals were seen that have minimal overlap. To total DNAzyme delivered was read by Cy5-detection probes and the cleaved DNAzyme with a Cy3-detection probe. As a positive control to verify that the method could detect nucleic acids in cells, an abundantly present mRNA transcript encoding for actin was imaged. The images show that high levels of pink dots depict high sodium pools, and the presence of only blue dots depict low sodium ion pools. The results strongly indicate that DNAzymes, combined with SNAIL probes, can identify metal ion pools within a biological environment.Methods

[0167] Barcode and probe design. Two unique 40-base barcodes with minimal secondary structures and interactions with the sodium DNAzyme were designed using UNAfold. One barcode was added to the three prime end of the active sodium DNAzyme, and the other was added to the inactive sodium DNAzyme at the same position. Each barcode had a 40-60% GC content, and it was confirmed that there were no homologous regions in other biological regions by blasting against the human genome. SNAIL probes composed of padlock probes (PLPs) and primer pairs were designed to read total and cleaved DNAzyme. To read metal ion abundances, (1) the barcodes were split into two equal parts of 19 bases; the 5' halves had complementarity with the PLPs and the 3' to the primer with two bases in between. The Complementarity region for both was designed to have a melting temperature of around 60°C. (2) The padlock probe contains a target sequence for specific bridge probes, which can facilitate the hybridization of the detector probe; this follows the principles of HYBISS. (3) To readDNAzyme cleavage, SNAIL probes were designed to bind to a partial section of the barcode in addition to the bases released after the cleavage reaction. (4) four SNAIL probes were designed to read DNAzyme cleavage and endogenous probe degradation. Two probes to read total DNAzyme delivered (targeting only the barcode sequence) and two to read cleavage (targeting only the bases released upon cleavage reaction.) All SNAIL probes were manufactured by Integrated DNA Technologies (IDT) and purified using PAGE in-house.

[0168] Synthesis of barcoded DNAzymes (BC-DNAzymes). A T4-mediated ligation reaction was performed to synthesize the inactive and active sodium-specific cis-DNAzyme. The reaction components included strand A, a five prime phosphate modified strand B, and a 30-base splint complementary to both strands A and B. The 30-base splint is necessary to increase the T4 ligation yield. Optimization was conducted to avoid forming side products that arise from a self- ligation event that occurs with strand B. To obtain the highest yield, a ratio of 4:1:1 strand A: strand B: splint where hybridized by heating to 95 °C for 5 minutes and cooled to room temperature for 15 minutes. Afterward, 5uL of T4 buffer and 5uL of T4 ligase enzyme were added to the strand solution to obtain a final volume of 50uL, and the ligation reaction was left to proceed at 16 °C for 16 hours. The final product was purified through a 10% denaturing PAGE. To avoid premature cleavage during the purification steps, buffers containing sodium salts were excluded and replaced with lithium chloride buffers.

[0169] Synthesis of photocaged barcoded DNAzymes (PC BC-DNAzymes). To synthesize the inactive and active sodium-specific PC-BC-DNAzymes, an ABI 392 solid-phase DNA synthesizer was utilized. Due to the large size of the strands (<100 bases) the synthesis was started with Glen UnySupport™ 2000 controlled-pore grass (CPGs) beads. The photocaging group was synthesized in-house using the previously published method and was added to the PC-BC-DNAzymes during its synthesis using a 15-minute coupling time. All syntheses were conducted at a Ipmol scale. The DNA was cleaved from the CPG beads with a 48-hour long 30% ammonium hydroxide treatment. A 20% denaturing polyacrylamide gel electrophoresis (PAGE) was run to purify the PC-BC-DNAzymes. All steps after the addition of the photocaging group were done in the dark, including the PAGE purification, to avoid premature activation of the DNAzyme. Similarly to the synthesis of the BC-DNAzymes, buffers containing sodium were avoided and replaced with lithium chloride-containing buffers.

[0170] Characterization of BC-DNAzyme. luM starting concentration of the synthesized DNAzymes (active and inactive sequences) in lOpL of 150mM TRIS wasprepared. To validate the activity of the sequences, the active and inactive sequences were incubated with no sodium, 10 mM sodium, 20 mM sodium, and 40 mM Na. The samples were incubated for 1 hour at 37C. After incubation, 200 nM were taken from the starting reaction and diluted with 2x denaturing loading dye. Samples were heated to 95 °C for 15 minutes before hot-loading into a large 10% denaturing PAGE gel. The gel ran for 45 minutes before staining with xl Sybr Au and imaging in the bio-rad imaging doc.

[0171] In vitro SNAIL probe ligation and amplification to read DNAzyme cleavage. The solution left over from the reaction carried out to characterize the DNAzyme's activity was split into two equal parts of 4uL. In one half, the on-target SNAIL probes were added, and in the other half, off-target SNAIL probes were added. The ratio between padlock probes, primer, and DNAzyme was 1:10:1, respectively. The off-target probes were added as a negative control to visualize any off-target ligation. The SNAIL probes were hybridized to the DNAzymes by heating the solutions to 95 °C for 5 minutes and cooling them to room temperature for 15 minutes. Afterward, 0.5 U / uL of T4 ligase, 1 uL of 10X T4 ligation buffer, and 0.2 mg ml BSA were added to the mixture and were left to incubate at room temperature in the dark for 45 minutes. To characterize the ligation of the PLPs, 200nmoles of the DNA were taken from the sample and mixed with x2 denaturing loading dye, heated to 95 °C for f5 minutes, and hot-loaded to a large prewarmed 10% denaturing PAGE gel. The gel was run for 45 minutes at 26 watts before staining it with SYBR Au and imaging it in a bio-rad gel doc imager. To perform the in vitro amplification reaction, the leftover solution of the ligation reaction was taken, and 0.2 U / uL Phi29 DNA polymerase, IX Phi29 reaction buffer, 250 p M dNTP mixture, and 0.2 mg ml-1 BSA were added. To visualize the formation of the amplicon, a 1.2% agarose gel with SYBR Au stain was run for 45 minutes at 120 volts. After, the gel was imaged using a bio-rad gel doc imager.

[0172] Delivery of PC-BC-DNAzymes into SY5Y cells. SY5Y cells were cultures in Poly-L lysing-coated 35mm imaging dishes at a 0.3 x 106 cell density. After reaching confluency, 200nM of PC-BC-DNAzyme was delivered using turbofect per manufacturer instructions for 4 hours. Afterward, the cells were washed with xl PBS three times, and fresh options-mem was added; immediately after replacing media, the cells were exposed to 365nm light for 30 minutes to activate the DNAzymes. Once the light treatment was over, the cells were incubated for an additional hour at 37°C.

[0173] Sample fixation and permeabilization. After the additional hour of incubation after DNAzyme delivery, the cells were fixed using 4% formaldehyde in xl Lithium base TRIS buffered solution for 15 minutes. After this step, keeping all washing and incubation steps sodium-free is crucial as the DNAzyme can react with exogenous sodium added after decaging. The fixation solution was removed, and immediately after ImL of ice cold, 100% methanol was added and incubated overnight. Samples were then quenched by adding 200pL of quenching solution (Img / mL yeast tRNA, 100 mM glycine, 0.1% tween 20, in xl Lithium TRIS buffered solution) and incubating for 10 minutes.

[0174] SNAIL probe hybridization, ligation, amplification, and fixation. Cells were then washed with xl Lithium base TRIS buffered solution 3 times before incubating with hybridization buffer solution (lOOmM Tris, 300mM LiCl, 10% formamide, 20mM ribonucleoside vanadyl complex, O.lmg / mL yeast tRNA, 0.1% tween-20, lOnM of PLPs, and lOOnM of primers) at 40°C for 12 hours in a humidifier over with a parafilm wrap. Samples were then washed twice with 0.1% Tween-20 in in xl Lithium base TRIS buffered solution for 20 minutes at 37°C and once with high salt washing buffer (lOOmM Tris and 300mM LiCl in xl Lithium base TRIS buffered solution) for also 20 minutes at 37°C. The solution was then removed, and one more rinse with 300p L xl Lithium base TRIS buffered solution was performed before adding the ligation mixture (0.25u / pL T4 DNA ligase, 0.5mg / mL BSA, xl T4 ligase buffer) and incubating for 2 hours with gentle shaking in the dark. After ligation, samples were washed twice with xl Lithium base TRIS buffered solution before adding the amplification mixture (0.2 U pl-1 Phi29 DNA polymerase, lx Phi29 reaction buffer, 250 pM dNTP mixture, 0.2 mg ml-1 BSA, 20 pM 5-(3-aminoallyl)-dUTP)) and incubating for 2 hours at 37°C. After, samples were rinsed with xl Lithium base TRIS buffered solution and crosslinked using 20mM of Methacrylic acid N-hydroxy succinimide ester in lOOmM potassium carbonate for 1 hour at room temperature.

[0175] Gel embedding. After crosslinking, the samples were washed with xl Lithium base TRIS buffered solution and incubated with monomer buffer (4% acrylamide, 0.2% bisacrylamide, lOOmM TRIS, and 300mM LiCl) for 30 minutes. Samples were then incubated once more with monomer buffer, but now, they contained 0.1% ammonium persulfate and 0.1% tetramethylethylenediamine to promote gel polymerization. Once the gel was completely polymerized, the samples were ready for the next steps. After, a proteinase K treatment was done to digest cell-gel hybrids over the course of 2 hours.

[0176] Sequence by hybridization. Samples were washed with xl Lithium base TRIS buffered solution twice and incubated with bridge probe hybridization mixture (lOOmM TRIS, 300mM LiCl, and lOOnM bridge probes) overnight at 37 °C. After samples were washed with xl Lithium base TRIS buffered solution twice for 20 min at 37°C; they were later incubated with imaging probe solution(100mM TRIS, 300mM LiCl, and lOOnM imaging probe) overnight at 37°C. The samples were washed thrice with xl Lithium base TRIS buffered solution for 20 minutes each before imaging.

[0177] Imaging. Imaging was performed in the Zeiss Observer 7 (Zen 3.1 pro) using a 20x objective, 5% laser power, and 800 ms exposure time.

[0178] Described in this example is a method to resolve metal ion abundances with mRNA spatially. By utilizing a metal-specific DNAzyme and adding a unique barcode, DNAzyme cleavage can be effectively read and total DNAzyme delivered into cells using SNAIL probes. This system is shown to work in vitro by running a SNAIL probe ligation assay demonstrating sodium-specific ligation. The ability to identify metal ion pools in SH-SY5Y cells has also been demonstrated. This method can be adopted for other spatial sequencing techniques, such as Bar-seq, which utilizes similar padlock probes to sequence mRNA in brain tissue slices.Example 6 - Using DNAzymes to investigate endogenous metal ion pools and correlate with the transcriptome

[0179] The method described herein can measure endogenous levels of metals in cells, therefore, the role of metals can be investigated without the need of metal supplementation which can affect basal biological processes. Additionally, the method is highly generalizable and can be adapted to any DNA based senor including DNAzymes and DNA aptamers. Spatial transcriptomic probes (SNAIL probes) are utilized to read DNAzyme cleavage. By using SNAIL probes, metal ion can be implemented to visualize changes in metal ion pools in addition to genes that might influence these pools

[0180] FISSEQ, Padlock, PLAYR, and SNAIL probes are all methods for multiplexed RNA imaging with rolling circle amplification: “in comparison with FISSEQ and padlock probes, SNAIL probes have overcome the efficiency-limiting step of reverse transcription and greatly simplified the experimental procedure; while PLAYR requires four probes, one additional step and two ligation sites, SNAIL only requires a pair of probes and one ligationsite. (H) Boxplots of RNAs per cell of 151 cell type gene markers measured by single-cell RNA sequencing (scRNA-seq; 25) and STARmap (extracting from 160-gene mapping of visual cortex). Box: the first and third quartiles; middle line: median; whisker: 5% and 95% data points. P value, rank-sum test. (I) Summary of single-cell RNA sequencing and RCA-based multiplexed RNA detection methods, numbers were extracted from references”. All these methods can be used to detect DNAzyme clevage however SNAIL probes were elected herein because their efficiency in detecting mRNA targets is like sc-RNA-seq. (Lein et al. 2017; Ke et al. 2013; Lee et al. 2015; Larsson et al 2010)Results and Discussion

[0181] Sodium-specific in vitro ligation and amplification of SNAIL probes with only padlock probes. To test the compatibility of DNAzymes with only padlock probes (PLP) and not SNAIL probes (padlock probes and primers) in vitro ligation assay was run with optimized PLP’s. The DNAzyme cleavage reaction was first performed and then the PLP’s were hybridized and added. Optimizations were conducted to allow high target specificity and minimize background ligation. Once hybridized, T4 DNA ligase enzyme was added to the DNAzyme and SNAIL probes mixture. The ligation reaction was monitored through denaturing PAGE as ligation yields a circularized product characterized by a mobility shift towards a heavier weight (shift up.) From the ligation assay, it was observed that the ligation with only the PLP mitigated any off-target effects (FIG. 10).

[0182] Using SNAIL probes, initial experiments attempted to read the DNAzyme’ s point mutation on the catalytic loop. These experiments did not distinguish between active and inactive DNAzyme, and thus advantages may be gained from targeting SNAIL probes to regions outside of the DNAzyme catalytic loop. Additionally, the conformational change upon metal addition was observed to also inhibit the binding of SNAIL probes trying to read total DNAzyme.

[0183] BC Aptamer Design. To apply the same method to aptamers and identify metabolite abundance using spatial transcriptomic methods, a way that will allow for base release upon binding to the target was needed. To do this, a unimolecular aptamer as described herein was investigated. When target is detected the capture strand gets released through a strand displacement reaction and the bases on this capture strand can be targeted by SNAILprobes. To read total aptamer vs aptamer bound to the target, a barcode sequence was added in between the aptamer and capture strand instead of a linker.

[0184] To apply this concept to Aptamer an intramolecular strand displacement can be used.ATP- ASP: Ce6-CACCTGGGGGAGTATTGCGGAGGAAGGTT-(cl 8 spacer)3 -GT CCA GCA GCT GAG CTG CAA CGT GTC TGT CTGCCAGGTG-BHQ2. (SEQ ID NO:8)To identify the binding of aptamer, the bases released (underlined bases) can be targeted, and to target the entire aptamer a target sequence can be introduced in place of a PEG linker. The bold section is the barcode used to identify total aptamer.

[0185] Characterization and validation of a Na-specific sensor for delivery into plant roots To first validate that DNA can be delivered into plant roots, a short ssDNA was synthesized to validate the delivery using thiol-mediated uptake. 5 -day old seedlings were incubated with fluorophore-labelled DNA as a negative control, and disulfide-modified fluorophore-labelled DNA to verify thiol-mediated uptake. It was seen that the disulfide- modified DNA emitted a greater fluorescence signal compared to the negative control with a ~1.4 fold turn-on by calculating the integrated density divided by the ROI area (FIG. 11).

[0186] Fluorescence Characterization of Na-Specific DNAzyme activity and selectivity After validated the delivery of DNA into plant roots, a modified DNAzyme was synthesized with disulfides to determine its activity inside plant roots as well. The DNAzyme was characterized in vitro using fluorescence-based characterization to show the activity of the active sequence, as well as the obliteration of activity in the inactive sequence. It was seen that with increasing levels of NaCl, the active sequence emitted higher fluorescence signal compared to the negative control, and that the inactive sequence remained inactive (FIG. 12) . Furthermore, it was also validated that the sensor was only active in the presence of NaCl by testing the fluorescence turn-on in the presence of other physiologically relevant metals, MgCb and KC1. It was seen that there was only fluorescence increase in the presence of NaCl (FIG. 12).

[0187] Design and synthesis of modified DNAzyme for delivery into Arabidopsis roots To determine the activity of the modified disulfide-DNAzyme into plant roots, 5-day old seedlings were grown on Vi MS growth media supplemented with either 0 mM NaCl, 25 mMNaCl, or 50 mM NaCl. The point-mutation inactive control DNAzyme was also delivered to account for any degradation during the delivery process. In the results it is seen that there is a higher fold turn-on signal from the active DNAzyme sequence compared to the negative control, and that with seedlings grown on increasing levels of NaCl, there was a higher fold turn-on fluorescent signal (FIG. 13).

[0188] Plant hybridization-based targeted observation of gene expression map PROOF-OF-CONCEPT To validate that published data can be reproduced utilizing spatial transcriptomic principles in plant roots, one gene was targeted that is highly expressed in xylem cells in plant roots. The plant roots were fixed and permeabilized according to previous protocols. Afterwards, the gene specific SNAIL probes were hybridized, ligated, and amplified in situ. Following that, bridge probes and detection probes containing fluorophores were incubated with samples to detect the amplicons generated from detecting the gene target inside cells. By following the published protocol described in the methods below, a clear dot signal was seen representing the expression of the targeted gene (FIG. 14). The results were validated by cross-comparing with the published data in the Nobori et al. (2023).Methods

[0189] Optimization using only the padlock probe for barcoded detection and DNAzyme cleavage The in vitro ligation using only the PLPs was done in a similar fashion as the ligation with SNAIL probes using both PLP’s and primers. luM starting concentration of the synthesized DNAzymes (active and inactive sequences) in lOpL of 150mM TRIS was prepared. To validate the activity of the sequences, the active and inactive sequences were incubated with no sodium, 10 mM sodium, 20 mM sodium, and 40 mM Na. The samples were incubated for 1 hour at 37C. After incubation, 200 nM were taken from the starting reaction and diluted with 2x denaturing loading dye. Samples were heated to 95 °C for 15 minutes before hot-loading into a large 10% denaturing PAGE gel. The gel ran for 45 minutes before staining with xl Sybr Au and imaging in the bio-rad imaging doc. The solution left over from the reaction carried out to characterize the DNAzyme's activity was split into two equal parts of 4uL. In one half, the on-target SNAIL probes were added, and in the other half, off-target SNAIL probes were added. The ratio between padlock probes and the DNAzyme was 1:1. The off-target probes were added as a negative control to visualize any off-target ligation. The PLP probes were hybridized to the DNAzymes by heating the solutions to 95 °C for 5 minutes and cooling them to room temperature for 15 minutes. Afterward, 0.5 U / uL of T4 ligase, 1 uL of10X T4 ligation buffer, and 0.2 mg ml BSA were added to the mixture and were left to incubate at room temperature in the dark for 45 minutes. To characterize the ligation of the PLPs, 200nmoles of the DNA were taken from the sample and mixed with x2 denaturing loading dye, heated to 95°C for 15 minutes, and hot-loaded to a large prewarmed 10% denaturing PAGE gel. The gel was run for 45 minutes at 26 watts before staining it with SYBR Au and imaging it in a bio-rad gel doc imager (FIG. 10). It was seen that the ligation with only the PLP mitigated any off-target effects. Design of barcode and synthesis of DNAzyme is additionally shown in FIG. 15.

[0190] Thiol-mediated uptake to deliver DNA into plants To facilitate the delivery of DNAzymes, a method to deliver ssDNA oligonucleotides into roots was developed. As a proof of concept, the delivery of short ssDNA nucleotides modified with Yakima Yellow (YY) through thiol-mediated uptake (TMU) was done first. TMU facilitates the delivery of sulfide containing molecules through dynamic covalent sulfur exchange interactions with cell-surface membrane proteins. Disulfide modifications to the YY labeled strand were added using a strain-promoted alkyne-azide cycloaddition (SPAAC). To accomplish this, a YY labeled strand was synthesized with a 5 prime azide modification containing a hybridization region to a helper strand that contained: 1. A 3 prime DBCO modification and 15 disulfide modifications. The reaction was carried with lOOmM TRIS and lOOmM KC1 with an excess (4 times) of DBCO disulfide containing helper strand. The reaction proceeded for 16 hours before the product was purified through HPEC. To deliver the now disulfide modified YY labelled short oligonucleotide into the roots 5-day old seedling were taken and incubated for 12 hours in luM of DNA. The samples were then washed 5 times for 5 minutes before imaging in a Zeiss fluorescent microscope.

[0191] Design and synthesis of modified DNAzyme for delivery into Arabidopsis roots As a proof of concept to demonstrate the DNAzyme’ s capability of detecting sodium ions in vivo, a trans-DNAzyme was used. Trans-DNAzymes are made of an enzyme strand and a substrate strand that can get cleaved upon the exposure of specific metal ions. The metal ion can coordinate with specific residues in the enzyme strand and catalyze a intramolecular transesterification reaction leading to the phosphodiester cleavage of the substrate strand. Using the catalytic beacon design, the DNAzyme was modified with fluorophores and quenchers such that there could be a fluorescent turn on upon cleavage of the substrate strand. Additional modifications were also made to the DNAzyme including 1) the addition of anitrobenzene to the 2 prime hydroxy group of the RNA base of the substrate strand to photocage the activity of the DNAzyme and 2) a disulfide linker to facilitate the delivery of the DNAzyme into plant roots. The photoaging group allows for temporal control as the DNAzyme can be “activated” once it is delivered into plant root by shining UV-light.

[0192] Fluorescence Characterization of Na-Specific DNAzyme activity All fluorescence -based characterization of our sensor was done using the ISS ChronosDFD fluorometer. Fluorescence activity of the photocaged disulfide-modified DNAzyme was monitored using the ISS fluorimeter to calculate fold change of the Na-specific DNAzyme with addition of increasing levels of NaCl in the active and the inactive sequence. Two samples for each DNAzyme were prepared to monitor the fluorescence of the sensor before and after decaging the DNAzyme. The DNAzyme samples were prepared by hybridizing 80 nM of the fluorescently labelled substrate strand to 400 nM of the enzyme strand by heating it up to 95 °C for five minutes and cooling it down to room temperature for 15 minutes. To decage the sensor, the sample was incubated with 365 nm UV light for 30 minutes. First, the fluorescence of the caged DNAzyme was read in the emission range of 540 to 640 nm, and excited at 520 nm based on the fluorophore Yakima yellow that was used. The increasing levels of NaCl were added in five-minute intervals to the same sample and stirred, and the fluorescence was read 5 minutes after addition of NaCl. Then, the same process was followed for the uncaged DNAzyme after incubating it with 365 nm UV light for 30 minutes. This was done for both the active and the inactive sequences to determine the activity of the active sequence and validate the obliteration of activity in the inactive sequence.

[0193] Fluorescence Characterization of Na-Specific DNAzyme selectivity Fluorescence-based activity of the photocaged DNAzyme was monitored using the ISS fluorimeter to calculate fold turn-on of the Na DNAzyme with different metals to determine selectivity of the sensor. A similar process was followed for these selectivity assays as the activity assays. 3 samples for each DNAzyme were prepared to monitor the fluorescence of the sensor in the presence of three different metals. To decage the sensor, the sample was incubated with 365 nm UV light for 30 minutes. First, the fluorescence of the uncaged DNAzyme sensor was read in the emission range of 540 to 640 nm, and excited at 520 nm based on the fluorophore Yakima yellow that was used without any metal added. Then, the metal was added in the same sample and stirred, and the fluorescence of the DNAzyme wasread 5 minutes after adding the metal. This was done for both the active and the inactive sequences.

[0194] Na-specific DNAzyme delivery and imaging in Arabidopsis roots To deliver the DNAzyme, 5 -day old seedling grown in 16 MS solid growth media were incubated for 8 hours in 2pM of hybridized disulfide modified sodium DNAzyme. Prior to incubation the DNAzyme was hybridized overnight with the photocaged substrate strand in lOOmM TRIS with lOOmM KC1. To avoid the degradation of the disulfides, the samples were not heated during the hybridization process. To account for any degradation of the DNAzyme sensor during the sensor’ s delivery, a negative control was include composed of an inactive DNAzyme sequence containing a point mutation that abolished the cleavage reaction upon the exposure of sodium. After the 8-hour incubation, the seedlings were washed with MilliQ water 5 times for 5 minutes and exposed to 365nm UV-light for 30 minutes to decage the DNAzyme and activate its activity. After decaging, the samples were incubated for 30 minutes at 37 C before imaging in a ZEIS fluorescent microscope (FIG. 14). The same process was done with 5-day old seedlings that were grown on 16 MS solid growth media supplemented with 0 mM, 25 mM, and 50 mM NaCl to determine the effects of increasing NaCl levels on the fluorescent turn-on of our sensor.

[0195] Plant hybridization-based targeted observation of gene expression map PROOF-OF-CONCEPT. Gene expression was mapped where the plant roots were fixed by immersing it in FAA: 16% v / v formaldehyde, 5% v / v acetic acid, and 50% ethanol for 1 hour at r.t. 200 uE per well. Samples were dehydrated in a series of 10-min washes once in 70% ethanol, once in 90% ethanol and twice in 100% ethanol, followed by two 10-min washes in 100% methanol, and then were stored in 100% methanol at -80 °C for overnight. Samples were rehydrated in a series of 5-min washes in 75%, 50% and 25% methanol in DPBS-T (0.1% Tween 20 in DPBS) at room temperature. The cell wall was partially digested by incubating samples in cell wall digestion solution (0.1% cellulase, 0.1% macerozyme, and 0.1% pectinase in DPBS-T) for 5 min on ice, and then for 1 hour at room temperature. (200 uE each sample). After two washes in DPBS-T samples were fixed in 10% formaldehyde for 30 min at room temperature and washed with DPBS-TR. (100 uL each sample). Proteins were digested by incubating samples in protein digestion buffer (0.1 M Tris-HCl pH 8, 50 mM EDTA pH 8) with a 1:100 volume of Proteinase K for 30 min at 37 °C. (300 uL each sample). After two washes in DPBS-TR, samples were fixed in 10% (v / v) formaldehyde for 30 min at roomtemperature and washed with DPBS-TR. A pool of SNAIL probes (5 pM each) was heated at 90 °C for 5 min and cooled at room temperature. For the proof-of-concept, one gene target was targeted which was Gene ID AT2G46570 to look at the xylem. Samples were incubated in hybridization buffer (2x SSC, 30% formamide, 1% Triton-X, 20 mM ribonucleoside vanadyl complex and pooled SNAIL probes at 100 nM per oligo) in a 40 °C humidified oven overnight. After hybridization, samples were washed twice in DPBS-TR and once in 4x SSC in DPBS- TR for 30 min at 37 °C and rinsed with DPBS-TR at room temperature. Samples were then incubated in a T4 DNA ligation mixture without T4 ligase (10X Ligation buffer, BSA (2 mg / mL), and NFH2O) on ice for 5 minutes. Then the samples were incubated with 200 pL ligation mixture with ligase at room temperature overnight. After ligation, samples were washed twice with DPBS-TR for 10 min at room temperature and incubated in a rolling circle amplification (RCA) mixture without polymerase (10X Phi29 DNA polymerase buffer, 250 pM dNTP, 0.1 pg pl -1 BSA, 1 mM dithiothreitol, and 20 pM aminoallyl dUTP) for 5 minutes. Then the samples were incubated with 200 pL RCA mixture with polymerase at 37 °C overnight. After RCA, samples were rinsed in DPBS-T and covalently cross-linked with 4.3 pg pl -1 BS(PEG)9 in DPBS-T. BS(PEG)9 was then quenched by incubating samples in 1 M Tris-HCl (pH 8) for 30 min at room temperature. After the fixation of DNA amplicons, samples were embedded in acrylamide gel by incubating in a polymerization mixture (4% acrylamide, 0.2% bis -acrylamide, 0.1% ammonium persulfate and 0.1% tetramethyl ethylenediamine in DPBS-T) for 1.5 h at room temperature. Samples were then rinsed in DPBS-T. Samples were incubated in ClearSee solution (ClearSee solutions were prepared by mixing xylitol powder [10% (w / v)J, sodium deoxycholate [15% (w / v)] and urea [ 25% (w / v)] in water overnight. Then, samples were washed with 2x SSC for 5 min at room temperature and then incubated in a bridge probe hybridization mixture (2x SSC, 20% formamide and the bridge probe at 100 nM per oligo in water) for overnight at room temperature. After washing twice in 2x SSC for 5 min at room temperature, samples were incubated in a detection probe hybridization mixture (2x SSC, 20% formamide, and the complementary fluorescent detection oligo (Cy3) at 100 nM per oligo in water) for overnight at room temperature. Samples were washed in 2x SSC for 5 min at room temperature. Imaging was performed with the fluorescent microscope. This protocol was a proof-of-concept experiment to replicate mapping gene expression in plant roots. This is based on the protocol described in Nobori et al. (2023).Example 7 - DNAzymes Using SNAIL ProbesT4 Titration with SNAIL Probes

[0196] To test which concentration of T4 Ligase was optimal in giving the least background ligation, a T4 ligase concentration titration was performed. We tested the titration on sequence with “on-target” binding, as well as sequences with “off-target” binding. The T4 ligase mixture contained T4 Ligase, T4 Ligase buffer, Bovine serum albumin, and ATP to aid with the ligation of the sequences. We can see that 0.25 U of T4 ligase gives the best result because in all of the sequences there is minimal background ligation product forming with only 0.25 U of ligase. This is indicated by the absence of a band forming at a higher molecular weight to the SNAIL probes on a denaturing PAGE gel. (FIG. 20)In vitro Ligation with Mock DNAzymes using SNAIL probes

[0197] Using the optimal T4 concentration determined in the concentration titration of 0.25 U, we then applied the SNAIL probes for ligation with the mock DNAzyme sequences. These sequences are “mock” because they do not have the RNA cleaving based, but they will fold into the correct secondary structure to imitate the real DNAzymes for binding with the SNAIL probes. To test this, we tried 4 conditions where we did the ligation for one hour at room temperatue, one hour at 28C, 16 hours at room temperature, and 16 hours at 28C. From this mock ligation, we can see that at 16H we reach an end point where there is ligation of the SNAIL probes in both full length and cleaved sequences and probes. In one hour we only see the ligation of the full-length probes. At 28C compared to room temperature, there is a reduction of background ligation. (FIGA. 21A-D)

[0198] The following methods were utilized in the experiments in this Example:T4 Titration with SNAIL Probes

[0199] The T4 concentration titration was done with padlock probes targeting the active cleaved DNAzyme, the inactive total DNAzyme, and the inactive cleaved DNAzyme. For the controls of just the PLP only, we dissolved 0.5 uL of 10 uM PLP into 4.5 uL of 150 mM tris buffer. For the samples we combined the active cleaved padlock probe with the active cleaved primer, the active cleaved padlock probe with the active total primer, the active cleaved padlock probe with the inactive total primer, the inactive total SNAIL probes, and the inactive cleavedSNAIL probes. Each of these sets of matched and mismatched SNAIL probes were made into stocks of 10:100 uM and 0.5 uL of each stock was dissolved in 4.5 uL of 150 mM tris. Then 5 uL of a T4 ligase mixture was added. The T4 ligase mixture for 0.25 U was composed of 7.2 uL BSA, 5 uL ATP, 36 uL of T4 Ligase Buffer, and 0.5 uL of T4 DNA Ligase in 131.3 uL of H2O, the T4 mixture for 0.5 U was composed of 7.2 uL BSA, 5 uL ATP, 36 uL of T4 Ligase Buffer, and 1 uL of T4 DNA Ligase in 130.8 uL of H2O, and the T4 mixture for 1 U of Ligase was composed of 7.2 uL BSA, 5 uL ATP, 36 uL of T4 Ligase Buffer, and 2 uL of T4 DNA Ligase in 130.3 uL of H2O. After letting the samples ligate for 1 hour at room temperature, 1.5 uL of the sample were taken out and dissolved in 2X Blue loading dye and hot loaded on to a 10% denaturing PAGE gel. The gel ran for 1 hour 30 minutes and was stained with SYBR gold and imaged on the BioRad GelDoc Imager.In vitro Ligation with Mock DNAzymes using SNAIL probes

[0200] The mock sequence ligation was was done using the SNAIL probes targeting the active total DNAzyme, the active cleaved DNAzyme, the inactive total DNAzyme, and the inactive cleaved DNAzyme. The snail probes were combined in a stock containing 10 uM:100 uM PLP to Primer. For the controls of just the PLP only, we dissolved 0.5 uL of 10 uM PLP into 4.5 uL of 150 mM tris buffer. For the samples with the total DNAzyme we dissolved 0.5 uL of the stock of PLP: P into 3.5 uL of 150 mM tris buffer. Then we added 1 uL of 5 uM total “mock” DNAzyme. For the samples with the cleaved DNAzyme we dissolved 0.5 uL of the stock of PLP:P into 3.5 uL of 150 mM tris buffer. Then we added 1 uL of 5 uM cleaved “mock” DNAzyme. We then added 5 uL of 2X ligase mixture containing 7.2 uL BSA, 5 uL ATP, 36 uL of T4 Ligase Buffer, and 0.5 uL of T4 DNA Ligase in 131.3 uL of H2O. The reaction for room temperature ligated for 16 hours and 1 hour on the bench and the samples for 28C were ligated for 16 hours and 1 hour in the benchmark dry bath. After ligation, 1.5 uL of the sample were taken out and dissolved in 2X Blue loading dye and hot loaded on to a 10% denaturing PAGE gel. The gel ran for 1 hour 30 minutes and was stained with SYBR gold and imaged on the BioRad GelDoc Imager.

[0201] By using a photoprotective diethylaminocoumarin (DEACM) group on the G50 position the inventors were able to deactivate the activity of the sodium DNAzyme and activate it once again by shinning 405nm wavelength light (60W) for 1 minute. DEACM group was added to the G50 position of the sodium-DNAzyme substrate strand labeled with fluorescein through solid phase synthesis. DEACM photoprotective dG monomers are commerciallyavailable. Substrate cleaving activity assays demonstrate that G50-DEACM modified substrate can deactivate the activity of the Na-DNAzyme and it is not until 405nm light is shined to the samples that the activity is recuperated. Results are shown in FIG. 22A.

[0202] Through a series of in silico studies we optimized the content of the barcode integrated to the DNAzymes. Using NuPack, a software that is free and available online, we predicted the hybridization probabilities and secondary structures between the barcoded DNAzyme and SNAIL probes. We rationalized the final barcoded DNAzyme sequence design to include: 1) an extended left binding arm that hybridizes up to the primer binding region of the cleaved SNAIL probe. 2) An extended right binding arm, and 3) a 4 dT loop to make the DNAzyme unimolecular. These optimizations are necessary to maintain the secondary structure of the DNAzyme required for catalytic activity and to minimize background ligation of the cleaved SNAIL probes. Results are shown in FIGS. 22B-E.

[0203] Nupack was also used to optimize the content of the SNAIL probes. In silico hybridization studies using NuPack showed that the probability of the cleaved padlock probe to displace the binding arm of the uncleaved DNAzyme and subsequently ligate causing background ligation is dependent on the number of bases the padlock probe binds to on the DNAzyme. By shortening the binding region of the cleaved padlock probe to the DNAzyme from 16 to 13 and conducting the hybridization studies at 22°C, we can eradicate any background ligation from the cleaved SNAIL probes. Results are shown in FIGS. 23A-B.

[0204] To verify that there were no interactions between the SNAIL probes themselves, we also conducted a “switch” of the binding regions between the padlock probes and primers that promote the ligation of the padlock probe upon the binding of the target sequence. This region was unique for total DNAzyme reading and cleaved DNAzyme reading SNAIL probes but remained the same between the probes detecting active and inactive barcoded DNAzymes. In vitro ligation assays confirmed that mismatch snail probe pairs did not lead to unspecific ligation, a problem we had with our past designs. In vitro ligation studies were carried by combining the barcoded DNAzyme, padlock probe, and primer pairs at a 1 : 1 : 1 ratio and adding 0.25U of T4 ligase. Reaction was carried at 28°C for 1 hour. Results are shown in FIG. 24.* * *

[0205] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. 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Claims

WHAT IS CLAIMED IS:

1. An in vitro method of detecting or measuring a cofactor in a cell comprising:(a) contacting the cell with a nucleic acid enzyme comprising: a cofactor binding site, optionally an effector binding site, and a nucleic acid affinity tag; wherein the nucleic acid enzyme can undergo a cleavage reaction in the presence of the cofactor; and(b) quantitatively amplifying and detecting portions of the nucleic acid enzyme using a first primer and a second primer; wherein the first primer that can selectively hybridize to the nucleic acid enzyme upstream of the cleavage site, and wherein the second primer can selectively hybridize to the nucleic acid enzyme downstream of the cleavage site.

2. The method of claim 1, wherein the method comprises purifying the nucleic acid enzyme by binding the nucleic acid affinity tag, optionally wherein the affinity tag is a polyadenine (poly-A).

3. The method of cany one of claims 1-2, wherein the nucleic acid enzyme comprises a barcode nucleic acid.

4. The method of any one of claims 1-3, wherein the method further comprises purifying and detecting one or more mRNA, wherein the purifying is by binding polyadenine (poly-A) in the mRNA.

5. The method of any one of claims 1-4, wherein the nucleic acid enzyme comprises a single-stranded DNAzyme, an RNAzyme, or an aptazyme.

6. The method of any one of claims 1-5, wherein the method further comprises detecting an mRNA in the cell.

7. The method of any one of claims 1-6, wherein the mRNA and the nucleic acid enzyme are substantially purified using dT-oligo magnetic beads or dT-oligo oil-based beads.

8. The method of any one of claims 1-7, wherein the cofactor is a metal ion, such as K, Pb, Hg, As, UO2, Fe, Zn, Ca, Cu, Co, Cd, Cr, Li, or Mn, or Mg(II), K(l+), Fe(II), Ag(I),Pb(II), Hg(II), As(III), Fe(III), Zn(II), Cd(II), Cu(II), Sr(II), Ba(II), Ni(II), Co(II), As(V), U(VI), or Cr(VI).

9. The method of any one of claims 1-8, wherein the DNAzyme is a 8-17 DNAzyme, EtNa DNAzyme, Li+-DNAzyme, 11-5 DNAzyme, or 10-23 DNAzyme.

10. The method of any one of claims 1-9, wherein the nucleic acid enzyme comprises a photocaged moiety, such as photocaged nitrobenzyl phosphoramidte.

11. An in vitro method of detecting or measuring a cofactor in a cell comprising:(a) contacting the cell with a nucleic acid enzyme comprising: a cofactor binding site, wherein the nucleic acid enzyme comprises a catalytic loop, and wherein the nucleic acid enzyme can self-hybridize upstream from the catalytic loop and downstream from the catalytic loop; wherein the nucleic acid enzyme can undergo a cleavage reaction in the presence of the cofactor; wherein the cleavage reaction releases a nucleic acid strand that was self-hybridized upstream or self-hybridized downstream to the nucleic acid enzyme, thereby providing a single- stranded primer site in the nucleic acid enzyme and(b) detecting the cleavage reaction by contacting a first primer that selectively binds the single-stranded primer site in the nucleic acid enzyme and amplifying a nucleic acid using a polymerase chain reaction (PCR) or rolling circle amplification (RCA), wherein the nucleic acid enzyme is preferably a DNAzyme; optionally wherein the first primer comprises a barcode.

12. The method of claim 11, wherein the first primer is a primer for rolling circle amplification (RCA), such as wherein the first primer is a SNAIL probe, a FISSEQ probe, or a PLAYR probe, or a Padlock probe, optionally wherein the SNAIL probe or padlock probe comprises a barcode. wherein the said amplifying comprises using rolling circle amplification (RCA) to generate a rolling circle amplification product, andwherein the rolling circle amplification product is detected with one or more detection probes that selectively bind the barcode.

13. The method of any one of claims 11-12, wherein the method comprises spatial transcriptomics detection and analysis of the amplification product in a plurality of cells, optionally wherein the spatial transcriptomics comprises a Spatially resolved Transcript Amplicon Readout Mapping (STARmap), merFISH, seqFISH, FISSEQ, non-gapfilling padlock probe, PLAYR, or gapfilling padlock / barSeq methodology.

14. The method of any one of claims 11-13, wherein the method further comprises detecting or measuring total nucleic acid enzyme levels by:(i) hybriding a second primer to the nucleic acid enzyme, wherein the second primer selectively binds a single-stranded portion of the nucleic acid enzyme that is not within the self-hybridized region upstream and is not within selfhybridized region downstream from the catalytic loop; and(ii) amplifying nucleic acid from the second primer using polymerase chain reaction (PCR) or rolling cycle amplification (RCA).

15. The method of any one of claims 11-14, wherein the nucleic acid enzyme comprises a photocaged moiety, optionally wherein the photocaged moiety is photocaged nitrobenzyl phosphoramidte or diethylaminocoumarin (DE ACM).

16. A composition comprising a nucleic acid enzyme, wherein the nucleic acid comprises: a cofactor binding site, optionally an effector binding site, and a nucleic acid affinity tag; and wherein the nucleic affinity tag is a polyadeninde (poly-A).

17. A composition comprising a nucleic acid enzyme, wherein the nucleic comprises a catalytic loop, and wherein the nucleic acid enzyme comprises a self-hybridized regions both upstream and downstream from the catalytic loop; whereinthe nucleic acid enzyme can self-cleave in the presence of a cofactor, and wherein the self-cleavage can release a nucleic acid from either the upstream self-hybridized region or the downstream self-hybridized region of the nucleic acid enzyme, thereby forming a singlestranded structure in the nucleic acid enzyme, optionally wherein the nucleic acid enzyme comprises a photocaged moiety, such as a photocaged nitrobenzyl phosphoramidte or diethylaminocoumarin (DEACM); optionally wherein the nucleic acid enzyme comprises a barcode nucleic acid.

18. A kit comprising the composition of any one of claims 16-17 in a container means.

19. A nucleic acid encoding the nucleic acid enzyme of any one of claims 1-17.

20. A cell comprising the nucleic acid claim 19.

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