Using tethered enzymes to detect nucleic acids

Abstract

To provide a method of detecting a target nucleic acid molecule in a sample, and compositions and kits useful for implementation.SOLUTION: The method includes providing a sample containing a target nucleic acid molecule and a capture oligonucleotide molecule. In one embodiment, the capture oligonucleotide molecule has (i) a length of 30-60 base pairs, (ii) a 4-8 base pair overhang on its 3' end, (iii) a 5' tail, (iv) a target-specific portion between the 3' end and the 5' tail, (v) a deoxy-adenosine diphosphate content of 40-50%, (vi) no deoxy thymidine phosphate in the 3' end or the 5' tail, and (vii) the 3' end and the 5' tail having an ATP content which is 40-50% of the ATP content of the capture oligonucleotide molecule. In another aspect of the detecting method, certain reagents are coupled to a solid support.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 810,448, filed Feb. 26, 2019, which is hereby incorporated by reference in its entirety.

[0002] Field This application relates to the use of tethered enzymes for detecting nucleic acids.

Background Art

[0003] Background Nucleic acid amplification can be used to determine whether a specific template nucleic acid is present in a sample. If an amplification product is generated, this indicates the presence of the template nucleic acid in the sample. Conversely, if no amplification product is generated, it indicates the absence of the template nucleic acid in the sample. Such techniques are very important for diagnostic applications, for example, when determining whether a pathogen is present in a sample.

[0004] Nucleic acids can be amplified by various thermocycling techniques and isothermal techniques. Thermocycling techniques such as polymerase chain reaction (PCR) use temperature cycling to repeat cycles of DNA synthesis, synthesizing large amounts of new DNA depending on the initial amount of template DNA. Recently, many isothermal techniques that do not rely on thermocycling to drive the amplification reaction have also been developed. For amplification reactions that do not involve an RNA synthesis step, isothermal techniques that utilize DNA polymerases with strand displacement activity have been developed. Similarly, for amplification reactions that involve an RNA synthesis step, isothermal techniques that use reverse transcriptase, RNase H, and DNA-dependent RNA polymerase have been developed.

[0005] Nevertheless, for the identification and classification of microorganisms, the diagnosis of infectious diseases, the detection and characterization of genetic abnormalities, the identification of genetic changes associated with cancer, the study of genetic susceptibility to diseases, and the measurement of responses to various types of treatment, the detection and / or quantification of specific nucleic acid sequences are important techniques. Such procedures are also useful for the detection and quantification of microorganisms in food, water, industrial and environmental samples, primary species, and other types of materials where it may be necessary to monitor the presence of specific microorganisms. Other applications are in forensic science, anthropology, archaeology, and biology, where measurements of nucleic acid sequence relatedness are used to identify criminal suspects, resolve paternity disputes, construct genealogical and phylogenetic trees, and assist in the classification of various forms of life.

[0006] Advances in the field of molecular biology over the past 20 years have made it possible to detect specific nucleic acid sequences in test samples obtained from patients and other subjects. Such test samples include serum, urine, feces, saliva, amniotic fluid, and other body fluids. Accordingly, many methods for detecting and / or quantifying nucleic acid sequences are well known in the art. However, the generation of by-products is an inherent result of high-sensitivity nucleic acid amplification systems. By-products include molecules that can interfere with the amplification reaction in some systems, thereby reducing specificity. This is because the limited amplification resources, including primers and enzymes required for primer extension and transcript formation, are diverted to the formation of by-products. In some cases, the appearance of by-products can complicate the analysis of amplicon generation by various molecular techniques. Furthermore, in many cases of interest, the specific nucleic acid sequence is present only at very low concentrations in the sample being tested for the desired nucleic acid sequence. In such cases, the presence of the desired molecule cannot be detected if the assay sensitivity cannot be increased.

[0007] The present application aims to overcome these and other deficiencies in the art. SUMMARY OF THE INVENTION

[0008] Summary One aspect of the present application relates to a method for detecting a target nucleic acid molecule in a sample. The method includes the steps of preparing a sample containing the target nucleic acid molecule, and contacting the sample with a capture oligonucleotide molecule that is complementary to at least a portion of the target nucleic acid molecule, such that the capture oligonucleotide molecule hybridizes to the complementary portion of the target nucleotide molecule to form a double-stranded nucleic acid molecule. The capture oligonucleotide molecule has (i) a length of 30 to 60 base pairs, (ii) an overhang of 4 to 8 base pairs at its 3' end, (iii) a 5' tail, (iv) a target-specific portion between the 3' end and the 5' tail, (v) a deoxyadenosine diphosphate content of 40 to 50%, (vi) no deoxythymidine phosphate at the 3' end or the 5' tail, and (vii) an ATP content that is 40 to 50% of the ATP content of the capture oligonucleotide molecule at the 3' end and the 5' tail. The double-stranded nucleic acid molecule, polymerase, and dNTP mixture are contacted with each other to form a polymerase extension mixture. The polymerase extension mixture is subjected to conditions under which the target nucleic acid molecule is extended to release free phosphate. Then adenosine triphosphate is generated from the released free phosphate, and the adenosine triphosphate generated from the free phosphate is metabolized by luciferase to generate a bioluminescence readout signal indicating the presence of the target nucleic acid molecule in the sample.

[0009] Another aspect of the present application relates to a method for detecting a target nucleic acid molecule in a sample. The method includes the steps of preparing a sample containing the target nucleic acid molecule, and contacting the sample with a capture oligonucleotide molecule that is complementary to at least a portion of the target nucleic acid molecule, such that the capture oligonucleotide molecule hybridizes to the complementary portion of the target nucleic acid molecule to form a double-stranded nucleic acid molecule. The double-stranded nucleic acid molecule, a polymerase, and a dNTP mixture are contacted with each other to form a polymerase extension mixture. The polymerase extension mixture is subjected to conditions under which the target nucleic acid molecule is extended to release free phosphate. Subsequently, adenosine triphosphate is enzymatically generated from the released free phosphate, and the adenosine triphosphate generated from the free phosphate is metabolized by luciferase to generate a bioluminescence readout signal indicating the presence of the target nucleic acid molecule in the sample. The DNA polymerase, the luciferase, and the enzyme that generates adenosine triphosphate are each bound to a solid support.

[0010] Another aspect of the present application relates to a kit for detecting a target nucleic acid molecule in a sample. The kit includes a capture oligonucleotide molecule that is complementary to at least a portion of the target nucleic acid molecule, such that the capture oligonucleotide molecule hybridizes to the complementary portion of the target nucleic acid molecule to form a double-stranded nucleic acid molecule, a polymerase bound to a solid support, a dNTP mixture, an enzyme bound to a solid support for generating adenosine triphosphate from the released free phosphate, and a luciferase bound to a solid support for generating a bioluminescence readout signal.

[0011] Another aspect of the present application relates to a kit for detecting a target nucleic acid molecule in a sample. The kit includes a capture oligonucleotide molecule that is complementary to at least a portion of the target nucleic acid molecule, and as a result, hybridizes to the complementary portion of the target nucleic acid molecule to form a double-stranded nucleic acid molecule. The capture oligonucleotide molecule has (i) a length of 30 to 60 base pairs (bp), (ii) an overhang of 4 to 8 base pairs at its 3' end, (iii) a 5' tail, (iv) a target-specific portion between the 3' end and the 5' tail, (v) a deoxyadenosine diphosphate content of 40 to 50%, (vi) no deoxythymidine phosphate at the 3' end or the 5' tail, and (vii) 3' and 5' tails having an ATP content that is 40 to 50% of the ATP content of the capture oligonucleotide molecule. The kit also includes a polymerase, a dNTP mixture, an enzyme for generating adenosine triphosphate from released free phosphate, and a luciferase for generating a bioluminescence readout signal.

[0012] Another final aspect of the present application relates to a composition comprising a capture oligonucleotide molecule, wherein the capture oligonucleotide molecule has (i) a length of 30 to 60 base pairs, (ii) an overhang of 4 to 8 base pairs at its 3' end, (iii) a 5' tail, (iv) a target-specific portion between the 3' end and the 5' tail, (v) a deoxyadenosine diphosphate content of 40 to 50%, (vi) no deoxythymidine phosphate at the 3' end or the 5' tail, and (vii) 3' and 5' tails having an ATP content that is 40 to 50% of the ATP content of the capture oligonucleotide molecule.

[0013] This application discloses significant progress in methods for detecting nucleic acids, for example, by using an enzymatic reaction in which the enzyme is tethered to a surface (e.g., a nanoparticle). The assays described herein are converted to a general luminescence output. That is, in certain embodiments, all of the assays are associated with a bioluminescence (BL) protein or substrate that can emit and be read for light, the amount of which correlates with the amount of target nucleic acid in the system or biological sample. This technique is suitable for generating qualitative as well as quantitative results for various nucleic acid molecules.

[0014] This application provides several advantages over other detection methods and systems. These advantages include the following: namely, 1) speed - assays using enzyme reactions are performed rapidly, and readout information can be obtained within minutes; 2) emission - based readout information is used, enabling stand - alone and highly portable systems and devices that do not require elements for large excitation (such as those required for fluorescence); 3) sensitivity - multiple species of free phosphate are released in each hybridization event, and due to the enzyme reaction assay, signal amplification is promoted in both the detection and readout steps; 4) low manufacturing cost - potential components of such systems, such as nanoparticles for example, can be manufactured from inexpensive materials and can be easily mass - produced; 5) multiplexing ability - by using a combination of biochemical reactions, in certain embodiments of this application, it may be possible to detect multiple species of nucleic acid molecules in a single system; 6) maximum enzyme stability and activity can be easily obtained by using tethered enzymes; 7) by using tethered enzymes, the reaction and readout are limited to a specific region of the system (e.g., a specific region of a card), reducing the size of the photodetector in the reader; 8) by using tethered enzymes, the reaction and readout are limited, enabling in - line negative controls and controls for background luminescence; 9) by using tethered enzymes, the reaction and readout are limited, reducing the contamination of light from the detection of other nucleic acid molecules within the same system; 10) the ability to detect multiple target oligonucleotides in a specific region of the system (e.g., a specific region of a card) is improved by immobilizing capture oligonucleotides; 11) by using isothermal amplification, detection at ambient temperature is possible without the need for temperature cycling; and 12) by the design of capture oligonucleotides, a single - step reaction without interference / inhibition by by - products is possible, and by being able to introduce a bioluminescent enzyme into the single - step reaction, further levels of signal amplification can occur from the free phosphate released from the metabolized AP molecules (returned to the ATP - generating reaction). [The present invention 1001] A method for detecting a target nucleic acid molecule in a sample, comprising: preparing a sample containing the target nucleic acid molecule; contacting the sample with a capture oligonucleotide molecule complementary to at least a part of the target nucleic acid molecule, so that the capture oligonucleotide molecule hybridizes to the complementary part of the target nucleotide molecule to form a double-stranded nucleic acid molecule, wherein the capture oligonucleotide molecule has (i) a length of 30 to 60 base pairs, (ii) an overhang of 4 to 8 base pairs at its 3' end, (iii) a 5' tail, (iv) a target-specific part between the 3' end and the 5' tail, (v) a deoxyadenosine diphosphate content of 40 to 50%, (vi) no deoxythymidine phosphate at the 3' end or the 5' tail, and (vii) an ATP content at the 3' end and the 5' tail that is 40 to 50% of the ATP content of the capture oligonucleotide molecule; contacting the double-stranded nucleic acid molecule, a polymerase, and a dNTP mixture to form a polymerase extension mixture; subjecting the polymerase extension mixture to conditions under which the target nucleic acid molecule is extended to release free phosphate; generating adenosine triphosphate from the released free phosphate; metabolizing the adenosine triphosphate generated from the free phosphate with luciferase to generate a bioluminescence readout signal indicating the presence of the target nucleic acid molecule in the sample; and the method as described above. [Invention 1002] The method of Invention 1001, wherein the DNA polymerase is bound to a solid support. [Invention 1003] The method of Invention 1002, wherein the DNA polymerase is bound to the solid support by a linker selected from the group consisting of His-Si, His, Si, biotin, streptavidin, Pt, Au, Ag, His-Pt, His-Au, His-Ag, GST, antibody, and epitope tag. [Invention 1004] The method of the present invention 1001, wherein the luciferase is bound to a solid support. [The present invention 1005] The method of the present invention 1004, wherein the luciferase is bound to the solid support by a linker selected from the group consisting of His-Si, His, Si, biotin, streptavidin, Pt, Au, Ag, His-Pt, His-Au, His-Ag, GST, antibody, and epitope tag. [The present invention 1006] The method of the present invention 1001, wherein the step of generating the adenosine triphosphate comprises subjecting the released free phosphate to an enzymatic reaction of the bound glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase to generate adenosine triphosphate. [The present invention 1007] The method of the present invention 1006, wherein the step of subjecting the released free phosphate to an enzymatic reaction of the bound glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase comprises contacting adenosine diphosphate, nicotinamide adenine dinucleotide, and glyceraldehyde 3-phosphate to achieve the enzymatic reaction of the bound glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase. [The present invention 1008] The method of the present invention 1007, wherein the glyceraldehyde 3-phosphate dehydrogenase and the phosphoglycerate kinase are bound to a solid support. [The present invention 1009] The method of the present invention 1001, wherein the step of generating the adenosine triphosphate comprises contacting the released free phosphate with adenosine 5'-phosphosulfate in the presence of adenosine triphosphate sulfurylase to generate adenosine triphosphate. [The present invention 1010] The method of the present invention 1009, wherein the adenosine triphosphate sulfurylase is bound to a solid support. [The present invention 1011] The method of the present invention 1001, wherein the target nucleic acid molecule is present in the sample at a concentration of less than 10 -5 moles per liter. [The present invention 1012] The method of the present invention 1001, wherein the target nucleic acid molecule is a microRNA. [The present invention 1013] The method of the present invention 1001, wherein the step of subjecting is carried out at a temperature of 0 to 100 °C. [The present invention 1014] The method of the present invention 1013, wherein the step of subjecting is carried out at a temperature of 25 to 40 °C. [The present invention 1015] The method of the present invention 1001, wherein the polymerase is full-length BST DNA polymerase, large fragment BST DNA polymerase, BST 2.0 DNA polymerase, Klenow fragment (3’→5’ exo), and DNA polymerase I (large Klenow fragment). [The present invention 1016] The method of the present invention 1001, further comprising the step of quantifying the bioluminescence readout signal to determine the presence or concentration of the target nucleic acid molecule in the sample. [The present invention 1017] The method of the present invention 1016, wherein the presence of the target nucleic acid molecule in the sample is determined. [The present invention 1018] The presence of the target nucleic acid molecule in the sample is determined by a procedure including calculating the initial rate of bioluminescence signal generation, calculating the period required to achieve peak bioluminescence, and calculating the magnitude of the peak of the bioluminescence signal or the integrated bioluminescence signal from time zero to peak bioluminescence in the method of the present invention 1017. [The present invention 1019] The method of the present invention 1016, wherein the concentration of the target nucleic acid molecule in the sample is determined. [The present invention 1020] The method of the present invention 1001, wherein deoxyadenosine triphosphate is excluded from the polymerase extension mixture. [The present invention 1021] The method of the present invention 1001, wherein the sample is selected from the group consisting of blood, urine, cerebrospinal fluid, saliva, tissue, and synthetic materials. [The present invention 1022] The method of the present invention 1001, which is carried out in a solution. [The present invention 1023] The method of the present invention 1001, wherein a plurality of capture oligonucleotide molecules are provided for detecting a plurality of target nucleic acid molecules. [The present invention 1024] A method for detecting a target nucleic acid molecule in a sample, comprising: preparing a sample containing the target nucleic acid molecule; contacting the sample with a capture oligonucleotide molecule that is complementary to at least a part of the target nucleic acid molecule, whereby the capture oligonucleotide molecule hybridizes to the complementary part of the target nucleic acid molecule to form a double-stranded nucleic acid molecule; contacting the double-stranded nucleic acid molecule, a polymerase, and a dNTP mixture to form a polymerase extension mixture; subjecting the polymerase extension mixture to conditions under which the target nucleic acid molecule is extended to release free phosphate; enzymatically generating adenosine triphosphate from the released free phosphate; metabolizing the adenosine triphosphate generated from the free phosphate with luciferase to generate a bioluminescence readout signal indicating the presence of the target nucleic acid molecule in the sample, wherein the DNA polymerase, luciferase, and the enzyme generating adenosine triphosphate are each bound to a solid support. The method as described above. [The present invention 1025] The method of the present invention 1024, wherein the capture oligonucleotide molecule has a length of 30 to 60 base pairs. [The present invention 1026] The method of the present invention 1024, wherein the capture oligonucleotide molecule has an overhang of 4 to 8 base pairs at its 3' end. [The present invention 1027] The method of the present invention 1026, wherein the capture oligonucleotide molecule has a 5' tail. [The present invention 1028] The method of the present invention 1027, wherein the capture oligonucleotide molecule has a target-specific portion between the 3' end and the 5' tail. [The present invention 1029] The method of the present invention 1024, wherein the capture oligonucleotide molecule has a deoxyadenosine diphosphate content of 40-50%. [The present invention 1030] The method of the present invention 1027, wherein the capture oligonucleotide molecule does not have deoxythymidine phosphate at the 3' end or the 5' tail. [The present invention 1031] The method of the present invention 1024, wherein the capture oligonucleotide molecule has a 3' end and a 5' tail that both have an ATP content that is 40-50% of the ATP content of the capture oligonucleotide molecule. [The present invention 1032] The method of the present invention 1024, wherein a plurality of capture oligonucleotide molecules are provided for detecting a plurality of target nucleic acid molecules. [The present invention 1033] A kit for detecting a target nucleic acid molecule in a sample, A capture oligonucleotide molecule that is complementary to at least a part of the target nucleic acid molecule, and as a result, hybridizes to the complementary part of the target nucleic acid molecule to form a double-stranded nucleic acid molecule, A polymerase bound to a solid support, A dNTP mixture, An enzyme bound to a solid support for generating adenosine triphosphate from the released free phosphate, A luciferase bound to a solid support for generating a bioluminescence readout signal The kit comprising the above. [The present invention 1034] The kit of the present invention 1033, comprising a plurality of capture oligonucleotide molecules for detecting a plurality of target nucleic acid molecules. [The present invention 1035] A kit for detecting a target nucleic acid molecule in a sample, a capture oligonucleotide molecule that is complementary to at least a part of the target nucleic acid molecule, and as a result, hybridizes to the complementary part of the target nucleic acid molecule to form a double-stranded nucleic acid molecule, (i) having a length of 30 to 60 base pairs, (ii) having an overhang of 4 to 8 base pairs at its 3'-end, (iii) having a 5'-tail, (iv) having a target-specific part between the 3'-end and the 5'-tail, (v) having a deoxyadenosine diphosphate content of 40 to 50%, (vi) having no deoxythymidine phosphate at the 3'-end or the 5'-tail, and (vii) having a 3'-end and a 5'-tail having an ATP content that is 40 to 50% of the ATP content of the capture oligonucleotide molecule, a polymerase, a dNTP mixture, an enzyme for generating adenosine triphosphate from the released free phosphate, a luciferase for generating a bioluminescence readout signal and the kit comprising the same. [Inventive Concept 1036] The kit of Inventive Concept 1035, comprising a plurality of capture oligonucleotide molecules for detecting a plurality of target nucleic acid molecules. [Inventive Concept 1037] A composition comprising a capture oligonucleotide molecule, wherein the capture oligonucleotide molecule (i) has a length of 30 to 60 base pairs, (ii) has an overhang of 4 to 8 base pairs at its 3'-end, (iii) has a 5'-tail, (iv) has a target-specific part between the 3'-end and the 5'-tail, (v) has a deoxyadenosine diphosphate content of 40 to 50%, (vi) has no deoxythymidine phosphate at the 3'-end or the 5'-tail, and (vii) has a 3'-end and a 5'-tail having an ATP content that is 40 to 50% of the ATP content of the capture oligonucleotide molecule.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0016] Detailed Description One aspect of the present application relates to a method for detecting a target nucleic acid molecule in a sample. The method includes the steps of preparing a sample containing the target nucleic acid molecule, and contacting the sample with a capture oligonucleotide molecule that is complementary to at least a portion of the target nucleic acid molecule, whereby the capture oligonucleotide molecule hybridizes to the complementary portion of the target nucleotide molecule to form a double-stranded nucleic acid molecule. The capture oligonucleotide molecule has (i) a length of 30 to 60 base pairs, (ii) an overhang of 4 to 8 base pairs at its 3'-end, (iii) a 5'-tail, (iv) a target-specific portion between the 3'-end and the 5'-tail, (v) a deoxyadenosine diphosphate content of 40 to 50%, (vi) no deoxythymidine phosphate at the 3'-end or the 5'-tail, and (vii) an ATP content that is 40 to 50% of the ATP content of the capture oligonucleotide molecule at the 3'-end and the 5'-tail. The double-stranded nucleic acid molecule, polymerase, and dNTP mixture are contacted with each other to form a polymerase extension mixture. The polymerase extension mixture is subjected to conditions under which the target nucleic acid molecule is extended to release free phosphate. Then adenosine triphosphate is generated from the released free phosphate, and the adenosine triphosphate generated from the free phosphate is metabolized by luciferase to generate a bioluminescence readout signal indicating the presence of the target nucleic acid molecule in the sample.

[0017] Another aspect of the present application relates to a method for detecting a target nucleic acid molecule in a sample. The method includes preparing a sample containing the target nucleic acid molecule, and contacting the sample with a capture oligonucleotide molecule complementary to at least a part of the target nucleic acid molecule, whereby the capture oligonucleotide molecule is hybridized to the complementary part of the target nucleic acid molecule to form a double-stranded nucleic acid molecule. The double-stranded nucleic acid molecule, a polymerase, and a dNTP mixture are contacted with each other to form a polymerase extension mixture. The polymerase extension mixture is subjected to conditions under which the target nucleic acid molecule is extended to release free phosphate. Then, adenosine triphosphate is enzymatically generated from the released free phosphate, and the adenosine triphosphate generated from the free phosphate is metabolized by luciferase to generate a bioluminescence readout signal indicating the presence of the target nucleic acid molecule in the sample. The DNA polymerase, the luciferase, and the enzyme that generates adenosine triphosphate are each bound to a solid support.

[0018] As shown in FIG. 1, a DNA polymerase is tethered to one nanoparticle, and GAPDH, PGK, and luciferase ("Luc") are tethered to another nanoparticle. The miRNA target anneals to a complementary insertion sequence within either the prepared, tethered, or solution capture oligonucleotide to generate a duplex. This allows the DNA polymerase to bind and initiate a polymerization reaction that releases free phosphate (PPi) through nucleotide incorporation. ADP, NAD+, and glyceraldehyde 3-phosphate are added to the assay. In the presence of these components, the free phosphate is used by tethered GAPDH and PGK to generate ATP. This ATP is then used by tethered Luc together with luciferin to generate a bioluminescence signal. The amount of light emitted is directly proportional to the amount of ATP in the system and thus corresponds to the amount of target miRNA in the system. The emitted light can be quantitatively and / or qualitatively read by a photodetector arranged to capture the emitted signal in one embodiment.

[0019] Alternatively, as shown in FIG. 2, the DNA polymerase is tethered to one nanoparticle, and the ATP sulfurylase (ATP-sul) and luciferase (“Luc”) are tethered to another nanoparticle. The miRNA target anneals to a complementary insertion sequence within either the prepared, tethered, or solution capture oligonucleotide, generating a double strand. This allows the DNA polymerase to bind and initiate a polymerization reaction, which releases free phosphate (PPi) through nucleotide incorporation. Adenosine 5'-phosphosulfate (APS) is added to the assay. In the presence of APS, the free phosphate is used by the tethered ATP-sul to generate ATP. This ATP is then used by the tethered Luc together with luciferin to generate a bioluminescence signal. The amount of light emitted is directly proportional to the amount of ATP in the system, and as a result corresponds to the amount of target miRNA in the system. The emitted light can be quantitatively and / or qualitatively read by a photodetector arranged to capture the emitted signal in one embodiment.

[0020] Suitable biological samples according to the present application include, but are not limited to, biological samples including blood, serum, plasma, cerebrospinal fluid, urine, saliva, and tissue. Industrial samples include food, beverages, and synthetic materials. Environmental samples include water, air, or surface samples.

[0021] The term “nucleic acid” refers to a polymer of nucleotides (e.g., ribonucleotides and deoxyribonucleotides, both natural and non-natural) including DNA, RNA, and their subcategories (such as cDNA, mRNA, miRNA, etc.). The nucleic acid may be single-stranded and typically contains 5'-3' phosphodiester bonds, although in some cases nucleotide analogs may have other bonds. The nucleic acid may include naturally occurring bases (adenosine, guanosine, cytosine, uracil, and thymidine) as well as non-natural bases.

[0022] As used herein, the "target nucleic acid" or "target" refers to a portion of a nucleic acid sequence in a sample that is the subject of detection or analysis. The term "target" includes all variants of the target sequence, such as variants due to one or more mutations and wild-type variants.

[0023] In one embodiment, the target nucleic acid molecule is a microRNA.

[0024] As used herein, a "capture oligonucleotide" refers to a nucleic acid fragment that specifically hybridizes to a target sequence in a target nucleic acid by standard base pairing. As used herein, "specifically hybridizes" means that under stringent hybridization assay conditions, the capture oligonucleotide hybridizes to those target sequences, or their replicas, to form stable capture oligonucleotide:target hybrids, while minimizing the formation of stable capture oligonucleotide:non-target hybrids. Thus, the capture oligonucleotide hybridizes to the target sequence, or its replica, to a much greater extent than to non-target sequences. Suitable hybridization conditions are well known in the art and can be predicted based on sequence composition or determined by conventional testing methods (e.g., see Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 nd nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 (which is hereby incorporated by reference in its entirety)).

[0025] Figure 3 shows an embodiment of the design of a capture oligonucleotide. As shown in Figure 3, the capture oligonucleotide may be 30 to 60 nucleotides in length, including a 5' extension tail, an internal complementary insertion sequence, and a 3' tail. The 5' extension tail sequence and the 3' tail sequence do not contain dTTP. The ATP content of the 5' extension tail and the 3' tail is 40 to 50% of the entire oligonucleotide. The optimal capture oligonucleotide sequence contains 4 to 8 additional nucleotides at the 3' tail. The capture oligonucleotide is preferably designed so as not to form a hairpin structure in a form that hybridizes with itself and interferes with hybridization to the target nucleic acid.

[0026] As used herein, "luciferase" refers to an oxygenase that catalyzes a luminescence reaction as follows. TIFF2025098095000002.tif12154

[0027] Accordingly, luciferase refers to an enzyme or a luminescent protein that catalyzes a bioluminescence reaction (a reaction that generates bioluminescence), and unless otherwise specified, is a naturally occurring luciferase, a recombinant luciferase, or a mutant luciferase. When naturally occurring, luciferase can be readily obtained from organisms by those skilled in the art. When the above luciferase is a naturally occurring luciferase, or a recombinant or mutant luciferase (e.g., a luciferase that retains the activity of a naturally occurring luciferase in the luciferase-luciferin reaction), a nucleic acid encoding the luciferase is expressed. Luciferase can be readily obtained from cultures such as bacteria, yeast, mammalian cells, insect cells, and plant cells. Furthermore, recombinant or mutant luciferase can be readily obtained from an in vitro cell-free system using a nucleic acid encoding luciferase. Luciferase is available from Promega Corporation, Madison, WI. A plurality of luciferases that are modified mutants or variants of luciferase are also known in the art, and they are described, for example, in Thorne et al, “Illuminating Insights into Firefly Luciferase and Other Bioluminescent Reporters Used in Chemical Biology,” Chemistry & Biology 17(6):646-657 (2010) (which is hereby incorporated by reference in its entirety).

[0028] The "polymerase extension" reaction according to the present application includes all forms of nucleic acid synthesis reactions catalyzed by template-directed polymerases. The conditions and reagents for the primer extension reaction are known in the art, and any standard methods, reagents, enzymes, etc. can be used at this stage (see, for example, Sambrook et al., (editors), Molecular Cloning: a Laboratory Manual (1989), Cold Spring Harbor Laboratory Press (this document is incorporated herein by reference in its entirety)). Thus, the above extension reaction in its most basic form is carried out in the presence of a primer, deoxynucleotides (dNTPs), and a suitable polymerase enzyme, such as Klenow, or actually any available and appropriate enzyme polymerase. By way of example, polymerases suitable for use in the methods of the present application are well known in the art and include, but are not limited to, full-length BST DNA polymerase, large fragment BST DNA polymerase, BST 2.0 DNA polymerase, Klenow fragment (3'→5' exo), and DNA polymerase I (large Klenow fragment). The above conditions can be optionally selected according to procedures known in the art.

[0029] The polymerase extension technique for use in the methods of the present application is an isothermal technique (i.e., a technique that is carried out at a single temperature or in which the major aspect of the amplification process is carried out at a single temperature). Such techniques rely on the ability of a polymerase to copy an amplified template strand and form a joined double strand. The isothermal techniques rely on a strand displacement polymerase to separate / displace the two strands of the double strand and re-copy the template. This well-known property has been the subject of numerous scientific papers (see, for example, Y. Masamute et al., J. Biol. Chem. 246:2692-2701 (1971); R. L. Lechner et al., J. Biol. Chem. 258:11174-11184 (1983); and R. C. Lundquist and B. M. Olivera, Cell 31:53-60 (1982), which are hereby incorporated by reference in their entirety).

[0030] Briefly, when used in the methods of the present application, polymerase extension occurs when a DNA polymerase binds to a capture oligonucleotide-target hybrid (i.e., double-stranded DNA) and extends a complementary DNA strand based on the capture oligonucleotide sequence. The extension reaction occurs using the available nucleotides provided in a deoxynucleotide (dNTP) mixture added to the reaction mixture. These dNTPs include deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP). In one embodiment, deoxyadenosine triphosphate is excluded from the polymerase extension mixture.

[0031] As described above, the polymerase extension technique for use in the method of the present application is an isothermal technique (i.e., a technique that is carried out at a single temperature or in which the main aspect of the amplification process is carried out at a single temperature). Thus, in certain embodiments, the polymerase extension reaction is carried out at a temperature of 0 to 100 °C, 5 to 100 °C, 10 to 100 °C, 15 to 100 °C, 20 to 100 °C, 25 to 100 °C, 30 to 100 °C, 35 to 100 °C, 40 to 100 °C, 45 to 100 °C, 50 to 100 °C, 55 to 100 °C, 60 to 100 °C, 65 to 100 °C, 70 to 100 °C, 75 to 100 °C, 80 to 100 °C, 85 to 100 °C, 90 to 100 °C, or 95 to 100 °C. In one embodiment, the polymerase extension reaction is carried out at a temperature of 25 to 40 °C.

[0032] For each nucleotide added to the DNA strand by the polymerase extension reaction, two phosphate groups (PPi) are released. In the method of the present application, the release of these free phosphates (PPi) can then be used to facilitate the detection of target nucleic acid molecules in a sample. Specifically, the presence or absence of target nucleic acid molecules can be detected by the conversion of PPi to ATP by an enzymatic reaction and subsequent bioluminescent detection of ATP using the signal transduction molecule luciferase.

[0033] Luciferase and luciferin are used in combination to identify the target nucleic acid. This is because the amount of light generated is substantially proportional to the amount of ATP generated, and in turn, is directly proportional to the amount of nucleotides incorporated and the amount of target nucleic acid present. Thus, the method includes providing luciferin and O2, and luciferin and O2 are added to the reaction mixture.

[0034] As described above, the method described herein involves subjecting a polymerase extension mixture to conditions under which a target nucleic acid molecule is extended and free phosphate is released. Next, adenosine triphosphate (ATP) is generated from the released free phosphate via an enzymatic reaction, and then the ATP is metabolized by luciferase to generate a bioluminescence readout signal. According to this embodiment, in one embodiment, generating adenosine triphosphate involves subjecting the released free phosphate to an enzymatic reaction of bound glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase (GAPDH-PGK) to generate adenosine triphosphate.

[0035] In this embodiment, the enzymatic reaction involves GAPDH reacting with PGK in the presence of PPi, adenosine diphosphate (ADP), nicotinamide adenine dinucleotide (NAD+), and glyceraldehyde 3-phosphate (GAP) to generate ATP. The ATP then reacts with luciferase to generate a measurable signal. The reaction scheme is shown below. TIFF2025098095000003.tif26137

[0036] In another embodiment, adenosine triphosphate can be generated by contacting the released free phosphate with adenosine 5'-phosphosulfate (APS) in the presence of adenosine triphosphate sulfurylase (ATP-sul) to generate adenosine triphosphate. The ATP then reacts with luciferase to generate a measurable signal. The reaction scheme is shown below. TIFF2025098095000004.tif26128

[0037] According to the above embodiments, the glyceraldehyde 3-phosphate dehydrogenase, the phosphoglycerate kinase, and / or the adenosine triphosphate sulfurylase may be bound to the solid support described above.

[0038] The amount of light generated can be easily determined using a device sensitive to appropriate light, such as a luminometer. Therefore, the luminometry method has the advantage of being quantifiable.

[0039] In one embodiment, the bioluminescence readout signal is quantified to determine the presence or concentration of a target nucleic acid molecule in a sample. The amount of target nucleic acid can be determined from the magnitude of the peak of the luminescence signal, and / or the time required for the signal to reach that peak magnitude, and / or the integrated amount of the signal emitted over a period of time, and / or the rate at which the luminescence is generated.

[0040] In one embodiment, the target nucleic acid molecule is present in the sample at a concentration of less than 10 -5 moles per liter.

[0041] In one embodiment, the presence of a target nucleic acid molecule in a sample is determined by a procedure that includes calculating the initial rate of bioluminescence signal generation, calculating the time required to reach the peak of the bioluminescence, and calculating the magnitude of the peak of the bioluminescence signal or the integrated bioluminescence signal from time zero to the peak of the bioluminescence.

[0042] These procedures may be used individually or as part of an analytical method incorporating two or more procedures for better quantification. For each procedure or combination of procedures, a threshold value may be determined in advance and calibrated against a known amount of target nucleic acid target. The predetermined value may also be useful for identifying sequences (i.e., mutations) that are similar but not identical to the desired target oligonucleotide. Further, the values obtained by these analytical procedures may be evaluated against a cut-off value to make a presence / absence measurement or evaluated as a scale to enable a quantitative readout.

[0043] In one embodiment, a plurality of capture oligonucleotide molecules are added to detect a plurality of target nucleic acid molecules.

[0044] The method contemplated according to this embodiment is a multiplex assay that utilizes multiple types of capture oligonucleotides to determine whether one or more of multiple types of predetermined nucleic acid target sequences are present or absent in a sample. A field in which such a multiplex assay is particularly useful is a screening assay that indicates the absence of the nucleic acid of interest with a normal analytical output.

[0045] In a multiplexed embodiment of the above method, the sample is mixed with multiple types of different capture oligonucleotides. In this embodiment, the analytical output regarding a particular result by one type of capture oligonucleotide is distinguishable from the analytical output regarding the opposite result by all capture oligonucleotides.

[0046] According to this embodiment, for example, one solid support may contain multiple types of capture oligonucleotides specific for multiple types of target nucleic acids. Each capture oligonucleotide may be localized at a defined position or region of the solid support, or may be synthesized in situ at a defined position or region on the surface of the solid support. Such a support facilitates the parallel analysis of multiple target nucleic acids bound with capture oligonucleotides. Such a support is also suitable for high-throughput screening.

[0047] In a particular embodiment, the reaction of the present application is carried out in solution. The term "in solution" refers to any assay in which the target nucleic acid is detected in solution or in suspension. For example, the first hybridization to the target nucleic acid can be carried out using the first capture oligonucleotide, and the second hybridization to the target nucleic acid can be carried out using the second capture oligonucleotide. Such multiple hybridizations may include a washing step to remove any unwanted (e.g., non-hybridizing sequences) components.

[0048] In certain embodiments, the enzyme of the method according to the present application may be bound to a solid support. In other embodiments, the enzyme of the method may remain in solution.

[0049] Suitable supports include organic or inorganic materials and may be materials of any and appropriate size or shape (e.g., scaffold sheets, platforms, and / or nanoparticles). Tethering or immobilizing the components of the assay according to the present application not only spatially confines the above components, but also, for example, helps to improve the stability and / or function of the above components when performing stepwise or sequential reactions as part of a particular assay. In certain embodiments, the support material includes, for example, nucleotide sequences or gels. In certain embodiments, the enzyme or component of the assay according to the present application may be immobilized on or tethered to the luminal surface of a support material such as a channel (e.g., a microfluidic channel) of a nanoparticle or a platform.

[0050] Several techniques can be used to immobilize the components (e.g., enzymes) of the assay according to the present application on the surface. For example, the components may be non-specifically bound or may be bound via a specific but non-oriented chemical reaction (such as a carboxy-amide bond). Oriented enzyme immobilization can also be used according to the method of the present application. Oriented enzyme immobilization provides several advantages, including, for example, arranging a binding tag (e.g., an affinity tag) such that the activity and stability of the tethered enzyme are optimized (see Mukai et al., “Sequential Reactions of Surface-Tethered Glycolytic Enzymes,” Chem. Biol. 16(9):1013-20 (2009), which is hereby incorporated by reference in its entirety).

[0051] An example of a method for tethering an enzyme involved in nucleic acid detection to a surface is the use of oriented immobilization. In certain embodiments of the assays according to the present application, a recombinant enzyme or assay component involved in the reaction of the assay is engineered using an affinity tag such that the enzyme can bind to a surface such as silica or nickel, or a component of a surface such as nickel-nitrilotriacetic acid. For example, the affinity tag can be bound to the amino or carboxy terminus of the protein to be immobilized, or can be embedded within the protein to be immobilized. The optimal position of the tethering domain will depend on the nature and position of the catalytic domain(s), substrate binding domain(s) of the enzyme, and any conformational changes that need not occur in the enzyme.

[0052] The use of affinity-tagged proteins is particularly convenient because the proteins used in the methods of the present application (i.e., DNA polymerase, luciferase, etc.) can be easily expressed as fusions with appropriate binding tags to facilitate immobilization to solid supports containing the corresponding capture binding moieties. Suitable capture moieties and binding tag partners that can be used according to this embodiment of the present application include, but are not limited to, His-Si, His, Si, biotin, streptavidin, Pt, Au, Ag, His-Pt, His-Au, His-Ag, GST, antibodies, and epitope tags. Methods for covalently attaching oligonucleotides to solid supports are well known in the art, see, for example, Gosh et al., “Covalent Attachment of Oligonucleotides to Solid Supports,” Nucleic Acids Res. 15(13): 5353-5372 (1987), Joos et al., “Covalent Attachment of Hybridizable Oligonucleotides to Glass Supports,” Anal. Biochem. 247(1):96-101 (1997); Lund et al., “Assessment of Methods for Covalent Binding of Nucleic Acids to Magnetic Beads, Dynabeads, and the Characteristics of the Bound Nucleic Acids in Hybridization Reactions,” Nucleic Acids Res. 16(22):10861-80 (1988), and these documents are hereby incorporated by reference in their entirety with this description.

[0053] In certain embodiments, the DNA polymerase and / or the luciferase is bound to a solid support.

[0054] According to this aspect of the present application, the above DNA polymerase and / or the above luciferase can be bound to a solid support using a linker selected from the group consisting of His-Si, His, Si, biotin, streptavidin, Pt, Au, Ag, His-Pt, His-Au, His-Ag, GST, antibody, and epitope tag.

[0055] Surfaces that function as supports, platforms, or scaffolds can take various forms, for example, including various nanoparticles or strands of nucleic acids, and can include various geometries.

[0056] In certain embodiments according to the present application, the above support is a nanoparticle. As used herein, the term "nanoparticle" refers to any particle whose average diameter is in the nanometer range, that is, the above particles with a maximum average diameter of 1 μm. The nanoparticles used can be manufactured from any and appropriate organic or inorganic substances known to those skilled in the art. For example, the nanoparticles may be composed of any polymer, iron (II, III) oxide, gold, silver, carbon, silica, CdSe, and / or CdS. In one embodiment, the above nanoparticles are magnetic nanoparticles. In another embodiment, the above nanoparticles are magnetic silica-coated nanoparticles ("MSP").

[0057] In addition to nanoparticles (NP), supports or scaffolds of various materials can take the form of rods, planes, graphene sheets, nanotubes, DNA scaffolds, gels, microspheres, or the inner channel walls of microchannels of larger supports. Quantum dots are also contemplated for use as supports according to the present application. Enzyme immobilization can be achieved by non-specific binding, chemical modification, affinity tags, or other complexation techniques.

[0058] In one embodiment according to the present application, the method further includes performing a positive control and / or a negative control. Detection of the diagnostic or prognostic amount of the target nucleic acid is performed by comparison with a control amount. The control amount of the target nucleic acid may be any amount or range of amounts that will be compared with the test amount of the target nucleic acid. The control amount may be the amount of the target nucleic acid in a positive control or negative control sample performed as part of the assay according to the present application. The control amount may be either an absolute amount (e.g., μg / ml) or a relative amount (e.g., relative intensity of the signal).

[0059] Exemplary negative controls for use in the method of the present application include blocking oligonucleotides targeting the target oligonucleotide (sequences that are fully or partially complementary to the target oligonucleotide), blocking oligonucleotides targeting the capture oligonucleotide (modified at the 3' / 5' end to inhibit extension), ribonuclease (RNase) added to the reaction mixture, a reaction mixture without the capture oligonucleotide, a reaction mixture without nucleotides (dNTPs), and a reaction mixture without any of the substrates. Exemplary positive controls for use in the method of the present application include DNA oligonucleotides having the same sequence as the target oligonucleotide derived from the sample in question (target oligonucleotide mimics) at various concentrations (including saturating amounts), and pre-annealed double-stranded DNA having a single-stranded sequence with an overhang that can be extended by the above polymerase.

[0060] In various related aspects, the present application also relates to devices and kits for implementing the methods described herein. Such kits can be utilized in a clinical or research environment, or can be adapted for either in-clinic testing or point-of-use, and include monitors, reagents, and procedures. In particular, kits containing the disclosed reagents used in implementing the methods described herein include any of several means for detecting captured target nucleic acid molecules and measuring the bioluminescence signals generated after capture of the target, along with appropriate instructions. Suitable kits include sufficient reagents to perform an assay for detecting target nucleic acid molecules.

[0061] It should be understood that such kits are useful for any method of the present application. The selection of specific components will vary depending on the specific method, and the method for which the kit is designed to implement that method. Additional components may be added to detect the analytical output measured by the release of ATP and detection of the bioluminescence signal.

[0062] As described above, the kit optionally further includes instructions for detecting target nucleic acids by the methods described herein. The instructions present in such kits explain to the user how to use the components of the kit for implementing various methods of the present application. These instructions may include an explanation of the detection methods of the present application, including detection by luminescence.

[0063] Accordingly, another aspect of the present application relates to a kit for detecting a target nucleic acid molecule in a sample. The kit includes a capture oligonucleotide molecule that is complementary to at least a portion of the target nucleic acid molecule, and as a result, hybridizes to the complementary portion of the target nucleic acid molecule to form a double-stranded nucleic acid molecule, and a polymerase bound to a solid support, and a dNTP mixture, and an enzyme bound to a solid support for generating adenosine triphosphate from the released free phosphate, and Luciferase bound to a solid support for generating a bioluminescence readout signal and comprise.

[0064] Another aspect of the present application relates to a kit for detecting a target nucleic acid molecule in a sample. The kit includes a capture oligonucleotide molecule that is complementary to at least a portion of the target nucleic acid molecule, and as a result, hybridizes to the complementary portion of the target nucleic acid molecule to form a double-stranded nucleic acid molecule. The capture oligonucleotide molecule has (i) a length of 30 to 60 base pairs, (ii) an overhang of 4 to 8 base pairs at its 3' end, (iii) a 5' tail, (iv) a target-specific portion between the 3' end and the 5' tail, (v) a deoxyadenosine diphosphate content of 40 to 50%, (vi) no deoxythymidine phosphate at the 3' end or the 5' tail, and (vii) an ATP content that is 40 to 50% of the ATP content of the capture oligonucleotide molecule, having the 3' end and the 5' tail. The kit also includes a polymerase, a dNTP mixture, an enzyme for generating adenosine triphosphate from the released free phosphate, and luciferase for generating a bioluminescence readout signal.

[0065] The above kit may also include a plurality of capture oligonucleotide molecules for detecting a plurality of target nucleic acid molecules. In a kit contemplating specific nucleic acid detection mediated by multiplexed capture oligonucleotides, the kit includes a plurality of capture oligonucleotides for a plurality of nucleic acid targets of interest. When the kit includes a plurality of capture oligonucleotides, each of the capture oligonucleotides is preferably designed to interrogate a different target nucleic acid sequence.

[0066] The last aspect of the present application is a composition comprising a capture oligonucleotide molecule, wherein the capture oligonucleotide molecule has (i) a length of 30 to 60 base pairs, (ii) an overhang of 4 to 8 base pairs at its 3' end, (iii) a 5' tail, (iv) a target-specific portion between the 3' end and the 5' tail, (v) a deoxyadenosine diphosphate content of 40 to 50%, (vi) no deoxythymidine phosphate at the 3' end or the 5' tail, and (vii) an ATP content of 40 to 50% of the ATP content of the capture oligonucleotide molecule, and relates to the above composition having the above 3' end and 5' tail.

Example

[0067] The following examples are intended to illustrate the implementation of the embodiments of the present disclosure, but are not intended to limit the scope thereof in any way.

[0068] Example 1 Improvement of the kinetics and sensitivity of the assay by excluding dATP from the reaction mixture A reaction mixture with a total volume of 100 μl was prepared by mixing 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl of Klenow (NEB, large fragment, M0210S, 5000 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide (T2 (SEQ ID NO: 7), 1 μM), 1.8 μl of dNTP Mix (each 33 mM), and + / - dATP. This reaction mixture was added to individual wells of a white 96-well plate containing 1 μl of target oligonucleotide (R6 (SEQ ID NO: 6), 1 μM). Then, this reaction mixture was immediately placed in a TECAN plate reader, and the luminescence signal was read for 2000 seconds at room temperature with an integration time of 400 milliseconds.

[0069] As shown in Fig. 4, two identical reaction mixtures were tested using the above conditions. However, the nucleotide mixture dATP (deoxyadenosine triphosphate) added to reaction mixture A was excluded. The binding of dATP to luciferase and the hydrolysis of dATP by luciferase result in attenuation of the initial luminescence signal (1), delay of the peak of the response phase (2), and decrease in the overall signal magnitude (3). This indicates the relationship between pure base sequencing and the tethered detection method used here, where bioluminescence generates the readout information. As a result of these data, the above supplementary oligonucleotide is designed to contain no dTPS in its sequence so that dATP need not be present in the above reaction mixture.

[0070] Example 2 Detection of mismatched nucleotides at the 3' end of the target oligonucleotide by the TET-miRNA assay A reaction mixture with a total volume of 100 μl was prepared by mixing 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl of Klenow (NEB, Large fragment, M0210S, 5000 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide (T2 (SEQ ID NO: 7), 100 μM), and 1.8 μl of dNTP Mix (33 mM each). This reaction mixture was added to individual wells of a white 96-well plate containing 1 μl of each target oligonucleotide (R1 - R6 (SEQ ID NO: 1 - 6), 100 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read at room temperature for 3500 seconds with an integration time of 400 milliseconds.

[0071] Figure 5A shows that as the proportion of mismatched nucleotides (indicated by underlined characters) at the 3'-end of the target oligonucleotide sequence shown in Figure 5B increases, the luminescence signal decreases. Two complementary target oligonucleotides (R1 (SEQ ID NO: 1) and R6 (SEQ ID NO: 6) that are shifted and complementary to overlapping sequences within the capture oligonucleotide) and four mismatch oligonucleotides (displayed with the proportion of mismatched nucleotides appended) were tested, demonstrating a strong inhibition of the TET reaction upon nucleotide mismatch, which is summarized in Figure 5C. Figure 5D shows a diagram of the proposed data analysis procedure for determining the presence of the target oligonucleotide and hybridization to the capture oligonucleotide (shown for the data regarding the target oligonucleotides R1 (SEQ ID NO: 1) and R6 (SEQ ID NO: 6) shown in Figure 5A), where a / a': calculation of the initial rate (gradient) of luminescence signal generation, b / b': time to the peak of luminescence, c / c': magnitude of the peak of the luminescence signal, d / d': luminescence signal integrated from time 0 to the peak.

[0072] Example 3 Determination of the sensitivity range of target oligonucleotide detection using the TET-miRNA assay 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl of Klenow (NEB, Large fragment, M0210S, 5000 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 0.5 μl of capture oligonucleotide (T2 (SEQ ID NO: 7) (see A of FIG. 7), 1 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (each 33 mM) were mixed to prepare a reaction mixture with a total volume of 100 μl. This reaction mixture was added to individual wells of a white 96-well plate containing target oligonucleotides (R6 (SEQ ID NO: 6), 0 mM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 1 μM) at decreasing concentrations, immediately placed in a TECAN plate reader, and the luminescence signal was read for 2000 seconds at room temperature with an integration time of 400 milliseconds.

[0073] A of FIG. 6 shows the luminescence signal in response to the decreasing concentration of the target oligonucleotide. A detection range of concentrations from 1 picomolar concentration (10 -12 mol / L) to 1 micromolar concentration (10 -6 mol / L) is shown. B of FIG. 6, although further optimization is required, shows that the summary of A of FIG. 6 calculated from the reaction kinetics (i.e., the slope of the initial reaction stage as a function of the concentration of the above oligonucleotide expressed in picoM) exhibits high sensitivity and a wide dynamic range.

[0074] Example 4 Design of the 5' and 3' tails of the capture oligonucleotide affects the TET-miRNA reaction rate 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl of Klenow (NEB, large fragment, M0210S, 5000 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of target oligonucleotide (R6 (SEQ ID NO: 6), 100 μM), and 1.8 μl of dNTP Mix (each 33 mM) were mixed to prepare a reaction mixture with a total volume of 100 μl. This reaction mixture was added to individual wells of a white 96-well plate containing 1 μl of each capture oligonucleotide (T2 - T6 (SEQ ID NO: 7 - 11), 100 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 1000 seconds at room temperature with an integration time of 400 milliseconds.

[0075] As shown in FIG. 7A, several designed capture oligonucleotides were generated (T2 - T6 (SEQ ID NO: 7 - 11)). All designs contain a complementary insertion sequence similar to (complementary to the R6 target oligonucleotide, shown in underlined text). FIG. 7B shows the luminescence signal in response to the addition of the target oligonucleotide measured in the presence of various capture oligonucleotides (presented in FIG. 7A). FIG. 7C is a summary of FIG. 7B, showing the differences in annealing kinetics, indicated by the reaction gradients calculated for various capture oligonucleotides.

[0076] Example 5 The content of capture oligonucleotide dATP affects the TET-miRNA reaction kinetics 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl of Klenow (NEB, large fragment, M0210S, 5000 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of target oligonucleotide (R6 (SEQ ID NO: 6), 100 μM), and 1.8 μl of dNTP Mix (33 mM each) were mixed to prepare a reaction mixture with a total volume of 100 μl. This reaction mixture was added to individual wells of a white 96-well plate containing 1 μl of each capture oligonucleotide (T1 (SEQ ID NO: 12) + T1A - T1E (SEQ ID NO: 13 - 17), 100 μM)), immediately placed in a TECAN plate reader, and the luminescence signal was read for 1000 seconds at room temperature with an integration time of 400 milliseconds.

[0077] As shown in FIG. 8A, for the detection of the above target oligonucleotide, hsa-let-7a-5p, six capture oligonucleotides containing increasing ratios of dATP (27% - 63%) were designed (all having similar complementary insertion sequences indicated by underlined characters). FIG. 8B is a summary of the data showing that the dATP content within the above capture oligonucleotide sequence exhibits the best activity at 40% - 50%. This finding is surprising and represents a significant improvement.

[0078] Example 6 The 3'tail length of the capture oligonucleotide affects the activity and reaction kinetics of the TET-miRNA reaction 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl of Klenow (NEB, large fragment, M0210S, 5000 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of target oligonucleotide (R6 (SEQ ID NO: 6), 100 μM), and 1.8 μl of dNTP Mix (each 33 mM) were mixed to prepare a reaction mixture with a total volume of 100 μl. This reaction mixture was added to individual wells of a white 96-well plate containing 1 μl of each capture oligonucleotide (T1 - T9 (SEQ ID NO: 12 and SEQ ID NO: 18 - 25), 100 μM)), immediately placed in a TECAN plate reader, and the luminescence signal was read for 1000 seconds at room temperature with an integration time of 400 milliseconds.

[0079] As shown in FIG. 9A, nine capture oligonucleotides were designed for the detection of the above target oligonucleotide, hsa-let-7a-5p. The nine capture oligonucleotides contain a decreasing number of nucleotides (shown in underlined characters) at the 3'-end of the sequence of the oligonucleotide following the complementary insertion site of the miRNA. FIG. 9B shows that when 5 - 8 nucleotides are added to the 3' of the complementary insertion sequence of the above target oligonucleotide, the optimal activity of the TET-miRNA assay is obtained.

[0080] Example 7 The TET-miRNA assay is sensitive to mutations in the sequence of the MIR-340 target oligonucleotide 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl of Klenow (NEB, Large fragment, M0210S, 5000 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide (T2, 100 μM), and 1.8 μl of dNTP Mix (33 mM each) were mixed to prepare a reaction mixture with a total volume of 100 μl. This reaction mixture was added to individual wells of a white 96-well plate containing 1 μl of each target oligonucleotide (MIR-340#1 - MIR-340#7 (SEQ ID NOs: 27 - 33), 100 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 1200 seconds at room temperature with an integration time of 400 milliseconds.

[0081] As shown in Figure 10A, the MIR340 target oligonucleotides (MIR-340#1 (SEQ ID NO: 27)) and various mutant sequences (MIR-340#2 - #7) (SEQ ID NOs: 28 - 33) were used in this experiment, and two controls were also used (one against unmutated MIR-340#2 and one that was a reaction mixture excluding any target oligonucleotides) (mutated nucleotides are shown as underlined characters). Figures 10B and C show the luminescence signals and kinetics measured for the various mutations shown in A. The data in Figure 10A show that all the mutations tested induced detectable differences in the measured signals.

[0082] Example 8 The TET-miRNA assay is capable of detecting naturally occurring miRNAs in human serum and human plasma 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl of Klenow (NEB, large fragment, M0210S, 5000 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of the capture oligonucleotide (100 μM) targeting the naturally occurring miRNA listed in Figure 10A, and 1.8 μl of dNTP Mix (33 mM each) were mixed to prepare a reaction mixture with a total volume of 40 μl. This reaction mixture was added to individual wells of a white 96-well plate containing 60 μl of human serum or human plasma, immediately placed in a TECAN plate reader, and the luminescence signal was read for 500 seconds at room temperature with an integration time of 400 milliseconds.

[0083] As shown in Figure 11A, six capture oligonucleotides were designed to detect six different naturally occurring miRNAs in commercially available human serum. It shows the kinetics of the TET-miRNA reaction in real time. Figure 11B is a summary of Figure 11A, integrating the bioluminescence signal for 500 seconds and showing the relative amounts of various miRNAs in the tested serum samples.

[0084] As shown in Figure 12, plasma samples from three human donors (collected at Guthrie Medical Center, Sayre PA) were tested using TET-miRNA for a panel of six naturally occurring miRNAs.

[0085] Example 9 Loss of TET-miRNA activity by ribonuclease (RNase) treatment 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl of Klenow (NEB, Large fragment, M0210S, 5000 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of a capture oligonucleotide (100 μM) targeting the naturally occurring miRNA described in FIG. 12, and 1.8 μl of dNTP Mix (33 mM each) were mixed to prepare a reaction mixture with a total volume of 40 μl. This reaction mixture was added to individual wells of a white 96-well plate containing 60 μl of human plasma, immediately placed in a TECAN plate reader, and the luminescence signal was read for 500 seconds at room temperature with an integration time of 400 milliseconds.

[0086] Panel A of FIG. 13 shows the results of testing plasma samples from three subjects (collected at Guthrie (Sayre, PA)) using TET-miRNA for a panel of four naturally occurring miRNAs. Panel B of FIG. 13 shows a significant decrease in the signal observed after treatment with RNase-A for 30 minutes at room temperature.

[0087] Example 10 Analysis of the Detection of Target Oligonucleotides by Reaction with Immobilized NPs and by Reaction in Solution On streptavidin-coated microspheres (500 nm SiO2, Bangs Laboratory, IN, USA), biotinylated enzymes were immobilized according to the manufacturer's instructions, and then unbound proteins were washed three times by centrifugation. Equal amounts of enzyme tethered to the NPs or untethered enzyme were added to a reaction mixture consisting of 5 μl of His-Si-luciferase (homemade in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide (Cap-HAS-MIR-451a, 1 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (each 33 mM) with a total volume of 100 μl per reaction mixture. This reaction mixture was added to individual wells of a white 96-well plate containing the target oligonucleotide (HAS-MIR-451a, 1 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 2000 seconds at room temperature with an integration time of 400 milliseconds.

[0088] Equal amounts of the target oligonucleotide, HSA-MIR-451a, were spiked into commercially available human serum samples, which were then added to a TET-miRNA reaction mixture containing soluble enzyme luciferase / ATP-sulfurylase / Klenow (Figure 14A, solution) or the above enzyme tethered to NPs (Figure 14B, NPs). Note that DNA polymerase and ATP-sulfurylase were tethered to the NPs using non-oriented immobilization by biotinylation. ATP sulfurylase (NEB, M0394S, 300 U / ml) and Klenow (NEB, large fragment, M0210S, 5000 U / ml) were biotinylated using the EZ-Link Sulfo-NHS-LC-biotinylation kit (Thermo Scientific, USA) according to the manufacturer's instructions.

[0089] Example 11 Inhibition of the TET-miRNA reaction assay by immobilization of commercially available BST2.0 to NPs via biotin-streptavidin binding BST2.0 (NEB, M0537S, 8000 U / ml) was biotinylated using the EZ-Link Sulfo-NHS-LC-biotinylation kit (Thermo Scientific, USA) according to the manufacturer's instructions. The biotinylated enzyme was immobilized onto streptavidin-coated microspheres (500 nm SiO2, Bangs Laboratory, IN, USA) according to the manufacturer's instructions, and then unbound proteins were washed three times by centrifugation. Equal amounts of BST2.0 tethered to NP or untethered BST2.0 were added to a reaction mixture containing 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide (Cap-HSA-MIR-451a, 1 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (each 33 mM) with a total volume of 100 μl per reaction mixture. This reaction mixture was added to individual wells of a white 96-well plate containing the target oligonucleotide (HSA-MIR-451a, 1 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 2000 seconds at room temperature with an integration time of 400 milliseconds.

[0090] Equal amounts of HSA-MIR-451a were spiked into commercially available human serum samples, which were then added to a TET-miRNA reaction mixture containing soluble enzyme luciferase / ATP-sulfurylase / Bst2.0 (Figure 15A, solution) or a replacement in which Bst2.0 was immobilized on NP via biotinylation (Figure 15B, NP). These data indicate that biotinylation had an adverse effect on its activity, and there was an even more adverse effect when Bst2.0 was tethered to NP via non-oriented immobilization.

[0091] Example 12 The TET-miRNA assay is only slightly affected by temperature 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl of Klenow (NEB, large fragment, M0210S, 5000 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide targeting Has-let-7a-5p miRNA (100 μM), 1.8 μl of dNTP Mix (33 mM each), and Has-let-7a-5p oligonucleotide were mixed to prepare a reaction mixture with a total volume of 100 μl. This reaction mixture was added to individual wells of a white 96-well plate and immediately placed in a TECAN plate reader, and the luminescence signal was read at various temperatures indicated. Luminescence was measured for 1000 seconds with an integration time of 400 milliseconds.

[0092] As shown in FIG. 16A, an equal amount of target oligonucleotide was spiked into a commercially available human serum sample, and this was added to the TET-miRNA reaction mixture at various temperatures (25°C to 40°C). Only slight differences were observed in the initial reaction kinetics parameters (measured from the gradient, FIG. 16B) and efficiency (measured from the integrated signal, FIG. 16C).

[0093] Example 13 Analysis of Two Different Capture Oligonucleotide Designs for Detecting miRNA in Human Serum 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl of Klenow (NEB, Large fragment, M0210S, 5000 U / ml), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of CAP1 (SEQ ID NO: 34) or CAP2 (SEQ ID NO: 35) capture oligonucleotide (100 μM), 1.8 μl of dNTP Mix (each 33 mM), and the test oligonucleotide were mixed to prepare a reaction mixture with a total volume of 100 μl. This reaction mixture was added to individual wells of a white 96-well plate, immediately placed in a TECAN plate reader, and the luminescence signal was read at room temperature. Luminescence was measured for 1000 seconds with an integration time of 400 milliseconds.

[0094] Figure 17A shows the design of two capture oligonucleotides tested for detecting six different naturally occurring miRNAs in human plasma. CAP1 is a random nucleotide sequence, and in CAP2, the 5’ tail and 3’ tail contain only adenosine. Figure 17B shows the real-time kinetics of the TET-miRNA reaction that detects both naturally occurring miRNAs and DNA-based target oligonucleotides (test oligonucleotides) with similar sequences. Figure 17C is an enlarged portion of the data shown in Figure 17B, showing the luminescence signals of various miRNA molecules. Figure 17D is a summary of Figure 17A, with the luminescence signal integrated for 500 seconds to show the relative amounts of various miRNAs. Figure 17E is a summary of the measured values for only the naturally occurring miRNAs (shown in Figure 17C).

[0095] Example 14 Comparison of various DNA polymerase activities in the TET-miRNA assay 0.05 μl of ATP sulfurylase (NEB, M0394S, 300 U / ml), DNA polymerase (shown in Figure 17A), 5 μl of His-Si-luciferase (self-made in the laboratory), 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide targeting hsa-let-7a-5p miRNA (100 μM), 1.8 μl of dNTP Mix (33 mM each), and hsa-let-7a-5p oligonucleotide were mixed to prepare a reaction mixture with a total volume of 100 μl. This reaction mixture was added to individual wells of a white 96-well plate and immediately placed in a TECAN plate reader, and the luminescence signal was read for 500 seconds at room temperature with an integration time of 400 milliseconds.

[0096] Figure 18A is a list of the DNA polymerases used in this experiment and some of their important characteristics. As shown in Figure 18B, equal amounts of miRNA oligonucleotides were spiked into commercially available human serum samples and added to the TET-miRNA reaction mixture in the presence of various DNA-polymerases (at room temperature). The bar graph of reaction kinetics (calculated from the initial slope of the reaction) shows that Bst, Klenow, and Terminator DNA-Poly give the fastest reaction kinetics under these experimental conditions. The reaction mixture of Control 1 (CTRL1) does not contain the capture oligonucleotide, while Control 2 (CTRL2) does not contain the DNA polymerase. The bars of the Bst and Klenow mutants used to obtain the data in the previous figure are highlighted.

[0097] Example 15 Comparison of the detection of target oligonucleotides when TET-miRNA is used in tethered form and in solution A reaction mixture consisting of an equal amount of NP tethered enzyme or untethered enzyme, 5 μl His-Si-luciferase, 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl APS (30 mM), 1 μl capture oligonucleotide (T2 (SEQ ID NO:7), 100 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (each 33 mM) was prepared. This reaction mixture was added to individual wells of a white 96-well plate containing increasing amounts of target oligonucleotide (R6 (SEQ ID NO:6), 200 nM, 500 nM, and 1 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 1400 seconds at room temperature with an integration time of 400 milliseconds.

[0098] Figure 19A is a schematic diagram of the design of His-Si-enzyme (ATPS: ATP-sulfurylase, DNA-Pol: DNA polymerase, Luc: luciferase). Genes encoding the following, namely, ATP-sulfurylase (MET3, adenylyl transferase sulfate, Saccharomyces cerevisiae), BST (Bacillus stearothermophilus DNA polymerase I (pol) gene), and Klenow (Escherichia coli strain LD93-1 DNA polymerase I) were fused with the His-Si tag and inserted into the pET17b vector to express the bacterial protein. The His-Si-protein was purified using Ni-NTA beads and stored in a native protein buffer (NPB) containing sorbitol until use. Figure 19B shows the measurement of enzyme activity in the case of tethering and in solution using the above reaction mixture. These data show the kinetics of individual reaction mixtures when the TET-miRNA enzyme is immobilized on 500 nm SiO2NP (NP, left figure) or in solution (Sol, right). Figure 19C is a bar graph of the reaction kinetics (calculated from the initial gradient of the reaction mixture in question), showing that when the TET-miRNA enzyme is tethered to the NP, the coupling reaction occurs with a faster reaction kinetics. Figure 19D is a summary of the NP TET-miRNA reaction in the detection of increasing concentrations of the R6 target oligonucleotide.

[0099] Example 16 Comparison of the activities of His-Si-Klenow and His-Si-BST in the TET-miRNA assay (comparison of tethered and untethered) Using a reaction buffer consisting of an equal amount of NP-tethered enzyme or non-tethered enzyme, 5 μl of His-Si-luciferase, 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide (T2 (SEQ ID NO: 7), 100 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (each 33 mM), the enzyme activities in the case of tethering and in solution were measured. This reaction mixture was added to individual wells of a white 96-well plate containing the target oligonucleotide (R6 (SEQ ID NO: 6), 1 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 1200 seconds at room temperature with an integration time of 400 milliseconds.

[0100] As shown in A of FIG. 20, the large Klenow fragment derived from E. coli (Escherichia coli strain LD93-1 DNA polymerase I) was expressed as a fusion protein downstream of the His-Si affinity tag. B of FIG. 20 shows the activity of His-Si-Klenow in solution compared with the activities of DNA-pol I and His-Si-BST2 (derived from Bacillus Stearo Thermophilus). His-Si-BST showed a slightly better kinetic rate in terms of the initial reaction rate and the overall activity. C of FIG. 20 shows a comparison of the activities of the two DNA polymerases when the reaction enzyme is tethered to 500 nm SiO2 nanoparticles. Again, His-Si-BST showed better activity. Furthermore, when the reaction mixture contained the tethered enzyme, both Klenow and BST showed improved activity.

[0101] Example 17 Comparison of Target Oligonucleotide Detection Using Various Ratios of His-Si-ATPS / His-Si-BST / NP The measurement of enzyme activity was carried out using a reaction mixture consisting of 20 μl of NP-His-Si-BST (of various enzyme / NP ratios shown in B of FIG. 20), 20 μl of NP-His-Si-ATPS, 5 μl of His-Si-luciferase, 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide (T2 (SEQ ID NO: 7), 100 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (each 33 mM). This reaction mixture was added to individual wells of a white 96-well plate containing 1 μl of target oligonucleotide (R6, 100 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 2000 seconds at room temperature with an integration time of 400 milliseconds.

[0102] FIG. 21A is a summary of experiments when the ratio of His-Si-BST2 (DNA-Pol):ATPS gradually increases from 0.1:1 to 2.5:1. FIG. 21B is a summary of experiments when the ratio of His-Si-BST2 (DNA-Pol):NP gradually increases from 0.1:1 to 10:1.

[0103] Example 18 Detection of miRNA (RNA oligonucleotide) using TET-miRNA (His-Si-enzyme) and DNA capture oligonucleotide The measurement of enzyme activity was carried out using a reaction mixture consisting of 20 μl of NP-His-Si-BST, 20 μl of NP-His-Si-ATPS, 5 μl of NP-His-Si-luciferase, 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide (T2 (SEQ ID NO: 7), 100 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (each 33 mM). This reaction mixture was added to individual wells of a white 96-well plate containing DNA or RNA target oligonucleotides (R5 (SEQ ID NOs: 38 and 39) or R6 (SEQ ID NOs: 36 and 37), 1 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 2500 seconds at room temperature with an integration time of 400 milliseconds.

[0104] Panel A of FIG. 22 shows the sequences of the target oligonucleotides (RNA and DNA) as provided by the manufacturer (IDT, San Diego CA) and the sequence of the capture oligonucleotide (T2) designed to detect them. Panel B of FIG. 22 shows the TET-miRNA reaction for detecting RNA and the corresponding DNA target oligonucleotides using a tethered enzyme. Panel C of FIG. 22 is a summary of the data shown in FIG. 22A and shows the calculated initial rate (gradient) of the reaction.

[0105] Example 19 Analysis Comparing the Sensitivity of Different Untethered DNA Polymerases (His-Si-Klenow and His-Si-BST) to Mismatches in Target Oligonucleotides The measurement of enzyme activity was carried out using a reaction mixture consisting of 20 μl of NP-His-Si-BST or NP-His-Si-Klenow, 20 μl of NP-His-Si-ATPS, 5 μl of NP-His-Si-luciferase, 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide (T2 (SEQ ID NO: 7), 100 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (each 33 mM). This reaction mixture was added to individual wells of a white 96-well plate containing 1 μl of each target oligonucleotide (1 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 2000 seconds at room temperature with an integration time of 400 milliseconds.

[0106] As shown in FIG. 23A, mismatched nucleotides to the sequence of the capture oligonucleotide T2 were introduced at an increasing ratio at the 3'-end of the target oligonucleotide sequence in the target oligonucleotides tested in this example (indicated by underlined characters). Both the R1 (SEQ ID NO: 1) and R6 (SEQ ID NO: 6) oligonucleotides match the capture oligonucleotide sequence, but their GC contents are at different levels. FIG. 23B is a summary of the initial rates of the TET-miRNA reaction for various oligonucleotides, and in both cases when using Klenow and when using BST, for the 100% match sequence, it shows a significantly faster kinetics calculated from the initial reaction gradient. FIG. 23C shows that by calculating the ratio of the reaction rates in the presence of various mismatched oligonucleotides and in the presence of the 100% match oligonucleotide, a measure of how sensitive the above two DNA polymerases are to the mismatch content in the target oligonucleotide is provided, and the BST polymerase becomes 2 to 5 times more sensitive depending on the ratio of mismatches.

[0107] Example 20 Comparison of the tethering of the TET-miRNA reaction when the capture oligonucleotide is in solution and when immobilized (non-oriented) Enzyme activity was measured using a reaction mixture consisting of 20 μl of NP-His-Si-BST or NP-His-Si-Klenow, 20 μl of NP-His-Si-ATPS, 5 μl of NP-His-Si-luciferase, 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of capture oligonucleotide (T2 (SEQ ID NO: 7), 100 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (each 33 mM). This reaction mixture was added to individual wells of a white 96-well plate containing 1 μl of target oligonucleotide (1 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 1500 seconds at room temperature with an integration time of 400 milliseconds.

[0108] The above TET-miRNA reaction was carried out using the capture oligonucleotide either in solution or immobilized on SiO2 NPs by non-specific adsorption (the capture oligonucleotide was incubated with SiO2 NPs at room temperature for 30 minutes and then washed by centrifugation and stored in native protein buffer). Figure 24 shows that adsorbing the above capture oligonucleotide onto the NPs significantly reduces the ability of the capture oligonucleotide to detect the target oligonucleotide.

[0109] Example 21 Analysis Comparing the TET-miRNA Reaction When the Capture Oligonucleotide Is in Solution and When It Is Immobilized on SiO2 NPs via Biotin-Streptavidin Enzyme activity was measured using a reaction mixture consisting of 20 μl of His-Si-BST, 20 μl of His-Si-ATPS, 5 μl of His-Si-luciferase, 5 μl of luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl of APS (30 mM), 1 μl of NP-capture oligonucleotide (T2 (SEQ ID NO: 7), 100 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (each 33 mM). This reaction mixture was added to individual wells of a white 96-well plate containing 1 μl of target oligonucleotide (R6 (SEQ ID NO: 6), 1 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 2000 seconds at room temperature with an integration time of 400 milliseconds.

[0110] Figure 25A identifies two immobilizable forms of the capture oligonucleotide generated, in which the biotin tag is attached to either the 5' or 3' end in the above oligonucleotide (the biotinylated oligonucleotide was purchased from IDT, CA, USA). The above 3' and 5' biotinylated oligonucleotides were immobilized on streptavidin-coated SiO2 NPs (500 nm, Bangs Laboratory, IN, USA) according to the manufacturer's instructions, and then unbound protein was washed three times by centrifugation. The TET-mRNA reaction showed low activity when the enzyme (DNA-Pol, ATP, and Luc) was in solution and the capture oligonucleotide tethered to 500 nm SiO2 NPs coated with streptavidin was used. Figure 25B shows that significantly higher activity was obtained when the TET-mRNA enzyme was also immobilized on the NPs (via the Si tag) in the presence of the 5'-biotinylated capture oligonucleotide, while the 3'-biotinylated capture oligonucleotide showed very low or no activity at all.

[0111] In this specification, the preferred embodiments have been described and illustrated in detail. However, those skilled in the relevant technical fields can make various modifications, additions, substitutions, etc. without departing from the spirit of the present invention. And thus, it will be apparent that these are considered to be within the scope of the present invention as defined in the appended claims.

[0112] Array information SEQUENCE LISTING <110> Cornell University <120> USING TETHERED ENZYMES TO DETECT NUCLEIC ACIDS <150> US 62 / 810,448 <151> 2019-02-26 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial <220> <223> R1 miRNA oligonucleotide <400> 1 atggggagca tttcctcttt 20 <210> 2 <211> 20 <212> DNA <213> Artificial <220> <223> R2 miRNA oligonucleotide <400> 2 atggggagca tttcctctta 20 <210> 3 <211> 20 <212> DNA <213> Artificial <220> <223> R3 miRNA oligonucleotide <400> 3 atggggagca tttcctctaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial <220> <223> R4 miRNA oligonucleotide <400> 4 atggggagca tttccttaaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial <220> <223> R5 miRNA oligonucleotide <400> 5 atggggagca ttaggataaa 20 <210> 6 <211> 20 <212> DNA <213> Artificial <220> <223> R6 miRNA oligonucleotide <400> 6 gagcatttcc tcttttattg 20 <210> 7 <211> 54 <212> DNA <213> Artificial <220> <223> T2 capture oligonucleotide <400> 7 cacaggccca ccaaaaagga aaaccccaat aaaagaggaa atgctccgaa aggg 54 <210> 8 <211> 46 <212> DNA <213> Artificial <220> <223> T3 capture oligonucleotide <400> 8 cacaggccca ccaaaaagga aaaccccaat aaaagaggaa atgctc 46 <210> 9 <211> 35 <212> DNA <213> Artificial <220> <223> T4 capture oligonucleotide <400> 9 caaaaaggaa aaccccaata aaagaggaaa tgctc 35 <210> 10 <211> 30 <212> DNA <213> Artificial <220> <223> T5 capture oligonucleotide <400> 10 gagcagacac caataaaaga ggaaatgctc 30 <210> 11 <211> 38 <212> DNA <213> Artificial <220> <223> T6 capture oligonucleotide <400> 11 gagcagacac caataaaaga ggaaatgctc cgaaaggg 38 <210> 12 <211> 54 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p -TARGET1 capture oligonucleotide <400> 12 cacaggccca ccaaaaagga aaacccctat acaacctact acctcacgaa aggg 54 <210> 13 <211> 54 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p -TARGET1A capture oligonucleotide <400> 13 cacaggccca aaaaaaagga aaacccctat acaacctact acctcacgaa aggg 54 <210> 14 <211> 54 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p -TARGET1B capture oligonucleotide <400> 14 cacaggaaaa aaaaaaagga aaacccctat acaacctact acctcacgaa aggg 54 <210> 15 <211> 54 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p - TARGET1C capture oligonucleotide <400> 15 aaaaaaaaaa aaaaaaagga aaacccctat acaacctact acctcacgaa aggg 54 <210> 16 <211> 54 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p - TARGET1D capture oligonucleotide <400> 16 aaaaaaaaaa aaaaaaaaaa aaaaaactat acaacctact acctcacgaa aggg 54 <210> 17 <211> 54 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p - TARGET1E capture oligonucleotide <400> 17 aaaaaaaaaa aaaaaaaaaa aaaaaactat acaacctact acctcaaaaa aaaa 54 <210> 18 <211> 53 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p - TARGET2 capture oligonucleotide <400> 18 cacaggccca ccaaaaagga aaacccctat acaacctact acctcacgaa agg 53 <210> 19 <211> 52 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p -TARGET3 capture oligonucleotide <400> 19 cacaggccca ccaaaaagga aaacccctat acaacctact acctcacgaa ag 52 <210> 20 <211> 51 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p -TARGET4 capture oligonucleotide <400> 20 cacaggccca ccaaaaagga aaacccctat acaacctact acctcacgaa a 51 <210> 21 <211> 50 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p -TARGET5 capture oligonucleotide <400> 21 cacaggccca ccaaaaagga aaacccctat acaacctact acctcacgaa 50 <210> 22 <211> 49 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p - TARGET6 capture oligonucleotide <400> 22 cacaggccca ccaaaaagga aaacccctat acaacctact acctcacga 49 <210> 23 <211> 48 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p - TARGET7 capture oligonucleotide <400> 23 cacaggccca ccaaaaagga aaacccctat acaacctact acctcacg 48 <210> 24 <211> 47 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p - TARGET8 capture oligonucleotide <400> 24 cacaggccca ccaaaaagga aaacccctat acaacctact acctcac 47 <210> 25 <211> 46 <212> DNA <213> Artificial <220> <223> hsa-let-7a-5p - TARGET9 capture oligonucleotide <400> 25 cacaggccca ccaaaaagga aaacccctat acaacctact acctca 46 <210> 26 <211> 22 <212> DNA <213> Artificial <220> <223> miRNA oligonucleotide MIR-340#2 <400> 26 ttataaagca atgagactga tt 22 <210> 27 <211> 22 <212> DNA <213> Artificial <220> <223> miRNA oligonucleotide MIR-340#1 <400> 27 tataaagcaa tgagactgat tg 22 <210> 28 <211> 22 <212> DNA <213> Artificial <220> <223> miRNA oligonucleotide MIR-340#2 1bp change <400> 28 gtataaagca atgagactga tt 22 <210> 29 <211> 22 <212> DNA <213> Artificial <220> <223> miRNA oligonucleotide MIR-340#3 <400> 29 ggataaagca atgagactga tt 22 <210> 30 <211> 22 <212> DNA <213> Artificial <220> <223> miRNA oligonucleotide MIR-340#4 <400> 30 ttataaagca atgagactga tg 22 <210> 31 <211> 22 <212> DNA <213> Artificial <220> <223> miRNA oligonucleotide MIR-340#5 <400> 31 ttataaagca atgagactga gg 22 <210> 32 <211> 22 <212> DNA <213> Artificial <220> <223> miRNA oligonucleotide MIR-340#6 <400> 32 ttataaagct atgagactga tt 22 <210> 33 <211> 22 <212> DNA <213> Artificial <220> <223> miRNA oligonucleotide MIR-340#7 <400> 33 ttataaagct ttgagactga tt 22 <210> 34 <211> 55 <212> DNA <213> Artificial <220> <223> Capture oligonucleotide CAP1 (hsa-miR-451a - TARGET1) <400> 34 cacaggccca ccaaaaagga aaacccactc agtaatggta acggtttcga aaggg 55 <210> 35 <211> 55 <212> DNA <213> Artificial <220> <223> Capture oligonucleotide CAP2 (hsa-miR-451a - TARGET2) <400> 35 aaaaaaaaaa aaaaaaaaaa aaaaaaactc agtaatggta acggtttaaa aaaaa 55 <210> 36 <211> 20 <212> RNA <213> Artificial <220> <223> Target oligonucleotide RNAR6 <400> 36 gagcauuucc ucuuuuauug 20 <210> 37 <211> 20 <212> DNA <213> Artificial <220> <223> Target oligonucleotide DNAR6 <400> 37 gagcatttcc tcttttattg 20 <210> 38 <211> 20 <212> RNA <213> Artificial <220> <223> Target oligonucleotide RNAR5 <400> 38 auggggagca uuaggauaaa 20 <210> 39 <211> 20 <212> DNA <213> Artificial <220> <223> Target oligonucleotide DNAR5 <400> 39 atggggagca ttaggataaa 20

Claims

1. 1. A method for detecting a target nucleic acid molecule in a sample, comprising: Providing a sample containing a target nucleic acid molecule; contacting the sample with a capture oligonucleotide molecule complementary to at least a portion of the target nucleic acid molecule, such that the capture oligonucleotide molecule hybridizes to the complementary portion of the target nucleic acid molecule and forms a double stranded nucleic acid molecule, the capture oligonucleotide molecule (i) having a length of 30-60 base pairs, (ii) having a 4-8 base pair overhang at its 3' end, (iii) having a 5' tail, (iv) having a target specific portion between the 3' end and the 5' tail, (v) having a deoxyadenosine diphosphate content of 40-50%, (vi) having no deoxythymidine phosphate at the 3' end or the 5' tail, and (vii) having an ATP content at the 3' end and the 5' tail that is 40-50% of the ATP content of the capture oligonucleotide molecule; contacting the double-stranded nucleic acid molecule, a polymerase, and a dNTP mixture to form a polymerase extension mixture; subjecting the polymerase extension mixture to conditions under which the target nucleic acid molecule is extended and releases a free phosphate; generating adenosine triphosphate from the released free phosphate; metabolizing the adenosine triphosphate generated from the free phosphate with luciferase to generate a bioluminescent readout signal indicative of the presence of the target nucleic acid molecule in the sample; The method comprising:

2. The method of claim 1 , wherein the DNA polymerase is bound to a solid support.

3. 3. The method of claim 2, wherein the DNA polymerase is attached to the solid support by a linker selected from the group consisting of His-Si, His, Si, biotin, streptavidin, Pt, Au, Ag, His-Pt, His-Au, His-Ag, GST, an antibody, and an epitope tag.

4. The method of claim 1 , wherein the luciferase is bound to a solid support.

5. 5. The method of claim 4, wherein the luciferase is attached to the solid support by a linker selected from the group consisting of His-Si, His, Si, biotin, streptavidin, Pt, Au, Ag, His-Pt, His-Au, His-Ag, GST, an antibody, and an epitope tag.

6. 2. The method of claim 1, wherein the step of generating adenosine triphosphate comprises subjecting the released free phosphate to a combined glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase enzymatic reaction to generate adenosine triphosphate.

7. 7. The method of claim 6, wherein the step of subjecting the released free phosphate to the enzymatic reaction of the combined glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase comprises contacting adenosine diphosphate, nicotinamide adenine dinucleotide, and glyceraldehyde 3-phosphate to effect the enzymatic reaction of the combined glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase.

8. The method of claim 7, wherein said glyceraldehyde 3-phosphate dehydrogenase and said phosphoglycerate kinase are bound to a solid support.

9. 2. The method of claim 1, wherein the step of generating adenosine triphosphate comprises contacting the released free phosphate with adenosine 5'-phosphosulfate in the presence of adenosine triphosphate sulfurylase to generate adenosine triphosphate.

10. The method of claim 9, wherein the adenosine triphosphate sulfurylase is bound to a solid support.

11. The target nucleic acid molecule is present in the sample at a concentration of 10 per liter. －５ 13. The method of claim 1, wherein the compound is present in a submolar concentration.

12. The method of claim 1 , wherein the target nucleic acid molecule is a microRNA.

13. 2. The method of claim 1, wherein the subjecting step is carried out at a temperature of from 0 to 100°C.

14. The method of claim 13, wherein the subjecting step is carried out at a temperature of from 25 to 40°C.

15. 2. The method of claim 1, wherein the polymerase is full length BST DNA polymerase, large fragment BST DNA polymerase, BST 2.0 DNA polymerase, Klenow fragment (3' to 5' exo), and DNA polymerase I (large Klenow fragment).

16. 10. The method of claim 1, further comprising quantifying the bioluminescent readout signal to determine the presence or concentration of the target nucleic acid molecule in the sample.

17. The method of claim 16 , wherein the presence of the target nucleic acid molecule in the sample is determined.

18. The presence of the target nucleic acid molecule in the sample calculating the initial rate of bioluminescent signal production; Calculating the period of time required to achieve peak bioluminescence; calculating the magnitude of the peak bioluminescence signal or the integrated bioluminescence signal from time zero to the peak bioluminescence; The method of claim 17, wherein the determination is made by a procedure comprising:

19. The method of claim 16, wherein the concentration of the target nucleic acid molecule in the sample is determined.

20. 10. The method of claim 1, wherein deoxyadenosine triphosphate is excluded from the polymerase extension mixture.

21. 2. The method of claim 1, wherein the sample is selected from the group consisting of blood, urine, cerebrospinal fluid, saliva, tissue, and synthetic material.

22. The method of claim 1 , which is carried out in solution.

23. The method of claim 1 , wherein multiple types of capture oligonucleotide molecules are provided for detecting multiple types of target nucleic acid molecules.

24. 1. A method for detecting a target nucleic acid molecule in a sample, comprising: Providing a sample containing a target nucleic acid molecule; contacting the sample with a capture oligonucleotide molecule that is complementary to at least a portion of the target nucleic acid molecule such that the capture oligonucleotide molecule hybridizes to the complementary portion of the target nucleic acid molecule and forms a double-stranded nucleic acid molecule; contacting the double-stranded nucleic acid molecule, a polymerase, and a dNTP mixture to form a polymerase extension mixture; subjecting the polymerase extension mixture to conditions under which the target nucleic acid molecule is extended and releases a free phosphate; enzymatically generating adenosine triphosphate from the released free phosphate; metabolizing the adenosine triphosphate generated from the free phosphate with luciferase to generate a bioluminescent readout signal indicative of the presence of the target nucleic acid molecule in the sample, wherein the DNA polymerase, the luciferase, and the adenosine triphosphate generating enzyme are each bound to a solid support; The method comprising:

25. 25. The method of claim 24, wherein the capture oligonucleotide molecule has a length of 30 to 60 base pairs.

26. 25. The method of claim 24, wherein the capture oligonucleotide molecule has a 4-8 base pair overhang at its 3' end.

27. 27. The method of claim 26, wherein the capture oligonucleotide molecule has a 5' tail.

28. 28. The method of claim 27, wherein the capture oligonucleotide molecule has a target-specific portion between the 3' end and the 5' tail.

29. 25. The method of claim 24, wherein the capture oligonucleotide molecules have a deoxyadenosine diphosphate content of 40-50%.

30. 28. The method of claim 27, wherein the capture oligonucleotide molecule does not have a deoxythymidine phosphate at the 3' end or the 5' tail.

31. 25. The method of claim 24, wherein the capture oligonucleotide molecule has a 3' end and a 5' tail, both of which have an ATP content that is 40-50% of the ATP content of the capture oligonucleotide molecule.

32. 25. The method of claim 24, wherein multiple types of capture oligonucleotide molecules are provided for detecting multiple types of target nucleic acid molecules.

33. 1. A kit for detecting a target nucleic acid molecule in a sample, comprising: a capture oligonucleotide molecule that is complementary to at least a portion of the target nucleic acid molecule such that it hybridizes to the complementary portion of the target nucleic acid molecule and forms a double-stranded nucleic acid molecule; a polymerase bound to a solid support; A dNTP mix; an enzyme bound to a solid support for generating adenosine triphosphate from the released free phosphate; Luciferase bound to a solid support to generate a bioluminescent readout signal. The kit comprising:

34. 34. The kit of claim 33, comprising a plurality of capture oligonucleotide molecules for detecting a plurality of target nucleic acid molecules.

35. 1. A kit for detecting a target nucleic acid molecule in a sample, comprising: a capture oligonucleotide molecule that is complementary to at least a portion of the target nucleic acid molecule such that it hybridizes to the complementary portion of the target nucleic acid molecule to form a double stranded nucleic acid molecule, the capture oligonucleotide molecule (i) having a length of 30-60 base pairs, (ii) having a 4-8 base pair overhang at its 3' end, (iii) having a 5' tail, (iv) having a target specific portion between the 3' end and the 5' tail, (v) having a deoxyadenosine diphosphate content of 40-50%, (vi) having no deoxythymidine phosphate at the 3' end or the 5' tail, and (vii) having an ATP content at the 3' end and the 5' tail that is 40-50% of the ATP content of the capture oligonucleotide molecule; A polymerase and A dNTP mix; an enzyme for producing adenosine triphosphate from the released free phosphate; Luciferase to generate a bioluminescent readout signal The kit comprising:

36. 36. The kit of claim 35, comprising a plurality of capture oligonucleotide molecules for detecting a plurality of target nucleic acid molecules.

37. 1. A composition comprising a capture oligonucleotide molecule, the capture oligonucleotide molecule (i) having a length of 30-60 base pairs, (ii) having a 4-8 base pair overhang at its 3' end, (iii) having a 5' tail, (iv) having a target specific portion between the 3' end and the 5' tail, (v) having a deoxyadenosine diphosphate content of 40-50%, (vi) having no deoxythymidine phosphate at the 3' end or the 5' tail, and (vii) having an ATP content at the 3' end and the 5' tail that is 40-50% of the ATP content of the capture oligonucleotide molecule.

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