WO2016134227A1

WO2016134227A1 - Dna/micro rna hybrids and determination of enzyme activity

Info

Publication number: WO2016134227A1
Application number: PCT/US2016/018620
Authority: WO
Inventors: David Surace KAPITULA; Gia-Ming HONG; Gan-Der HO
Original assignee: Jhl Biotech, Inc.
Priority date: 2015-02-20
Filing date: 2016-02-19
Publication date: 2016-08-25

Abstract

The present disclosure describes methods, assays, kits, compositions, and mixtures for determining enzyme activity. The disclosure provides methods, assays, kits, and mixtures for determining the cleavage rate of enzymes, including the the quantification and kinetics of enzyme activity. For example, the present disclosure provides clipping assays for determination of the cleavage rate of enzymes such as DNase.

Description

DNA/MICRO RNA HYBRIDS AND DETERMINATION OF ENZYME ACTIVITY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/118,801, filed February 20, 2015, which is hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present disclosure describes methods, assays, kits, compositions, and mixtures for the quantification of enzyme activity. For example, the present disclosure provides clipping assays for determination of the cleavage rate of enzymes such as DNase.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0003] The content of the text file submitted electronically herewith is incorporated herein by reference in its entirety: A computer readable format copy of the Sequence Listing (filename: JHBI_002_01WO_SeqList_ST25.txt); date recorded: February 12, 2016; file size 3 KB).

BACKGROUND

[0004] There is a need in the art for assays capable of providing a measurement of the activity of enzymes, such as DNase, restriction enzymes, or other enzymes.

[0005] For example, the currently known assays for determination of DNase activity are lengthy, difficult, imprecise, require organic extraction of DNA substrate, cannot be performed in the presence of light (RED A), and/or are unable to quantify the DNase assay. Deoxyribonuclease (DNase) is a non-specific nuclease that binds to the minor groove and cleaves phosphodiester linkages of double-stranded DNA. In the presence of divalent cations, such as Ca²⁺ and Mg²⁺, DNase acquires its maximum enzymatic activity for DNA hydrolysis, yielding di-, tri, and oligonucleotides with 5 '-phosphorate and 3' -OH fragments (Sutton et al. Biochem. J. (1997)).

[0006] In molecular biology, DNase has been used as a powerful tool for DNA fingerprinting, genome mapping, and removal of residual DNA for reverse transcription. In clinical application, recombinant human DNase (rhDNase) has been successfully used for treatment of cystic fibrosis (CF). Additionally, DNase is also under consideration for other diseases such as non-CF pediatric lung disease and systemic lupus erythematosus (Boogaard et al. Pediatr. Pulmonol. (2007) and Gaipl et al. Curr. Dir. Autoimmun (2006)).

[0007] There is a need in the art for a simple, precise, quantitative, high throughput assay for determining enzymatic activity. In particular, there is a need in the art for methods for determining the kinetics of enzymes, such as DNase.

SUMMARY OF INVENTION

[0008] The present disclosure provides compositions, methods, assays, kits, and mixtures for determining enzyme activity in a sample. In one aspect, the disclosure provides PCR-based clipping assays for quantification of enzyme activity, utilizing hairpin-like DNA-RNA hybrids as substrates. In one aspect, the disclosure provides compositions, methods, assays, kits, and mixtures for determining DNase activity using hairpin-like DNA-RNA hybrids.

[0009] The structure of the DNA-RNA hybrids, in one embodiment, allows the enzyme to cleave the DNA portion of the DNA-RNA hybrid in a specific and controlled manner, such that the RNA portion of the DNA-RNA hybrid is clipped from the hybrid structure. Total released RNA molecules (i.e., the RNA that has been clipped from the DNA-RNA hybrid by enzyme activity) can be detected to quantitate enzyme activity. For example, released RNA can be reverse transcribed for cDNA synthesis, and cDNA can be quantified using quantitative polymerase chain reaction (PCR). In certain embodiments, the RNA is microRNA.

[0010] The DNA-RNA hybrid molecules disclosed herein, in one embodiment, comprise a first single stranded DNA, a second single stranded DNA that is complementary to the first single stranded DNA, and a RNA molecule. In one embodiment, the first and second single stranded DNAs pair to form a double stranded DNA comprising a single DNase cleavage site. In another embodiment, the RNA molecule is located between the first and second single stranded DNAs. In a further embodiment, the first and second single stranded DNAs pair so that the DNA-RNA hybrid molecule forms a stable stem-loop structure. In a yet further embodiment, the stable stem- loop structure is formed at a temperature at or below 50°C. In another embodiment, the RNA molecule is released from the DNA molecule upon cleavage at the DNase cleavage site. In a further embodiment, the RNA released from the DNA-RNA hybrid molecule upon DNase cleavage can be quantified, for example, by a PCR assay. [0011] In one embodiment, the DNA-RNA hybrid is structured to provide a single cleavage site for a particular enzyme, such that upon clipping the RNA from the DNA at the single cleavage site, a single RNA molecule is released. In a further embodiment, the release of a RNA molecule due to the single cleavage site allows quantification of enzyme activity. In a further embodiment, the single cleavage site is provided by the length of the double stranded DNA (e.g., 11 base pairs), the conformation of the double stranded DNA, or the specific sequence of the double stranded DNA. In a further embodiment, the 11 base pair DNA provides a single cleavage site for DNase; or the conformation or sequence of the double stranded DNA provides a specific cleavage site for a particular enzyme, such as a restriction enzyme.

[0012] In one embodiment, the first and single stranded DNAs are each 11 bases, such that the DNA-RNA hybrid molecule, once formed as a stem-loop structure, comprises an 11 base-pair DNA portion and a RNA portion. Thus, in one embodiment, the DNA-RNA hybrid molecule allows precise measurement of DNase activity, since DNase specifically cleaves double stranded DNA molecules having 11 base pairs.

[0013] In another embodiment, the first and second single stranded DNAs are each 11 bases long, less than 11 bases long, or more than 11 bases long. In some embodiments, the first and second single stranded DNAs pair to form a double stranded DNA molecule that may be specifically recognized by a particular enzyme, such as a restriction enzyme. For example, in one embodiment, the restriction enzyme is an EcoRI restriction enzyme, which cleaves a 6 base pair sequence having the following 5' to 3' sequence: GAATTC. As another example, the Sfil restriction enzyme cleaves a 13 base pair DNA sequence having a 5' to 3' sequence according to SEQ ID NO: 8 (O JCCNNNNNO JCC). In one embodiment, the restriction enzyme specifically cuts the double stranded DNA portion of the molecule, releasing the RNA portion. In a further embodiment, the release of the RNA portion at the single cleavage site of the DNA sequence allows for quantification of the enzyme.

[0014] In one embodiment, the RNA molecule in the DNA-RNA hybrid molecules provided herein is any type of RNA molecule. In one embodiment, the RNA molecule is a microRNA molecule. In a further embodiment, the microRNA is MIR234 (SEQ ID NO: 5). In one embodiment, the RNA molecule comprises about 10 to about 50, about 15 to about 30, or about 19 to about 21 bases. [0015] In one embodiment, the DNA-RNA hybrid molecule comprises a reporter dye and a quencher dye. The reporter dye may be selected from the group consisting of cyanine dyes, fluorescein dyes, and rhodamine dyes. In one embodiment, the cyanine dye is selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5. In another embodiment, the quencher dye is IAbRQSp.

[0016] In one embodiment, the present disclosure provides a DNA-RNA hybrid molecule comprising a first single stranded DNA, a second single stranded DNA that is complementary to the first single stranded DNA, and a RNA molecule, wherein the first and second single stranded DNAs comprise sequences according to SEQ ID NO: 1 (CGAAAATTTTC) and SEQ ID NO: 2 (GAAAATTTTCG), respectively; or wherein the first and second single stranded of DNAs comprise sequences according to SEQ ID NO: 3 (CGCGAATTCGC) and SEQ ID NO: 4 (GCGAATTCGCG), respectively. In one embodiment, the DNA-RNA hybrid molecule comprises, from 5' to 3': SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 2. In another embodiment, the DNA-RNA hybrid molecule comprises, from 5' to 3': SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 4. In some embodiments, the DNA-RNA hybrid molecule comprise a reporter dye at the 5'terminus and a quencher dye at the 3' terminus.

[0017] In one aspect, methods for detecting enzyme activity in a sample are provided herein, the methods comprising the following steps:

(a) preparing a reagent comprising a DNA-RNA hybrid molecule and a buffer, wherein the DNA portion of the DNA-RNA hybrid molecule is designed to be cleaved by the enzyme of interest in a predictable way;

(b) subjecting the reagent preparation of step (a) to an activation step to allow the enzyme activity to clip the RNA molecule from the DNA-RNA hybrid, and an inactivation step to stop the reaction; and

(c) detecting the clipped RNA present in the sample.

[0018] In one embodiment, the methods provided are useful for detecting DNase activity. For example, in one embodiment, the methods comprise the following steps:

(a) preparing a reagent comprising a DNA-RNA hybrid molecule and DNase reaction buffer; (b) subjecting the reagent preparation of step (a) to an activation step to allow DNase activity to clip the RNA molecule from the DNA-RNA hybrid, and an inactivation step to stop the reaction; and

(c) detecting the clipped RNA present in the sample.

[0019] In one embodiment, the reagent prepared in step (a) is subjected to a hold step prior to the initiation of the activation step of step (b). In one embodiment, the hold step is carried out at about 4°C for about 5 minutes. In another embodiment, the activation step is carried out at a temperature of about 10°C to about 37°C for about 10 minutes. For example, in one embodiment, the activation step is carried out at a temperature of about 25°C or about 37°C. In another embodiment, the inactivation step is carried out at about 80°C or about 85°C for about 10 minutes.

[0020] In one embodiment, the clipped RNA is detected by PCR, such as reverse transcription (RT)-PCR. In a further embodiment, the clipped RNA is detected by RT-PCR and quantitative PCR (qPCR). For example, in one embodiment, the clipped RNA is reverse transcribed to cDNA, and then qPCR is performed to quantitate the clipped RNA in the sample. In a further embodiment, the enzyme activity in the sample is determined by calculating the threshold cycle value (Ct) of the qPCR.

[0021] In one embodiment, the present disclosure provides compositions, methods, assays, kits, and mixtures for determining the enzymatic activity of DNase in the sample. The DNase binds to the minor groove of the double stranded DNA during the activation step. In one embodiment, the double stranded DNA is 11 base pairs. In a further embodiment, since the DNA is 11 base pairs, the DNase binds at a single DNase binding stop and therefore the single cleavage event for each DNA-RNA hybrid molecule provides a means for quantitating the enzymatic activity of the DNase in the sample.

[0022] Thus, the present disclosure, in one aspect, provides methods of determining DNase activity in a sample, wherein the methods comprise using the DNA-RNA hybrid molecules disclosed herein to quantify DNase activity in a sample.

[0023] The present disclosure also provides assays, kits, and mixtures comprising the DNA- RNA hybrid molecules disclosed herein. For example, in one embodiment, the present disclosure provides a mixture comprising a DNA-RNA hybrid molecule. In further embodiments, the mixture further comprises a RNAse inhibitor. In still further embodiments, the mixture further comprises the enzyme and a reaction buffer. In yet further embodiments, the mixture comprises RNA, e.g., clipped RNA. In one embodiment, the mixture comprises a DNA-RNA hybrid molecule comprising an 11 base pair DNA as described herein. In a further embodiment, the mixture comprises a RNAse inhibitor, DNase, and a DNase reaction buffer. In a yet further embodiment, the mixture comprises RNA, e.g., clipped RNA. The DNase reaction buffer, in one embodiment, comprises magnesium and calcium.

[0024] In one embodiment, the present disclosure provides compositions comprising the DNA- RNA hybrid molecules described herein. In another embodiment, the present disclosure provides kits for detecting enzyme activity in a sample, the kits comprising DNA-RNA hybrid molecules as described herein and a reaction buffer as described herein. In one embodiment, the kits comprise DNA-RNA hybrid molecules comprising an 11 base pair DNA as described herein, and a DNase reaction buffer.

BRIEF DESCRIPTION OF THE FIGURES

[0025] Figure 1 provides a schematic depiction of the experimental design of the clipping assay. The DNA/microRNA hybrids are shown on the left side of the figure, where the upper portion is a microRNA (miRNA) molecule and the lower portion is a double-stranded DNA helix. "R" denotes a reporter dye, and "Q" denotes a dark quencher. The figure also provides a schematic depiction of the reverse transcription reaction and an exemplary QPCR amplification curve.

[0026] Figure 2 shows the sequence of both DNA and microRNA-234 of the engineered DNA/microRNA hybrid A4T4-miR234-Cy5 and the IDT Oligo ESI chromatographic analysis of the A4T4-miR234-Cy5 hybrid. The sequence of the hybrid is: 5'-Cy5- CGAAAATTTTCrUrUrArUrUrGrCrUrCrGrArGrArArUrArCrCrCrUrUGAAAATTTTCG- IAbRQSp-3' (SEQ ID NO: 6). "r" denotes RNA; "Cy5" denotes a Cy5 reporter; and "IAbRQSp" denotes a quencher.

[0027] Figure 3 shows the melting curve analysis of the A4T4-miR234-Cy5.

[0028] Figure 4 shows the sequence of both DNA and microRNA-234 of the engineered

DNA/microRNA hybrid A2T2-miR234-Cy5 and the IDT Oligo ESI chromatographic analysis of the A2T2-miR234-Cy5 hybrid. The sequence of the hybrid is: 5'-Cy5-

CGCGAATTCGCrUrUrArUrUrGrCrUrCrGrArGrArArUrArCrCrCrUrUGCGAATTCGCG- IAbRQSp-3' (SEQ ID NO: 7). "r" denotes RNA; "Cy5" denotes a Cy5 reporter; and "IAbRQSp" denotes a quencher.

[0029] Figure 5 shows the melting curve analysis of the A2T2-miR234-Cy5.

[0030] Figure 6 provides the real-time monitoring of DNase enzymatic activity using serial- diluted A4T4-MIR234-CY5 as substrates.

[0031] Figure 7 depicts the real-time monitoring of DNase enzymatic activity using serial- diluted A2T2-MIR234-CY5 as substrates.

[0032] Figure 8 shows the quantification of clipped/released miR234 molecules (from A2T2- mir234 oligomers or from A4T4-mir234 oligomers) using qPCR

[0033] Figure 9 shows the time-dependent study of the clipping assay. Clipping was performed at room temperature for 30 sec, 1 minute, 2 minutes, and 3 minutes, using A4T4-miR234-Cy5 as substrates. The threshold values for each clipping reaction, determined by qPCR, are provided in Table 1.

DETAILED DESCRIPTION

[0034] In one aspect, the present disclosure provides methods, assays, compositions, kits, and mixtures for determining the activity of a molecule in a sample, or for determining the absence of activity of a molecule in a sample. For example, in one embodiment, the present disclosure provides methods, assays, compositions, kits, and mixtures for quantifying enzyme activity in a sample. In one embodiment, the disclosure provides hybrid molecules comprising a RNA portion that can be released from the hybrid molecule and quantified. In one embodiment, the hybrid molecule comprises a RNA portion and a DNA portion, wherein the DNA portion comprises a single cleavage site for an enzyme. In a further embodiment, upon cleavage of the DNA portion at the single cleavage site, the RNA portion is released from the DNA portion.

[0035] In one embodiment, the disclosure provides methods for determining enzyme activity in a sample using hairpin-like DNA-RNA hybrid molecules. In another embodiment, the disclosure provides hairpin-like DNA-RNA hybrids as substrates for the determination of enzyme activity. In one embodiment, the disclosure provides hairpin-like DNA-RNA hybrids as substrates for the determination of the absence of enzyme activity, such as the absence of nuclease activity.

[0036] In one embodiment, the enzyme activity is DNase activity. Thus, in one aspect, the present disclosure provides methods and DNA-RNA hybrid molecules wherein the DNase activity in a sample clips the RNA molecule from the DNA-RNA hybrid molecule, such that the clipped RNA molecule can be quantified as a measure of DNase activity in the sample.

Structure of DNA/R A hybrids.

[0037] In some embodiments of the present disclosure, engineered stem-loop (hairpin-like) DNA-RNA hybrids are used as DNase substrates for the quantification of DNase activity. In one embodiment, each DNA-RNA hybrid is made up of two complimentary strands of DNA and a RNA molecule. In some embodiments, the complimentary strands of DNA are each 11 bases long. In other embodiments, the complimentary strands are each 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more bases long. In one embodiment, the two complimentary strands of DNA comprise a single cleavage site for a particular enzyme. In a further embodiment, the single cleavage site is an 11 base pair cleavage site for DNase. In another embodiment, the cleavage site is a specific cleavage site for a particular enzyme, such as a particular DNA conformation or sequence. In a yet further embodiment, the single cleavage site is a sequence that is specifically recognized by an enzyme, such as a restriction enzyme.

[0038] In some embodiments, the DNA sequence may be any sequence, so long as the strands are capable of complimentary pairing to form a double stranded DNA. In further embodiments, the DNA sequence may be any 11 base pair sequence. In other embodiments, the DNA sequence may be a specific DNA sequence that provides for specific recognition by an enzyme, including any enzyme or substrate with enzymatic activity known in the art.

[0039] In some embodiments, the RNA molecule is located between the single stranded DNA sequences, such that once the complimentary DNA sequences hybridize to form a double stranded DNA sequence, the DNA portion of the DNA-RNA hybrid forms a stem-like DNA double helix and the adjacent RNA molecule forms a loop structure. This stem-loop (hairpin) structure remains stable when the temperature is below 50°C. This ensures that double-stranded DNA is present during the DNA cleavage step in the methods disclosed herein.

[0040] Given that DNase non-specifically cleaves DNA by targeting its minor groove, and, a complete turn of double helix is made up with 10.5 base pairs, a DNA-RNA hybrid having an 11 base pair DNA ensures that the DNA portion in the hybrid has only one minor groove to be targeted by DNases. Therefore, the cleavage of the DNA-RNA hybrids disclosed herein comprising an 11 base pair double-stranded DNA is a specific and controlled cleavage. In one embodiment, the 11 base pair double-stranded DNA may be any random 11 base pairs.

[0041] The skilled person will understand that the activity (or absence of activity) of any enzyme may be determined using the compositions and methods provided herein, by designing the DNA portion of the DNA-RNA hybrid to be specifically recognized by a particular enzyme. For example, in some embodiments, the DNA portion of the DNA-RNA hybrid molecule is cleaved by a restriction enzyme. The restriction enzyme may be any restriction enzyme known in the art including, but not limited to, EcoRI, EcoRII, EcoRV, Sfil, Bamffl, Hindlll, Xhol, Notl, Smal, Haelll, Xbal, Sacl, SacII, and Taql. For example, in one embodiment, the DNA portion of the DNA-RNA hybrid molecule is an EcoRI restriction enzyme cleavage sequence, which comprises the following 5' to 3' sequence: GAATTC. In another embodiment, the DNA portion of the DNA-RNA hybrid molecule is a Sfil restriction enzyme cleavage sequence, which comprises a 5' to 3' sequence according to SEQ ID NO: 8 (GGCCNNNNNGGCC). The skilled artisan will understand that any DNA sequence corresponding to a restriction enzyme site may be used to form the DNA portion of the DNA-RNA hybrid molecule. Thus, in one embodiment, the present disclosure provides methods for quantifying restriction enzyme activity in a sample.

[0042] Thus, in some embodiments, the DNA-RNA hybrid comprises a double stranded DNA that is 11 base pairs, longer than 11 base pairs, or shorter than 11 base pairs, and that is specifically recognized by a particular enzyme. In one embodiment, the DNA portion of the DNA-RNA hybrid comprises a single cleavage site for a particular enzyme. In one embodiment, the single cleavage site provides for release of a single RNA molecule such that the activity of the enzyme can be quantified. In one embodiment, the methods, kits, mixtures, and DNA-RNA hybrids provided herein allow for the determination of the absence of enzyme activity, such as the absence of nuclease activity.

[0043] The RNA molecule in the DNA-RNA hybrid, in some embodiments, is any RNA molecule, such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA) synthetic RNA or microRNA. MicroRNAs (used interchangeably herein with "miRNA") are small, non-coding RNA molecules that generally regulate gene expression and are known in the art and described, for example, in Bartel Cell 116; 281 (2004), which is incorporated herein by reference in its entirety. MIR234 is one microRNA gene family. Other microRNAs or RNA molecules may be used in the invention. The RNA molecule may be about 10 to about 50 bases, or about 15 to about 40 bases, or about 18 to about 25 bases, or about 19 to about 21 bases.

[0044] Thus, in some embodiments, the DNA-RNA hybrids provided herein are DNA- microRNA hybrids comprising an 11 base pair DNA and a microRNA such as, for example, MIR234.

[0045] In some embodiments, the DNA-RNA hybrid further comprises a reporter dye and/or a quencher dye for detection of the clipped RNA. Thus, in some embodiments, each DNA-RNA hybrid comprises a double-stranded DNA sequence having 11 base pairs, a RNA molecule having 19-21 bases, a reporter dye (e.g., Cy5) modified at its 5'end, and a dark quencher modified at its 3' end.

[0046] As described above, the DNA sequence may be any DNA sequence. In some embodiments, the DNA sequence is an 11 -base pair DNA sequence. It has been suggested that the catalytic efficiency of DNase is sensitive to the width of the minor groove (Sutton et al, 1997). The present disclosure encompasses modulation of the width of the minor groove by changing the DNA sequence to characterize the DNase in greater detail. For example, the present disclosure provides methods for determining the effect of the width of the minor groove on the catalytic efficiency of DNase.

[0047] In one embodiment, the DNA sequence is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4. In one embodiment, The A4T4 hybrids disclosed herein comprise a DNA sequence according to SEQ ID NO: 1 and the reverse complement (SEQ ID NO: 2). In another embodiment, the A2T2 hybrids disclosed herein comprise a DNA sequence according to SEQ ID NO: 3 and its reverse complement (SEQ ID NO: 4). In another embodiment, the microRNA is MIR234, having a sequence according to SEQ ID NO: 5. In one embodiment, the DNA-RNA hybrid is an A4T4-MIR234 hybrid, having the following sequence: CGAAAATTTTCrUrUrArUrUrGrCrUrCrGrArGrArArUrArCrCrCrUrUGAAAATTTTCG (SEQ ID NO: 6). In other embodiments, the DNA-RNA hybrid is an A2T2-MIR234 hybrid, having the following sequence:

CGCGAATTCGCrUrUrArUrUrGrCrUrCrGrArGrArArUrArCrCrCrUrUGCGAATTCGCG (SEQ ID NO: 7). Cleavage of the DNA molecule and release of the RNA molecule

[0048] DNase non-specifically cleaves DNA molecules by targeting their minor grooves; DNase does not cleave RNA. In some embodiments, the 11 base pair length of the double-stranded DNA ensures the DNA portion in the hybrid has only one minor groove for the DNA cleavage by DNase, because DNase non-specifically cleaves DNA by targeting its minor groove, and a complete turn of a double helix is 10.5 base pairs. Thus, DNase cleaves the DNA-RNA hybrid molecule at the single DNase cleavage site, releasing, or "clipping," the RNA molecule from the hybrid. In some embodiments, the DNA portion of the hybrid is longer or shorter than 11 base pairs, and comprises a single cleavage site for an enzyme of interest, such that the RNA molecule is released or clipped from the hybrid.

[0049] The DNA-RNA hybrids disclosed herein form hairpin-like structures at temperatures below about 50°C. In some embodiments, the hairpin structure becomes completely linearized when the temperature is above 80°C. Therefore, the clipping reaction (i.e., the cleavage of the DNA molecule and release of the RNA molecule) is carried out, in some embodiments, at a temperature from between about 10°C to about 45°C to ensure that the hairpin structure is stabilized and that the enzyme cleaves only double-stranded DNA, and not single stranded DNA. In some embodiments, the clipping reaction is carried out at about 10°C. In some embodiments, the clipping reaction is carried out at about 25°C. In some embodiments, the clipping reaction is carried out at about 37°C.

[0050] The specific cleavage reaction provides predictable and controlled release of the RNA molecule. Thus, the compositions and methods provided herein enable the determination of enzyme kinetics, i.e. Vmax or Km. Further, the compositions and methods provided herein provide the ability to detect enzyme kinetics or enzyme activity due to change in protein structure or protein post-translational modifications.

Detection of clipped RNA

[0051] Once cleaved, the RNA molecule is disconnected from the hybrid. Total released RNA (also referred to herein as "clipped RNA") can then be detected by any method known in the art. In one embodiment, RT-PCR is used to detect clipped RNA. Thus, in one embodiment, the total clipped RNA is reverse transcribed to synthesize cDNA. In one embodiment, the synthesized cDNA is quantified by quantitative PCR (qPCR). In one embodiment, reverse transcription and qPCR are performed using Life Technologies Taqman® MicroRNA assay.

[0052] In PCR, determining the transition point at the end of the baseline region (the cycle threshold, or "CT") is useful for measuring the efficiency of the PCR process. The CT value can be used to measure the absolute or relative copy numbers of the molecule.

Clipping reagent and clipping assay

[0053] In one aspect, provided herein are compositions, mixtures, methods, kits, and assays for detecting enzyme activity in a sample. In some embodiments, the compositions, mixtures, methods, kits, and assays comprise clipping reagents, and the use of clipping reagents to detect enzyme activity in a sample. In some embodiments, the methods, kits, and assays provided herein comprise a clipping assay (also referred to herein as "clipping reaction") in which the RNA molecule is clipped, or disconnected, from the DNA-RNA molecule described herein. In one embodiment, the clipping assay comprises three steps: DNA cleavage, reverse transcription of released/clipped RNA molecules, and quantification of RNA molecules by qPCR. Figure 1 provides a schematic depiction of the three steps of the clipping reaction.

[0054] In one embodiment, the clipping reagent comprises DNA-RNA hybrids disclosed herein. In a further embodiment, the clipping reagent further comprises a reaction buffer. In one embodiment, the reaction buffer is a DNase reaction buffer, wherein the DNase reaction buffer comprises magnesium and calcium. In one embodiment, the clipping reagent is in PCR tubes. In another embodiment, the preparation of the clipping reagent is at about 4°C. In a further embodiment, preparation of the clipping reagent at about 4°C ensures that the enzymes remain inactive during preparation of the clipping reagent.

DNA cleavage

[0055] Once the clipping reagent has been prepared, in one embodiment, the clipping assay is performed using any temperature adjustable machine. For example, in one embodiment, the assay is performed on a PCR machine or thermocycler. In one embodiment, the clipping reagent is subjected to a hold step at about 4°C, followed by an activation step at about 10°C to about 37°C, followed by an inactivation step at about 80°C to about 85°C. [0056] In one embodiment, the hold step is performed on a cold plate, to ensure the enzyme is inactive during reagent preparation. In one embodiment, the hold step is for about 1 minute to about 30 minutes. In a further embodiment, the hold step is from about 2 minutes to about 15 minutes. In a further embodiment, the hold step is for about 5 minutes.

[0057] In one embodiment, the clipping reaction is initiated (activation step) by placing the tubes on a temperature adjustable machine such as a thermocycler or PCR machine. In one embodiment, the clipping reaction is initiated by raising the temperature to a temperature in the range of about 10°C to about 40°C. In one embodiment, the clipping reaction is initiated by raising the temperature to about 10°C, about 15°C, about 20°C, about 25°C, about 30°C , about 35°C, about 37°C, or about 40°C. In one embodiment, the clipping reaction is initiated by raising the temperature to about 25°C. In one embodiment, the clipping reaction is initiated by raising the temperature to about 37°C. In one embodiment, the activation step is for about 1 minute to about 60 minutes. In a further embodiment, the activation step is for about 2 minutes to about 30 minutes. In a further embodiment, the activation step is for about 4 minutes to about 15 minutes. In a further embodiment, the activation step is for about 10 minutes. In one embodiment, the whole clipping time is about 3 minutes.

[0058] In one embodiment, the clipping reaction is stopped (inactivation step) following the activation step. In another embodiment, the inactivation step is for about 1 minute to about 60 minutes. In a further embodiment, the inactivation step is for about 2 minutes to about 30 minutes. In a further embodiment, the inactivation step is for about 4 minutes to about 15 minutes. In a further embodiment, the inactivation step is for about 5 minutes. In another embodiment, the inactivation step is for about 10 minutes.

[0059] In one embodiment, the hold step is for about 5 minutes, the activation step is for about 10 minutes, and the inactivation step is for about 10 minutes.

Reverse transcription of released/clipped RNA molecules

[0060] After the clipping reaction, clipped RNA molecules are free from the hybrids. In one embodiment, total released RNA molecules are then reverse transcribed to cDNA. In one embodiment, the RNA molecules are microRNA molecules, which are reverse transcribed using Life Technologies' TAQMAN® microRNA assay kit. The TAQMAN® miRNA assay kit utilizes a stem-loop primer that specifically targets microRNA for cDNA synthesis. The stem- loop structure in the tail of the primer provides specific detection of microRNA molecules. Reverse transcription of non-cleaved microRNA molecules would be inhibited since the 3 ' end of the non-cleaved microRNA is hinged by the 11 bases of DNA. Thus, the TAQMANI® miRNA assay kit specifically reverse transcribes clipped microRNA.

Quantification of RNA molecules by qPCR

[0061] In one embodiment, the next step of clipping assay is to quantify total cDNA molecules. Quantification may be by any method known in the art, including by qPCR. In one embodiment, the Life Technologies' TAQMAN® microRNA assay kit is used for quantification. This kit utilizes a miRNA-specific forward primer, reverse primer, and dye-labeled TaqMan probes for specific quantification. In one embodiment, the cycle threshold (Ct) value is used to quantify enzyme activity, since it is proportional to the clipped RNA molecules. For example, in a given reaction time, higher enzyme activity has lower CT value.

Kits and mixtures

[0062] In some embodiments, the present disclosure also provides kits and mixtures for use in the methods disclosed herein. The kits and mixtures provided herein comprise any one or more of the components disclosed herein. For example, the present disclosure provides kits and mixtures comprising the DNA-RNA hybrids disclosed herein. The kits and mixtures may further comprise a RNase inhibitor and a reaction buffer. The mixtures may further comprise RNA (e.g., clipped RNA). RNase inhibitors are known in the art and include, without limitation, reagents such as RNAlater (Ambion) and RNA protect Bacteria Reagent (Qiagen), chemicals such as guanidinium isothiocynate and diethyl-pyrocarbonate, recombinant RNase inhibitors, human placental RNase inhibitors, porcine RNase inhibitors, rat RNase inhibitors, and anti-nuclease antibodies. In some embodiments, the mixtures provided herein comprise a DNA-RNA hybrid, DNase reaction buffer, and a RNAse inhibitor. In further embodiments, the mixtures provided herein further comprise DNase. In still further embodiments, the mixtures provided herein further comprise clipped RNA. DNase reaction buffers are known in the art and may comprise magnesium and calcium.

[0063] In some embodiments, the kits provided herein comprise a DNA-RNA hybrid and a reaction buffer. In further embodiments, the kits comprise DNA-RNA hybrid molecules comprising an 11 base pair DNA as described herein, and a DNase reaction buffer. Kit components may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube. In some embodiments, the kits disclosed herein comprise one or more reagents for use in the embodiments disclosed herein. For example, a kit may provide one or more buffers, such as, for example, storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). Suitable buffers include, but are not limited to, phosphate buffered saline, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. As used herein, the terms "clip" and "clipping" refer to the cleavage of the DNA from the RNA molecule of the DNA-RNA hybrids provided herein. Thus, the clipped RNA or clipped microRNA is RNA or microRNA that has been cleaved from the DNA structure by an enzyme. For example, the clipped RNA or clipped microRNA is a RNA or microRNA that has been cleaved from the DNA structure by DNase activity.

[0064] In one aspect, the present disclosure provides methods and compositions for determining enzyme kinetics. For example, in one embodiment, the Vmax and Km values of enzyme activity can be assessed using the compositions and methods provided herein. In one aspect, the present disclosure provides compositions and methods that allow detection of enzyme kinetics or enzyme activity that is due to changes in protein structure or protein post-translational modification. For example, in one embodiment, the provided compositions and methods allow the differentiation of the difference between deamidated DNase and non-deaminated DNase, with regard to enzyme activity.

[0065] In one aspect, the disclosure provides methods for synthesizing the DNA-RNA hybrids disclosed herein, as well as methods for assessing the identity of DNA-RNA hybrids and oligonucleotides. DNA-RNA hybrids and other hybrid molecules and oligonucleotides may be generated and assessed by any method known in the art. In one embodiment, the DNA-RNA hybrids provided herein are synthesized and analyzed by mass spectrometry. In further embodiments, the mass spectrometry is Integrated DNA Technologies (IDT) electrospray ionization mass spectrometry (ESI) or matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In particular embodiments, the DNA-RNA hybrids are analyzed by ESI.

[0066] The use of the singular includes the plural unless specifically stated otherwise. The word "a" or "an" means "at least one" unless specifically stated otherwise. The use of "or" means "and/or" unless stated otherwise. The meaning of the phrase "at least one" is equivalent to the meaning of the phrase "one or more." Furthermore, the use of the term "including," as well as other forms, such as "includes" and "included," is not limiting. Also, terms such as "element" or "component" encompass both elements or components comprising one unit and elements or components comprising more than one unit unless specifically stated otherwise. The term "about," as used herein, refers to an amount or value that one of skill in the art would understand to be close to the stated amount or value. For example, the term "about" may refer to an amount or value that is within 20% of the stated amount or value.

[0067] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.

EXAMPLES

Example 1. Synthesis of the DNA-microRNA hybrid A4T4-MIR234-Cy5

[0068] The DNA/microRNA hybrid A4T4-MIR234-Cy5 was synthesized and analyzed by IDT Oligo Electrospray Ionization Mass Spectrometry (ESI). The sequence of this DNA-microRNA hybrid is : 5 ' -Cy 5 -CGAAAATTTTCrUrUr ArUrUrGrCrUrCrGr ArGr Ar ArUr ArCrCrCrUrUG AAAATTTTCG-IAbRQSp-3 ' , where r denotes RNA, Cy5 denotes a Cy5 reporter dye, and IAbRQSp denotes a quencher dye. The IDT Oligo EST analysis is provided in Figure 2.

[0069] To ensure the A4T4-MIR234-Cy5 could form a hairpin structure, melting curve analyses were performed to monitor the photon release. The photon is quenched when the A4T4-MIR234- Cy5 is in the hairpin structure. The results of the study are shown in Figure 3, and indicate that A4T4-MIR234-Cy5 formed a hairpin structure at temperatures below 50°C. In addition, the hairpin structure becomes completely linearized when the temperature is above 80°C, as shown by the release of photons from the reporter dye, in Figure 3. Since the clipping reaction is carried out at 37°C, the results of the study show that the stabilized hairpin structure at this temperature ensures that the DNase only targets the double-stranded DNA, and not single-stranded DNA. Example 2. Synthesis of the DNA-microRNA hybrid A2T2-MIR234-Cy5

[0070] The DNA/microRNA hybrid A2T2-MIR234-Cy5 was synthesized and analyzed by IDT Oligo Electrospray Ionization Mass Spectrometry (ESI). The sequence of this DNA-microRNA hybrid is: 5'-Cy5-CGCGAATTCGCrUrUrArUrUrGrCrUrCrGrArGrArArUrArCrCrCrUrUGCG AATTCGCG-IAbRQSp-3 ', where r denotes RNA, Cy5 denotes a Cy5 reporter dye, and IAbRQSp denotes a quencher dye. The IDT Oligo EST analysis is provided in Figure 4.

[0071] To ensure the A2T2-MIR234-Cy5 could form a hairpin structure, melting curve analyses were performed to monitor the photon release. The photon is quenched when the A2T2-MIR234- Cy5 is in the hairpin structure. The results of the study are shown in Figure 5, and indicate that, similar to A4T4-MIR234-CY5, the A2T2-MIR234-Cy5 was linearized at temperatures above 80°C, and that it formed a stable hairpin structure at temperatures below 50°C.

Example 3. Real-time activity monitoring of clipping assay

[0072] To ensure that the A4T4-MIR234-Cy5 and A2T2-MIR234-Cy5 could be cleaved by DNase, clipping reactions with 2, 0.02, 0.002, or 0 units (U) of DNase were performed at 25°C on a thermocycler. Specifically, 2, 0.02, 0.002, or 0 U DNase was added to a mixture containing DNase reaction buffer and either A4T4-MIR234-Cy5 or A2T2-MIR234-Cy5, and the clipping reaction was carried out at 25°C. Upon cleavage, the photon was released, and therefore could be monitored by the thermocycler in real time.

[0073] The results of the study are provided in Figure 6 (A4T4-MIR234-Cy5) and Figure 7 (A2T2-MIR234-Cy5). Both A4T4-MIR234-Cy5 and A2T2-MIR234-Cy5 were cleaved by DNase in a dose-dependent fashion.

Example 4. Quantification of clipped MIR234 molecules

[0074] Clipping reactions with A4T4-MIR234-Cy5 or A2T2-MIR234-Cy5 in the presence of 2 U of DNase were conducted as described above in Example 3. Clipped MIR234 molecules were reverse transcribed to cDNA using Life Technologies' TAQMAN® microRNA assay kit. The TAQMAN® miRNA assay kit utilizes stem-loop primer that specifically targets MIR-234 for cDNA synthesis. The stem-loop structure in the tail of the primer provides specific detection of miR-234 molecules. Quantification of miR-234 was also accomplished by use of Life Technologies' TAQMAN® microRNA assay kit, in which utilizes miRNA-specific forward primer, reverse primer, and a dye-labeled TaqMan probes for specific quantification

[0075] The results of the study are shown in Figure 8. Both A4T4-MIR234-Cy5 and A2T2- MIR234-Cy5 were clipped, reverse transcribed, and quantified, indicating that the DNA-RNA hybrids were effective for the quantification of DNase activity. The no-template control showed no detectable signal.

Example 5. Time-dependent study of clipping assay

[0076] A time-dependent study of the clipping reaction was conducted, using A4T4-MIR234- Cy5 as substrates. Clipping was performed at room temperature for 30 seconds, 1 minute, 2 minutes, or 3 minutes. Clipped RNA was reverse transcribed and quantified by qPCR as described above in Examples 3 and 4. After quantification, threshold values for each clipping reaction were determined. The results of the study are provided in Figure 9 and Table 1. The data indicated that clipping for 3 minutes results in more detectable (i.e., lower threshold value) microRNA molecules.

Table 1. Threshold value for varying clipping reaction times

Example 6. Comparison of clipping assay to methyl green method for DNase activity

[0077] Recombinant human DNase 1 (pulmozyme) was used as a reference standard for the determination of relative activity of 5 different samples, as measured by the clipping assay disclosed herein or the standard methyl green method. The results of the study are provided in Table 2. The study showed that the clipping assay provided herein is comparable to the methyl green assay, but that it exhibits better sensitivity. The clipping assay provided herein also requires less DNase input for DNA cleavage, and therefore has better resolution. Therefore, clipping assay has the ability to detect enzymatic kinetics or enzyme activity, due to changes in protein structure of protein post-translational modifications. Thus, the compositions, methods, kits, and assays provided herein provide a more efficient, reliable method for testing the activity of DNase in a sample.

Table 2. Sensitivity of the Clipping Assay versus Methyl Green Assay

[0078] The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference for all purposes. References

[0079] Sutton et al; "The dependence of DNasel activity on the conformation of oligodeoxy- nucleotides "; Biochem. J. 321 : 481-486 (1997)

[0080] Boogaard et al; "Recombinant Human Deoxyribonuclease in Infants with Respiratory Syncytial Virus Bronchiolitis"; Pediatr. Pulmonol. 131 :3 (2007)

[0081] Gaipl et al; "Clearance of apoptotic cells in human SLE." Curr. Dir. Autoimmun. 9: 173- 183 (2006)

[0082] Tolun and Myers; "A real-time DNase assay (ReDA) based on PicoGreen fluorescence" Nuc. Acid Res. 31 :el l l (2003)

[0083] Shak et al; "Colorimetric determination of DNase I activity with a DNA-methyl green substrate." Anal. Biochem. 222:351-358 (1994)

Claims

1. A DNA-RNA hybrid molecule comprising a first single stranded DNA, a second single stranded DNA that is complementary to the first single stranded DNA, and a RNA molecule, wherein the first and second single stranded DNAs are capable of pairing to form a double stranded DNA comprising an enzyme cleavage site, and wherein the RNA molecule is released from the DNA molecule upon cleavage at the enzyme cleavage site.

2. The DNA-RNA hybrid molecule of claim 1 , wherein the RNA molecule is located between the first and second single stranded DNAs, and wherein the first and second single stranded DNAs pair to form a stable stem-loop structure at a temperature at or below 50°C.

3. The DNA-RNA hybrid molecule of claim 1, wherein the RNA molecule is a microRNA molecule.

4. The DNA-RNA hybrid molecule of claim 3, wherein the microRNA is MIR234 (SEQ ID NO: 5).

5. The DNA-RNA hybrid molecule of claim 1, wherein the RNA molecule comprises about 10 to about 50 bases.

6. The DNA-RNA hybrid molecule of claim 4, wherein the RNA molecule comprises about 15 to about 30 bases.

7. The DNA-RNA hybrid molecule of claim 6, wherein the RNA molecule comprises about 19 to about 21 bases.

8. The DNA-RNA hybrid molecule of claim 1, wherein the molecule further comprises a reporter dye and a quencher dye.

9. The DNA-RNA hybrid molecule of claim 8, wherein the reporter dye is selected from the group consisting of cyanine dyes, fluorescein dyes, and rhodamine dyes.

10. The DNA-RNA hybrid molecule of claim 9, wherein the cyanine dye is selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5.

11. The DNA-RNA hybrid molecule of claim 8, wherein the reporter dye is Cy5 and the quencher dye is IAbRQSp.

12. The DNA-RNA hybrid molecule of claim 1, wherein the first and second single stranded DNAs are each 11 bases long.

13. The DNA-RNA hybrid molecule of claim 12, wherein the first and second single stranded DNAs comprise sequences according to SEQ ID NO: 1 (CGAAAATTTTC) and SEQ ID NO: 2 (GAAAATTTTCG), respectively; or wherein the first and second single stranded of DNAs comprise sequences according to SEQ ID NO: 3 (CGCGAATTCGC) and SEQ ID NO: 4 (GCGAATTCGCG), respectively.

14. The DNA-RNA hybrid molecule of claim 12, wherein the DNA-RNA hybrid molecule comprises, from 5' to 3': SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 2.

15. The DNA-RNA hybrid molecule of claim 12, wherein the DNA-RNA hybrid molecule comprises, from 5' to 3': SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 4.

16. The DNA-RNA hybrid molecule of claim 14 or 15, wherein the molecule further comprises Cy5 dye at the 5' terminus and IAbRQSp dye at the 3' terminus.

17. A mixture comprising

(i) a DNA-RNA hybrid molecule comprising a first single stranded DNA, a second single stranded DNA that is complementary to the first single stranded DNA, and a RNA molecule, wherein the first and second single stranded DNAs are capable of pairing to form a double stranded DNA comprising an enzyme cleavage site;

(ii) RNase inhibitor

(iii) an enzyme; and

(iv) a reaction buffer.

18. The mixture of claim 17, further comprising RNA.

19. The mixture of claim 17, wherein the RNA molecule is located between the first and second single stranded DNAs, and wherein the first and second single stranded DNAs pair to form a stable stem-loop structure at a temperature at or below 50°C.

20. The mixture of claim 17, wherein the RNA molecule is a microRNA molecule.

21. The mixture of claim 20, wherein the microRNA is MIR234 (SEQ ID NO: 5).

22. The mixture of claim 17, wherein the RNA molecule comprises about 10 to about 50 bases.

23. The mixture of claim 22, wherein the RNA molecule comprises about 15 to about 30 bases.

24. The mixture of claim 23, wherein the RNA molecule comprises about 19 to about 21 bases.

25. The mixture of claim 17, wherein the DNA-RNA hybrid molecule further comprises a reporter dye and a quencher dye.

26. The mixture of claim 25, wherein the reporter dye is selected from the group consisting of cyanine dyes, fluorescein dyes, and rhodamine dyes.

27. The mixture of claim 26, wherein the cyanine dye is selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5.

28. The mixture of claim 27, wherein the reporter dye is Cy5 and the quencher dye is IAbRQSp.

29. The mixture of claim 17, wherein the first and second DNAs are each 11 bases long, wherein the enzyme is DNase, and wherein the reaction buffer is DNase reaction buffer.

30. The mixture of claim 29, wherein the first and second single stranded DNAs comprise sequences according to SEQ ID NO: 1 (CGAAAATTTTC) and SEQ ID NO: 2

(GAAAATTTTCG), respectively; or wherein the first and second single stranded of DNAs comprise sequences according to SEQ ID NO: 3 (CGCGAATTCGC) and SEQ ID NO: 4 (GCGAATTCGCG), respectively.

31. The mixture of claim 29, wherein the DNA-RNA hybrid molecule comprises, from 5 ' to 3 ' : SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 2.

32. The mixture of claim 29, wherein the DNA-RNA hybrid molecule comprises, from 5' to 3': SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 4.

33. The mixture of claim 31 or 32, wherein the molecule further comprises Cy5 dye at the 5' terminus and IAbRQSp dye at the 3' terminus.

34. A method for detecting enzyme activity in a sample, the method comprising the steps of:

(a) preparing a reagent comprising (i) a DNA-RNA hybrid molecule, wherein the DNA- RNA hybrid molecule comprises a RNA molecule and a double stranded DNA comprising an enzyme cleavage site; and (ii) reaction buffer;

(b) placing the reagent preparation of step (a) in a thermo cycler and subjecting the reagent preparation to a hold step, an activation step, and an inactivation step to obtain the RNA that has been clipped from the DNA-RNA hybrid molecule; and

(c) detecting the clipped RNA, thereby determining the enzymatic activity of the enzyme in the sample.

35. The method of claim 34, wherein the hold step of step (b) is at 4°C for about 5 minutes; the activation step is at about 37°C for about 10 minutes; and the inactivation step is at 85°C for about 10 minutes.

36. The method of claim 34, wherein the enzyme is DNase and the reaction buffer is DNase reaction buffer.

37. The method of claim 36, wherein the DNase reaction buffer comprises magnesium and calcium.

38. The method of claim 36, wherein the DNase binds to the minor groove of the double stranded DNA during the activation step of step (b).

39. The method of claim 36, wherein the clipped RNA is detected in step (c) by obtaining and reverse transcribing to cDNA the RNA that is clipped from the DNA-RNA hybrid molecule, and performing quantitative PCR to quantitate the clipped RNA; and wherein the DNase activity in the sample is determined by calculating the threshold value of the quantitative PCR.

40. A kit for detecting enzyme activity in a sample, the kit comprising (i) a DNA-RNA hybrid molecule, wherein the DNA-RNA hybrid molecule comprises a RNA molecule and a double stranded DNA comprising an enzyme cleavage site; and (ii) reaction buffer.

41. The kit of claim 40, wherein the enzyme is DNase.

42. A method for synthesizing a DNA-RNA hybrid molecule comprising a first single stranded DNA, a second single stranded DNA that is complementary to the first single stranded DNA, and a RNA molecule;

wherein the first and second single stranded DNAs are capable of pairing to form a double stranded DNA comprising an enzyme cleavage site, and wherein the RNA molecule is released from the DNA molecule upon cleavage at the enzyme cleavage site; and

wherein the DNA-RNA hybrid molecule is analyzed by mass spectrometry.

43. The method of claim 42, wherein the mass spectrometry is electrospray ionization mass spectrometry (ESI).