Detection of nucleic acids using tethered enzymes

文档序号：246218 发布日期：2021-11-12 浏览：10次中文

阅读说明：本技术 使用拴系酶检测核酸 (Detection of nucleic acids using tethered enzymes ) 是由 R·科恩 A·特拉维斯于 2020-02-26 设计创作，主要内容包括：本申请涉及检测样品中靶核酸分子的方法。所述方法包括提供含有靶核酸分子的样品和捕获寡核苷酸分子。在一个实施方案中,所述捕获寡核苷酸分子(i)长度为30-60个碱基对,(ii)在其3’端上具有4-8个碱基对的突出端,(iii)具有5’尾,(iv)在所述3’端和所述5’尾之间具有靶特异性部分,(v)脱氧二磷酸腺苷含量为40-50％,(vi)在所述3’端或所述5’尾中没有脱氧磷酸胸苷,并且(vii)所述3’端和所述5’尾的ATP含量为所述捕获寡核苷酸分子的ATP含量的40-50％。在检测方法的另一方面中,某些试剂与固体支持物偶联。本申请还涉及可用于进行本申请方法的组合物和试剂盒。(The present application relates to methods for detecting a target nucleic acid molecule in a sample. The method includes providing a sample containing a target nucleic acid molecule and a capture oligonucleotide molecule. In one embodiment, the capture oligonucleotide molecule (i) is 30-60 base pairs in length, (ii) has an overhang of 4-8 base pairs on its 3 'end, (iii) has a 5' tail, (iv) has a target-specific moiety between the 3 'end and the 5' tail, (v) has an adenosine diphosphate content of 40-50%, (vi) has no thymidine diphosphate in the 3 'end or the 5' tail, and (vii) has an ATP content of the 3 'end and the 5' tail that is 40-50% of the ATP content of the capture oligonucleotide molecule. In another aspect of the detection method, certain reagents are coupled to a solid support. The present application also relates to compositions and kits useful for performing the methods of the present application.)

1. A method of detecting a target nucleic acid molecule in a sample, the method 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 nucleotide molecule and forms a double-stranded nucleic acid molecule, wherein the capture oligonucleotide molecule (i) is 30-60 base pairs in length, (ii) has an overhang of 4-8 base pairs on its 3 'end, (iii) has a 5' tail, (iv) has a target-specific portion between the 3 'end and the 5' tail, (v) has an adenosine diphosphate content of 40-50%, (vi) has no thymidine deoxyphosphate in either the 3 'end or the 5' tail, and (vii) has an ATP content of 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 that extend the target nucleic acid molecule and release free phosphate;

generating adenosine triphosphate from the released free phosphate; and

metabolizing the adenosine triphosphate produced by the free phosphate with luciferase to produce a bioluminescent readout signal indicating the presence of the target nucleic acid molecule in the sample.

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

3. The method of claim 2, wherein the DNA polymerase is coupled to the solid support with 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.

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

5. The method of claim 4, wherein the luciferase is coupled to the solid support with 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. The method of claim 1, wherein the generating adenosine triphosphate comprises:

subjecting the released free phosphate to a coupled glyceraldehyde-3-phosphate dehydrogenase-phosphoglycerate kinase enzymatic reaction to produce adenosine triphosphate.

7. The method of claim 6, wherein said subjecting said released free phosphate to a coupled glyceraldehyde-3-phosphate dehydrogenase-phosphoglycerate kinase enzymatic reaction comprises:

contacting adenosine diphosphate, nicotinamide adenine dinucleotide and glyceraldehyde-3-phosphate to effect the coupled glyceraldehyde-3-phosphate dehydrogenase-phosphoglycerate kinase enzymatic reaction.

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

9. The method of claim 1, wherein the generating adenosine triphosphate comprises:

contacting the released free phosphate with adenosine 5' -phosphosulfate in the presence of adenosine triphosphate sulfurylase to produce adenosine triphosphate.

10. The method of claim 9, wherein the atpase is coupled to a solid support.

11. The method of claim 1, wherein the target nucleic acid molecule is less than 10^-5The concentration of moles/liter is present in the sample.

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

13. The method of claim 1, wherein the subjecting is performed at a temperature of 0 to 100 ℃.

14. The method of claim 13, wherein the subjecting is performed at a temperature of 25 to 40 ℃.

15. The method of claim 1, wherein the polymerase is full-length BST DNA polymerase, large fragment BST DNA polymerase, BST2.0 DNA polymerase, Klenow fragment (3 'to 5' exo), and DNA polymerase I (large Klenow fragment).

16. 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 method of claim 17, wherein the presence of the target nucleic acid molecule in the sample is determined by a procedure comprising:

calculating an initial rate of bioluminescent signal generation;

calculating the time period required for reaching the peak bioluminescence; and

the peak amplitude of the bioluminescent signal or the integrated bioluminescent signal from time zero to peak bioluminescence is calculated.

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

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

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

22. The method of claim 1, wherein the method is performed in solution.

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

24. A method of detecting a target nucleic acid molecule in a sample, the method 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 that extend the target nucleic acid molecule and release free phosphate;

enzymatically producing adenosine triphosphate from the released free phosphate; and

metabolizing the adenosine triphosphate produced by the free phosphate with luciferase to produce 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 producing enzyme are each coupled to a solid support.

25. The method of claim 24, wherein the capture oligonucleotide molecule is 30-60 base pairs in length.

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

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

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

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

30. The method of claim 27, wherein the capture oligonucleotide molecule is free of deoxythymidine triphosphate in the 3 'end or the 5' tail.

31. The method of claim 24, wherein the ATP content of the 3 'end and the 5' tail of the capture oligonucleotide molecule is 40-50% of the ATP content of the capture oligonucleotide molecule.

32. The method of claim 24, wherein a plurality of capture oligonucleotide molecules are provided for detecting a plurality of target nucleic acid molecules.

33. A kit for detecting a target nucleic acid molecule in a sample, the kit comprising:

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;

a polymerase coupled to a solid support;

a dNTP mixture;

an enzyme for producing adenosine triphosphate from the released free phosphate, coupled to a solid support; and

a luciferase for generating a bioluminescent readout signal, the luciferase being coupled to a solid support.

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

35. A kit for detecting a target nucleic acid molecule in a sample, the kit comprising:

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 a complementary portion of the target nucleic acid molecule and forms a double-stranded nucleic acid molecule, wherein the capture oligonucleotide molecule (i) is 30-60 base pairs in length, (ii) has an overhang of 4-8 base pairs on its 3 'end, (iii) has a 5' tail, (iv) has a target-specific portion between the 3 'end and the 5' tail, (v) has an adenosine deoxydiphosphate content of 40-50%, (vi) has no thymidine deoxyphosphate in the 3 'end or the 5' tail, and (vii) has an ATP content of 40-50% of the ATP content of the capture oligonucleotide molecule;

a polymerase;

a dNTP mixture;

an enzyme that produces adenosine triphosphate from the released free phosphate; and

luciferase for use in generating a bioluminescent readout signal.

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

37. A composition, comprising:

a capture oligonucleotide molecule, wherein the capture oligonucleotide molecule (i) is 30-60 base pairs in length, (ii) has an overhang of 4-8 base pairs on its 3 'end, (iii) has a 5' tail, (iv) has a target-specific moiety between the 3 'end and the 5' tail, (v) has an adenosine diphosphate content of 40-50%, (vi) has no thymidine phosphate in the 3 'end or the 5' tail, and (vii) has an ATP content of the 3 'end and the 5' tail that is 40-50% of the ATP content of the capture oligonucleotide molecule.

Technical Field

The present application relates to the detection of nucleic acids using tethered enzymes.

Background

Nucleic acid amplification can be used to determine whether a particular template nucleic acid is present in a sample. If an amplification product is produced, this indicates the presence of template nucleic acid in the sample. In contrast, the absence of any amplification product indicates that the template nucleic acid is not present in the sample. These techniques are important in diagnostic applications, for example, for determining the presence of pathogens in a sample.

Nucleic acids can be amplified by a variety of thermal cycling and isothermal techniques. Thermal cycling techniques, such as Polymerase Chain Reaction (PCR), use temperature cycling to drive repeated cycles of DNA synthesis, resulting in large amounts of new DNA being synthesized in proportion to the original amount of template DNA. Recently, a number of isothermal techniques have also been developed that do not rely on thermal cycling to drive the amplification reaction. Isothermal techniques using DNA polymerases with strand displacement activity have been developed to achieve amplification reactions that do not involve RNA synthesis steps. Similarly, isothermal techniques using reverse transcriptase, rnase H and DNA-dependent RNA polymerase have been developed for amplification reactions involving RNA synthesis steps.

However, the detection and/or quantification of specific nucleic acid sequences is an important technique for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to diseases, and measuring response to various types of treatment. These procedures can also be used to detect and quantify microorganisms in food, water, industrial and environmental samples, seed stores, and other types of materials that may require monitoring for the presence of particular microorganisms. Other applications are found in forensic science, anthropology, archaeology and biology, where measurements of nucleic acid sequence relatedness have been used to identify criminal suspects, resolve paternity determinations, construct pedigrees and phylogenetic trees and help classify various life forms.

Advances in the field of molecular biology over the last 20 years have allowed the detection of 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 bodily fluids. Thus, many methods for detecting and/or quantifying nucleic acid sequences are well known in the art. However, an inherent result of highly sensitive nucleic acid amplification systems is the presence of by-products. The inclusion of byproducts in some systems may interfere with the amplification reaction molecules, thereby reducing specificity. This is because limited amplification resources, including primers and enzymes required for primer extension and formation of transcription products, are transferred to the formation of byproducts. In some cases, the presence of by-products may also complicate the analysis of amplicon production by various molecular techniques. Furthermore, in many cases of interest, specific nucleic acid sequences are present in very low concentrations in samples tested for the desired nucleic acid sequence. In this case, if the assay sensitivity cannot be increased, the presence of the desired molecule cannot be detected.

The present application is directed to overcoming these and other deficiencies in the art.

Disclosure of Invention

One aspect of the present application relates to a method of detecting a target nucleic acid molecule in a sample. The method comprises the following steps: providing a sample containing a 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 and forms a double-stranded nucleic acid molecule. A capture oligonucleotide molecule (i) 30-60 base pairs in length, (ii) an overhang (overlap) with 4-8 base pairs on its 3 'end, (iii) a 5' tail, (iv) a target-specific moiety between the 3 'end and the 5' tail, (v) an adenosine diphosphate content of 40-50%, (vi) no thymidine diphosphate in the 3 'end or the 5' tail, and (vii) an ATP content of the 3 'end and the 5' tail is 40-50% of the ATP content of the capture oligonucleotide molecule. The double-stranded nucleic acid molecule, polymerase and dNTP mixture are contacted together to form a polymerase extension mixture. The polymerase extension mixture is subjected to conditions that cause extension of the target nucleic acid molecule and release of free phosphate. Adenosine triphosphate is then produced from the released free phosphate and the adenosine triphosphate produced from the free phosphate is metabolized with luciferase to produce a bioluminescent readout signal, indicative of the presence of the target nucleic acid molecule in the sample.

Another aspect of the present application relates to a method of detecting a target nucleic acid molecule in a sample. The method comprises the following steps: providing a sample containing a 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 and forms a double-stranded nucleic acid molecule. The double-stranded nucleic acid molecule, polymerase and dNTP mixture are contacted together to form a polymerase extension mixture. The polymerase extension mixture is subjected to conditions that cause extension of the target nucleic acid molecule and release of free phosphate. Adenosine triphosphate is then enzymatically produced from the released free phosphate and metabolized by luciferase to produce a bioluminescent readout signal indicative of the presence of the target nucleic acid molecule in the sample. The DNA polymerase, luciferase and adenosine triphosphate producing enzyme are each coupled to a solid support.

Another aspect of the present application relates to a kit for detecting a target nucleic acid molecule in a sample. The kit comprises a capture oligonucleotide molecule that is complementary to at least a portion of a 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; a polymerase coupled to a solid support; a dNTP mixture; an enzyme for producing adenosine triphosphate from the released free phosphate, coupled to a solid support; and a luciferase for generating a bioluminescent readout signal, wherein the luciferase is coupled to a solid support.

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 and forms a double-stranded nucleic acid molecule. A capture oligonucleotide molecule (i) 30-60 base pairs in length, (ii) having a 4-8 base pair overhang at its 3 'end, (iii) having a 5' tail, (iv) having a target-specific moiety between the 3 'end and the 5' tail, (v) having an adenosine diphosphate content of 40-50%, (vi) having no thymidine diphosphate in either the 3 'end or the 5' tail, and (vii) having an ATP content of the 3 'end and the 5' tail of 40-50% of the ATP content of the capture oligonucleotide molecule. The kit further comprises a polymerase, a dNTP mix, an enzyme for generating adenosine triphosphate from the released free phosphate and a luciferase for generating a bioluminescent readout signal.

A final aspect of the present application relates to a composition comprising a capture oligonucleotide molecule, wherein the capture oligonucleotide molecule (i) is 30-60 base pairs in length, (ii) has an overhang of 4-8 base pairs on its 3 'end, (iii) has a 5' tail, (iv) has a target-specific portion between the 3 'end and the 5' tail, (v) has an adenosine deoxydiphosphate content of 40-50%, (vi) has no thymidine deoxyphosphate in the 3 'end or the 5' tail, and (vii) has an ATP content of the 3 'end and the 5' tail that is 40-50% of the ATP content of the capture oligonucleotide molecule.

The present application discloses significant advances in methods for detecting nucleic acids by using, for example, enzymatic reactions in which enzymes are tethered to a surface (e.g., nanoparticles). The assays described herein are converted to a common luminescence output. That is, in certain embodiments, they may all be linked to a Bioluminescent (BL) protein or substrate, which will allow for the emission and reading of light in an amount that correlates to 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.

The present application has several advantages over other detection methods and systems. These include: 1) speed-assays using enzymatic reactions occur rapidly, providing a readout within minutes; 2) using luminescence-based readout, it enables independent, highly portable systems and devices that do not require bulky excitation elements (such as those required for fluorescence); 3) sensitivity-signal amplification due to release of multiple free phosphates for each hybridization event and enzymatic reaction assay, facilitating detection and readout steps; 4) manufacturing costs are reduced-possible components of such systems, including for example nanoparticles, can be made of inexpensive materials and can be easily mass produced; 5) multiplexing capability-in certain embodiments of the present application, coupled biochemical reactions can detect multiple nucleic acid molecules in a single system; 6) the use of tethered enzymes promotes maximum enzyme stability and activity; 7) the use of tethered enzymes limits the reaction and readout to specific areas of the system (e.g., specific areas of the card), reducing the size of the photodetectors in the reader; 8) the use of the tethered enzyme limits the reaction and reading, and realizes on-line negative control and background luminescence control; 9) the use of the tethered enzyme limits reaction and read-out, reducing light contamination for detection of other nucleic acid molecules in the same system; 10) immobilization of the capture oligonucleotide enhances the ability to detect multiple target oligonucleotides in a specific region of the system (e.g., a specific region of the card); 11) the use of isothermal amplification enables detection at ambient temperature without temperature cycling; and 12) the design of the capture oligonucleotide enables a single step reaction without interference/inhibition of by-products, and the ability to incorporate a bioluminescent enzyme into the single step reaction enables an additional level of signal amplification from the release of free phosphate from the metabolized AP molecule, which is fed back into the ATP-generating reaction.

Drawings

Figure 1 shows an embodiment of the Tethered Enzyme Technology (TET) -miRNA reaction. The miRNA (or other single-stranded nucleic acid polymer) anneals to a complementary insertion sequence within the capture oligonucleotide and generates a double strand. This allows DNA-polymerase to bind and initiate a polymerization reaction, which releases free phosphate (PPi) by nucleotide incorporation (isothermal replication). These PPi groups are then enzymatically reacted by coupled GAPDH-PGK in the presence of ADP, NAD +, and GAP to generate ATP. Finally, luciferase hydrolyses ATP to generate a bioluminescent signal.

Figure 2 shows another embodiment of the TET-miRNA reaction. The miRNA (or other single-stranded nucleic acid polymer) anneals to a complementary insertion sequence within the capture oligonucleotide and generates a double strand. This allows DNA-polymerase to bind and initiate a polymerization reaction, which releases free phosphate (PPi) by nucleotide incorporation (isothermal replication). These PPi groups are then used by the enzyme ATP-sulfurylase to generate ATP in the presence of APS. Finally, luciferase hydrolyses ATP to generate a bioluminescent signal.

FIG. 3 shows the design and characteristics of the capture oligonucleotide (Cap-Oligo).

Figure 4 shows that exclusion of dATP from the reaction mixture improves assay kinetics and sensitivity.

Fig. 5A to 5D show that TET-miRNA assay can detect mismatched nucleotides in the 3' end of the target oligonucleotide, as well as various analyses or quantifications of the data.

Fig. 6A to 6B show the testing of the sensitivity range of TET-miRNA assay for target oligonucleotide detection.

Fig. 7A to 7C show that the design of the 5 'and 3' tails of the capture oligonucleotides affects TET-miRNA reaction kinetics.

Fig. 8A to 8B show that capture oligonucleotide dATP content affects TET-miRNA reaction kinetics.

Fig. 9A to 9B show that the 3' tail length of the capture oligonucleotide affects TET-miRNA reactivity and kinetics.

Fig. 10A to 10C show that the TET-miRNA assay is sensitive to mutations in the MIR-340 target oligonucleotide (lung cancer-associated microrna) sequence.

Fig. 11A-11B show that TET-miRNA assay can detect miRNA naturally occurring in human serum.

Figure 12 shows that the TET-miRNA assay can detect mirnas naturally present in human plasma.

Fig. 13A to 13B show that ribonuclease (rnase) treatment abolished TET-miRNA activity.

Fig. 14A-14B show detection of target oligonucleotides relative to reactions in solution using (non-directional) NP immobilization.

Fig. 15A-15B show inhibition of TET-miRNA reaction assay by immobilization of commercial BST2.0 onto NP via biotin-streptavidin binding.

Fig. 16A to 16C show that TET-miRNA assay is only little affected by temperature.

Fig. 17A to 17E show two different capture oligonucleotide designed assays (not tethered) for detection of mirnas in human serum.

FIGS. 18A-18B show activity comparisons of various DNA polymerase activities in TET-miRNA assays.

Fig. 19A to 19D show target oligonucleotide detection using TET-miRNA when tethered versus in solution.

Fig. 20A-20C show a comparison of His-Si-Klenow to His-Si-BST activity in the TET-miRNA assay, tethered versus untethered.

FIGS. 21A-21B show a comparison of target oligonucleotide detection using various ratios of His-Si-ATPS/His-Si-BST/NP.

Fig. 22A to 22C show miRNA (RNA oligonucleotide) detection using TET-miRNA (His-Si-enzyme) and DNA capture oligonucleotide.

Fig. 23A-23C show the target oligonucleotide mismatch sensitivity (His-Si-Klenow versus His-Si-BST) for testing different non-tethered DNA polymerases.

Figure 24 shows testing TET-miRNA responses when capture oligonucleotides are in solution versus immobilized (non-oriented).

FIGS. 25A-25B show when the capture oligonucleotide is immobilized to SiO in solution relative to by biotin-streptavidin₂Test TET-miRNA response at NP.

Detailed Description

One aspect of the present application relates to a method of detecting a target nucleic acid molecule in a sample. The method comprises the following steps: providing a sample containing a 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 and forms a double-stranded nucleic acid molecule. A capture oligonucleotide molecule (i) 30-60 base pairs in length, (ii) having a 4-8 base pair overhang at its 3 'end, (iii) having a 5' tail, (iv) having a target-specific moiety between the 3 'end and the 5' tail, (v) having an adenosine diphosphate content of 40-50%, (vi) having no thymidine diphosphate in either the 3 'end or the 5' tail, and (vii) having an ATP content of the 3 'end and the 5' tail of 40-50% of the ATP content of the capture oligonucleotide molecule. The double-stranded nucleic acid molecule, polymerase and dNTP mixture are contacted together to form a polymerase extension mixture. The polymerase extension mixture is subjected to conditions that cause extension of the target nucleic acid molecule and release of free phosphate. Adenosine triphosphate is then produced from the released free phosphate and the adenosine triphosphate produced from the free phosphate is metabolized with luciferase to produce a bioluminescent readout signal, indicative of the presence of the target nucleic acid molecule in the sample.

As shown in fig. 1, 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 the provided capture oligonucleotide in tether or solution, and a double strand is generated. This allows the DNA polymerase to bind and initiate a polymerization reaction, releasing free phosphate (PPi) by nucleotide incorporation. ADP, NAD + and glyceraldehyde 3-phosphate are provided for the assay. In the presence of these components, GAPDH and PGK, tethered to free phosphate, are used to generate ATP. The ATP tethered Luc is then used to generate a bioluminescent signal in conjunction with fluorescein. 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. In one embodiment, the emitted light may be read quantitatively and/or qualitatively by a photodetector positioned to capture the emitted signal.

Alternatively, as shown in fig. 2, a DNA polymerase is tethered to one nanoparticle, and an ATP sulfurylase (ATP-sul) and luciferase ("Luc") are tethered to another nanoparticle. The miRNA target anneals to a complementary insertion sequence within the provided capture oligonucleotide in tether or solution, and a double strand is generated. This allows the DNA polymerase to bind and initiate a polymerization reaction, releasing free phosphate (PPi) by nucleotide incorporation. Adenosine 5' -phosphosulfate (APS) is provided for the assay. Free phosphate-tethered ATP-sul is used to generate ATP in the presence of APS. The ATP tethered Luc is then used to generate a bioluminescent signal in conjunction with fluorescein. 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. In one embodiment, the emitted light may be read quantitatively and/or qualitatively by a photodetector positioned to capture the emitted signal.

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

The term "nucleic acid" refers to a polymer of nucleotides (e.g., natural and non-natural ribonucleotides and deoxyribonucleotides), including DNA, RNA, and their subclasses, such as cDNA, mRNA, miRNA, and the like. Nucleic acids can be single-stranded and typically contain 5'-3' phosphodiester linkages, but in some cases, nucleotide analogs can have other linkages. Nucleic acids may include naturally occurring bases (adenosine, guanosine, cytosine, uracil and thymidine) as well as non-natural bases.

As used herein, the term "target nucleic acid" or "target" refers to a portion of a nucleic acid sequence to be detected or analyzed in a sample. The term target includes all variants of the target sequence, such as one or more mutant variants and wild-type variants.

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

As used herein, "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 hybridize" refers to the hybridization of capture oligonucleotides to their target sequences or copies thereof under stringent hybridization assay conditions 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 counterpart to a much greater extent than to non-target sequences. Suitable hybridization conditions are well known in the art, can be predicted based on sequence composition, or can be determined by using conventional testing methods (see, e.g., Sambrook et al, Molecular Cloning, a Laboratory Manual, 2 nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, n.y.,1989, which is incorporated by reference herein in its entirety).

FIG. 3 depicts an embodiment of the design of the capture oligonucleotide. As shown in FIG. 3, the capture oligonucleotide may be 30-60 nucleotides in length, including a 5 'extension tail, an internal complementary insert, and a 3' tail. The 5 'extension tail and 3' tail sequences do not contain dTTP. The ATP content of the 5 'extension tail and the 3' tail is 40-50% of the total oligonucleotide. The optimal capture oligonucleotide sequence comprises 4 to 8 additional nucleotides at the 3' tail. Preferably, the capture oligonucleotide is designed not to hybridize to itself to form a hairpin structure, such that hybridization to the target nucleic acid is interfered.

As used herein, "luciferase" refers to an oxygenase that catalyzes the luminescence reaction as follows:

thus, unless otherwise specified, a luciferase refers to an enzyme or photoprotein that catalyzes a bioluminescent reaction (a reaction that produces bioluminescence), and is a naturally occurring, recombinant, or mutant luciferase. When naturally occurring, the luciferase can be readily obtained from the organism by one of ordinary skill in the art. If the luciferase is a naturally-occurring luciferase, or a recombinant or mutant luciferase (e.g., a luciferase that retains activity in the luciferase-luciferin reaction of the naturally-occurring luciferase), then a nucleic acid encoding the luciferase is expressed. It can be readily obtained from cultures of transformed bacteria, yeast, mammalian cells, insect cells, plant cells, and the like. In addition, recombinant or mutant luciferases can be readily obtained from in vitro cell-free systems using nucleic acids encoding the luciferases. Luciferase was purchased from Promega Corporation, Madison, WI.. Luciferases, modified mutants or variants thereof are also known in the art and are described, for example, in Thorne et al, "Illuminating instruments to Firefly Luciferase and Other bioluminescence Reporters Used in Chemical Biology," Chemistry & Biology 17(6): 646-.

"polymerase extension" reactions according to the present application include all forms of template-directed polymerase-catalyzed nucleic acid synthesis reactions. The conditions and reagents for the primer extension reaction are known in the art, and any standard methods, reagents, enzymes, and the like can be used at this stage (see, e.g., Sambrook et al, (ed.), Molecular Cloning: a Laboratory Manual (1989), Cold Spring Harbor Laboratory Press, which is incorporated herein by reference in its entirety). Thus, in its most basic form, the extension reaction is carried out in the presence of primers, deoxynucleotides (dNTPs) and a suitable polymerase (e.g., Klenow) or indeed any useful and suitable 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, BST2.0 DNA polymerase, Klenow fragment (3 'to 5' exo), and DNA polymerase I (large Klenow fragment). The conditions may be selected according to choice according to procedures known in the art.

The polymerase extension technique used in the methods of the present application is an isothermal technique (i.e., a technique that is performed at a single temperature or a technique in which a major aspect of the amplification process is performed at a single temperature). These techniques rely on the ability of the polymerase to replicate the amplified template strand to form a binding duplex. Isothermal techniques rely on strand displacing polymerases to separate/displace both strands of a duplex and to replicate the template. This well-known property has been the subject of many scientific articles (see, e.g., 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 incorporated herein by reference in their entirety).

Briefly, as 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 is performed using available nucleotides provided in a deoxynucleotide (dNTP) mixture added to the reaction. These dNTPs include deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP). In one embodiment, the deoxyadenosine triphosphate is excluded from the polymerase extension mixture.

As mentioned above, the polymerase extension technique used in the methods of the present application is an isothermal technique (i.e., a technique that is performed at a single temperature or a technique in which a major aspect of the amplification process is performed at a single temperature). Thus, in certain embodiments, the polymerase extension reaction is performed at a temperature of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 to 100 ℃. In one embodiment, the polymerase extension reaction is performed at a temperature of 25 to 40 ℃.

Polymerase extension reactions release two phosphates (PPi) per nucleotide added to the DNA strand. In the methods of the present application, the release of these free phosphates (PPi) can then be used to facilitate the detection of a target nucleic acid molecule in a sample. Specifically, the presence or absence of a target nucleic acid molecule can be detected by enzymatic reaction to convert PPi to ATP and subsequent bioluminescent detection of ATP using the signal transduction molecule luciferase.

The luciferase and luciferin are used in combination to identify the target nucleic acid, as the amount of light generated is substantially proportional to the amount of ATP generated, and in turn proportional to the amount of nucleotide incorporated and the target nucleic acid present. Thus, the method further comprises providing fluorescein and O₂Wherein fluorescein and O are mixed₂Is added to the reaction.

As described above, the methods described herein involve subjecting a polymerase extension mixture to conditions that extend the target nucleic acid molecule and release the free phosphate. Adenosine Triphosphate (ATP) is then produced from the free phosphate released by the enzymatic reaction and then metabolized with luciferase to produce a bioluminescent readout signal. According to this aspect, in one embodiment, generating adenosine triphosphate comprises subjecting the released free phosphate to a coupled glyceraldehyde-3-phosphate dehydrogenase-phosphoglycerate kinase (GAPDH-PGK) enzymatic reaction to generate adenosine triphosphate.

In this embodiment, the enzymatic reaction involves reacting GAPDH 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 produce a measurable signal. The reaction scheme is shown below:

in another embodiment, adenosine triphosphate can be produced by contacting the released free phosphate with adenosine 5' -phosphosulfate (APS) in the presence of adenosine triphosphate sulfurylase (ATP-sul) to produce adenosine triphosphate. The ATP then reacts with luciferase to produce a measurable signal. The reaction scheme is shown below:

according to the above embodiments, the glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and/or adenosine triphosphate sulfurylase may be coupled to the solid support as described above.

The amount of light produced can be readily determined using a sensitive device such as a photometer under appropriate light. Thus, luminescence assays offer the advantage of being able to quantify.

In one embodiment, the bioluminescent read signal is quantified to determine the presence or concentration of the target nucleic acid molecule in the sample. The amount of target nucleic acid can be determined from the peak amplitude of the luminescence signal, and/or the time it takes for the signal to reach its peak amplitude, and/or the integrated amount of signal emitted over a period of time, and/or the rate at which luminescence is produced.

In one embodiment, the target nucleic acid molecule is less than 10^-5The concentration of moles/liter is present in the sample.

In one embodiment, the presence of a target nucleic acid molecule in a sample is determined by a procedure comprising: calculating an initial rate of bioluminescent signal generation; calculating the time period required for reaching the peak bioluminescence; and calculating the peak amplitude of the bioluminescent signal or an integrated bioluminescent signal from time zero to peak bioluminescence.

These procedures can be used alone or as part of an analytical method that combines two or more procedures for better quantitation. For each procedure or combination of procedures, a threshold can be predetermined and calibrated against a known amount of target nucleic acid target. Predetermined values may also be used to identify sequences (i.e., mutations) that are similar but not identical to the desired target oligonucleotide. In addition, the values provided by these analytical procedures can be evaluated against cut-off values to provide a measure of presence/absence, or as a measure that enables quantitative readings.

In one embodiment, a plurality of capture oligonucleotide molecules are provided for detecting a plurality of target nucleic acid molecules.

A contemplated method according to this embodiment is a multiplex assay in which a plurality of capture oligonucleotides are utilized to determine whether one or more of a plurality of predetermined nucleic acid target sequences are present in a sample. One particularly useful field of such multiplex assays is screening assays where the usual analytical output indicates the absence of the nucleic acid sought.

In a multiplex embodiment of the above method, the sample is mixed with a plurality of different capture oligonucleotides. In this embodiment, the analytical output of a certain result using one capture oligonucleotide is distinguishable from the analytical output of the opposite result using all capture oligonucleotides.

According to this embodiment, for example, the solid support can contain a plurality of capture oligonucleotides specific for a plurality of target nucleic acids. Each capture oligonucleotide may be localized at a defined location or region of the solid support or synthesized in situ on the surface of a defined location or region of the solid support. Such supports facilitate parallel analysis of a plurality of capture oligonucleotide-bound target nucleic acids. These supports are also suitable for high throughput screening.

In certain embodiments, the reaction of the present application is carried out in solution. The term "in solution" refers to any assay that detects a target nucleic acid in solution or suspension. For example, a first hybridization to a target nucleic acid can be performed with a first capture oligonucleotide and a second hybridization to the target nucleic acid can be performed with a second capture oligonucleotide. Such multiple hybridizations may include a washing step to remove any undesired (e.g., non-hybridizing sequence) components.

In certain embodiments, the enzyme according to the methods of the present application may be coupled to a solid support. In other embodiments, the enzymes of the process may remain in solution.

Suitable supports include organic or inorganic materials, and can be of any suitable size or shape (e.g., scaffold sheets, platforms, and/or nanoparticles). Tethering or immobilizing components of an assay according to the present application serves to, for example, spatially confine them and enhance their stability and/or function in performing, for example, a cascade or sequential reaction as part of a particular assay. In certain embodiments, the support material comprises, for example, a nucleotide sequence or a gel. In certain embodiments, an enzyme or component of an assay according to the present application can be immobilized or tethered to a luminal surface of a channel (e.g., a microfluidic channel) such as a nanoparticle or a support material such as a platform.

Several techniques may be used to immobilize a component (e.g., an enzyme) of an assay according to the present application on a surface. For example, the components may be non-specifically attached or bound by specific but non-directed chemical reactions (e.g., carboxy-amide binding). According to the method of the present application, directed enzyme immobilization may also be used. Directed enzyme immobilization confers several advantages, including, for example, the localization of binding tags (e.g., affinity tags), such that the activity and stability of Tethered Enzymes are optimized (see Mukai et al, "Sequential Reactions of Surface-treated genomic Enzymes," chem. biol.16(9):1013-20(2009), which is incorporated by reference herein in its entirety).

One example of how enzymes involved in nucleic acid detection are tethered to a surface is the use of directional immobilization. In certain embodiments of assays according to the present application, the recombinase or assay components involved in the reaction of the assay are engineered with affinity tags such that they are capable of binding to a surface such as silica or nickel or a component of a surface such as nickel-nitrilotriacetic acid. For example, the affinity tag may be attached to the amino or carboxy terminus of the protein to be immobilized, or embedded within the protein to be immobilized. The optimal location of the tethering domain will depend on the nature and location of the enzyme catalytic domain, the substrate binding domain, and any conformational changes that the enzyme must produce.

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 readily expressed as fusions with appropriate binding tags to facilitate immobilization to a solid support containing the corresponding capture binding moiety. Suitable capture moieties and binding tag partners that can be used in accordance with 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, e.g., Gosh et al, "compatible attachments of Oligonucleotides to Solid Supports," Nucleic Acids Res.15(13): 5353-; joos et al, "value Attachment of Hybridizable Oligonucleotides to Glass Supports," anal. biochem.247(1):96-101 (1997); lund et al, "Assessment of Methods for equivalent Binding of Nucleic Acids to Magnetic Beads, Dynabeads, and the characterization of the Bound Nucleic Acids in Hybridization Reactions," Nucleic Acids Res.16(22):10861-80(1988), which are incorporated herein by reference in their entirety.

In certain embodiments, the DNA polymerase and/or luciferase are coupled to a solid support.

According to this aspect of the application, the DNA polymerase and/or luciferase may be coupled to the solid support with 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 surface used as a support, platform, or scaffold can take a variety of forms including, for example, various nanoparticles or nucleic acid strands, and can include various geometries.

In certain embodiments according to the present application, the support is a nanoparticle. As used herein, the term "nanoparticle" refers to any particle having an average diameter in the nanometer range (i.e., an average diameter of at most 1 μm). The nanoparticles used may be made of any suitable organic or inorganic substance known to those of ordinary skill 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 nanoparticle is a magnetic nanoparticle. In another embodiment, the nanoparticles are magnetic silica-coated nanoparticles ("MSPs").

In addition to Nanoparticles (NPs), supports or scaffolds of different materials may be in the form of rods, flat surfaces, graphene sheets, nanotubes, DNA scaffolds, gels, microspheres, or inner channel walls of microchannels of larger supports. Quantum dots are also contemplated for use as supports according to the present application. Enzyme immobilization may be achieved by non-specific binding, chemical modification, affinity tags, or other conjugation techniques.

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

Exemplary negative controls for use in the methods of the present application include blocking oligonucleotides that target the target oligonucleotide (sequences that are fully or partially complementary to the target oligonucleotide), blocking oligonucleotides that target the capture oligonucleotide (modified at the 3'/5' end to inhibit extension), ribonucleases (rnases) added to the reaction mixture, reaction mixtures lacking the capture oligonucleotide, reaction mixtures lacking nucleotides (dntps), and reaction mixtures lacking any substrate. Exemplary positive controls for use in the methods of the present application include various concentrations (including saturating amounts) of a target oligonucleotide mimic (a DNA oligonucleotide having the same sequence as the target oligonucleotide from the sample) and a pre-annealed double-stranded DNA having a overhanging single-stranded sequence that is capable of being extended by a polymerase.

In various related aspects, the present application also relates to devices and kits for performing the methods described herein. Such kits comprise monitors, reagents and procedures that can be used in a clinical or research setting or are suitable for field laboratory or field use. In particular, kits comprising the disclosed reagents for performing the methods described herein are contemplated to include any of a variety of means for detecting captured target nucleic acid molecules and measuring the bioluminescent signal generated upon target capture, along with appropriate instructions. Suitable kits include reagents sufficient to perform an assay to detect a target nucleic acid molecule.

It is understood that such kits may be used in any of the methods of the present application. The selection of a particular component depends on the particular method for which the kit is designed to perform. Additional components may be provided for detecting the assay output as measured by the release of ATP and detection of the bioluminescent signal.

As described above, the kit optionally further comprises instructions for detecting the target nucleic acid by the methods described herein. Instructions present in such kits instruct the user how to use the components of the kit to perform the various methods of the present application. These instructions may include a description of the detection methods of the present application, including detection by luminescence.

Thus, another aspect of the present application relates to a kit for detecting a target nucleic acid molecule in a sample. The kit comprises a capture oligonucleotide molecule that is complementary to at least a portion of a 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; a polymerase coupled to a solid support; a dNTP mixture; an enzyme for producing adenosine triphosphate from the released free phosphate, coupled to a solid support; and a luciferase for generating a bioluminescent readout signal, wherein the luciferase is coupled to a solid support.

The kit can further comprise a plurality of capture oligonucleotide molecules for detecting a plurality of target nucleic acid molecules. In contemplated kits for multiplex capture oligonucleotide-mediated detection of specific nucleic acids, the kit contains a plurality of capture oligonucleotides directed against a nucleic acid target of interest. Preferably, when the kit contains a plurality of capture oligonucleotides, each capture oligonucleotide is designed to interrogate a different target nucleic acid sequence.

Examples

The following examples are intended to illustrate the practice of embodiments of the present disclosure, but are in no way intended to limit the scope thereof.

Example 1-exclusion of dATP from the reaction mixture improves assay kinetics and sensitivity

Mu.l ATP sulfurylase (NEB, M0394S, 300U/ml), 0.25. mu.l Klenow (NEB, Lg fragment, M0210S, 5000U/ml), 5. mu.l His-Si-luciferase (prepared in the laboratory), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl. RTM.) were mixed together₂) Mu.l APS (30mM), 1. mu.l capture oligonucleotide (T2(SEQ ID NO:7), 1. mu.M), 1.8. mu.l dNTP mix (33 mM each) and +/-dATP to prepare 100ul of a total volume reaction mixture. The reaction mixture was added to individual wells of a white 96-well plate containing 1. mu.l of the target oligonucleotide (R6(SEQ ID NO:6), 1. mu.M). The reaction mixture was then immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 2000 seconds with a 400 millisecond integration time.

As shown in FIG. 4, two identical reactions were tested using the conditions described above, excluding dATP (adenosine triphosphate) from the nucleotide mixture added to reaction A. Luciferase binding and hydrolysis of dATP caused attenuation of the initial luminescent signal (1), delay in peak response phase (2), and overall lower signal amplitude (3). This shows the relationship between pure sequencing and the tethered detection method used here, where bioluminescence is generated as a readout. As a result of these data, the capture oligonucleotide was designed not to include any dTPS in its sequence to avoid the need for dATP in the reaction mixture.

Example 2 TET-miRNA assay mismatch nucleotides in the 3' end of detectable target oligonucleotides

Mu.l ATP sulfurylase (NEB, M0394S, 300U/ml), 0.25. mu.l Klenow (NEB, Lg fragment, M0210S, 5000U/ml), 5. mu.l His-Si-luciferase (prepared in the laboratory), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl. RTM.) were mixed together₂) Mu.l APS (30mM), 1. mu.l capture oligonucleotide (T2(SEQ ID NO:7), 100uM) and 1.8. mu.l dNTP mix (33 mM each) to prepare a 100. mu.l total volume reaction. The reaction mixture was added to individual wells of a white 96-well plate containing 1. mu.l of each target oligonucleotide (R1-R6(SEQ ID NO:1-6), 100. mu.M) and immediately placed in a TECAN plate reader to read the luminescence signal for 3500 seconds at room temperature with a 400 millisecond integration time.

FIG. 5A shows that the luminescence signal decreases with increasing percentage of mismatched nucleotides at the 3' end of the target oligonucleotide sequence as shown in FIG. 5B (in underlined font). Two complementary target oligonucleotides (R1(SEQ ID NO:1) and R6(SEQ ID NO:6), complementary to the shifted and overlapping sequences in the capture oligonucleotide) and 4 mismatched oligonucleotides (expressed as a percentage of mismatched nucleotides) were tested, demonstrating strong inhibition of the TET reaction to nucleotide mismatches, and are summarized in FIG. 5C. FIG. 5D shows an illustration of a possible data analysis procedure for determining the presence and hybridization of target oligonucleotides to capture oligonucleotides (data for target oligonucleotides R1(SEQ ID NO:1) and R6(SEQ ID NO:6) shown in FIG. 5A are provided): a/a' -calculating the initial rate (slope) of luminescence signal generation; b/b' -peak light emission time; c/c' -peak amplitude of the luminescence signal; and d/d' -the integrated luminescence signal from time 0 to the peak.

Example 3 determination of sensitivity Range of target oligonucleotide detection Using TET-miRNA assay

Mu.l ATP sulfurylase (NEB, M0394S, 300U/ml), 0.25. mu.l Klenow (NEB, Lg fragment, M0210S, 5000U/ml), 5. mu.l His-Si-luciferase (prepared in the laboratory), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl. RTM.) were mixed together₂) Mu.l of APS (30mM), 0.5. mu.l of capture oligonucleotide (T2(SEQ ID NO:7) (see FIG. 7A), 1. mu.M) and 1.8. mu.l of dCTP/dTTP/dGTP mixtures (33 mM each) were used to prepare a total volume of 100. mu.l of reaction mixture. The reaction mixture was added to individual wells of a white 96-well plate containing decreasing concentrations of the target oligonucleotide (R6(SEQ ID NO:6), 0mM, 1pM, 10pM, 100pM, 1nM, 10nM, 1. mu.M) and immediately placed in a TECAN plate reader to read the luminescence signal at 400 msec integration time for 2000 sec at room temperature.

FIG. 6A shows the luminescent signal in response to decreasing concentrations of target oligonucleotide. Shows 1 picomole (10)^-12mol/L) to 1 micromole (10)^-6mol/L) concentration detection range. Figure 6B shows that the summary of figure 6A as calculated from the reaction kinetics (i.e. the slope of the initial reaction phase as a function of oligonucleotide concentration (in picoM)) indicates high sensitivity and wide dynamic range, although further optimization is required.

Example 4 design of the 5 'and 3' tails of the Capture oligonucleotides influences the kinetics of the TET-miRNA reaction

By mixing 0.05. mu.l ATP sulfurylase (NEB, M0394S, 300U/ml), 0.25. mu.l Klenow (NEB, Lg fragment, M0210S, 5000U/ml),5ul His-Si-luciferase (laboratory prepared), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl)₂) Mu.l APS (30mM), 1. mu.l target oligonucleotide (R6(SEQ ID NO:6), 100. mu.M) and 1.8. mu.l dNTP mix (33 mM each) to prepare a total volume of 100. mu.l reaction mixture. The reaction mixture was added to individual wells of a white 96-well plate containing 1. mu.l of each capture oligonucleotide (T2-T6(SEQ ID NO:7-11), 100. mu.M) and immediately placed in a TECAN plate reader to read the luminescence signal for 1000 seconds at room temperature with a 400 millisecond integration time.

As shown in FIG. 7A, several designs of capture oligonucleotides were generated (T2-T6(SEQ ID NOS: 7-11)). All designs included similar complementary inserts (complementary to the R6 target oligonucleotide, indicated in underlined font). Figure 7B shows the luminescence signal as measured in response to the addition of the target oligonucleotide in the presence of various capture oligonucleotides (as provided in figure 7A). Figure 7C is a summary of figure 7B and shows the differences in annealing kinetics as indicated by the calculated reaction slopes of the various capture oligonucleotides.

Example 5-Capture oligonucleotide dATP content influencing the kinetics of the TET-miRNA reaction

Mu.l ATP sulfurylase (NEB, M0394S, 300U/ml), 0.25. mu.l Klenow (NEB, Lg fragment, M0210S, 5000U/ml), 5. mu.l His-Si-luciferase (prepared in the laboratory), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl. RTM.) were mixed together₂) Mu.l APS (30mM), 1. mu.l target oligonucleotide (R6(SEQ ID NO:6), 100. mu.M) and 1.8. mu.l dNTP mix (33 mM each) to prepare a total volume of 100. mu.l reaction mixture. The reaction mixture was added to individual wells of a white 96-well plate containing 1. mu.l of each capture oligonucleotide (T1(SEQ ID NO:12) + T1A-T1E (SEQ ID NO:13-17), 100. mu.M) and immediately placed in a TECAN plate reader to read the luminescence signal at 400 msec integration time for 1000 sec at room temperature.

As shown in FIG. 8A, 6 capture oligonucleotides containing increasing percentages of dATP (27% -63%) were designed for detection of the target oligonucleotide hsa-let-7a-5p (all with similar complementary insertion sequences, in underlined font). Figure 8B is a summary of data showing optimal activity with 40% -50% dATP content within the capture oligonucleotide sequence. This experimental value is surprising and represents a significant enhancement.

Example 6-3' Tail Length of Capture oligonucleotides affects TET-miRNA reactivity and kinetics

Mu.l ATP sulfurylase (NEB, M0394S, 300U/ml), 0.25. mu.l Klenow (NEB, Lg fragment, M0210S, 5000U/ml), 5. mu.l His-Si-luciferase (prepared in the laboratory), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl. RTM.) were mixed together₂) Mu.l APS (30mM), 1. mu.l target oligonucleotide (R6(SEQ ID NO:6), 100. mu.M) and 1.8. mu.l dNTP mix (33 mM each) to prepare a total volume of 100. mu.l reaction mixture. The reaction mixture was added to individual wells of a white 96-well plate containing 1. mu.l of each capture oligonucleotide (T1-T9(SEQ ID NO:12 and SEQ ID NO:18-25), 100. mu.M) and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 1000 seconds with a 400 millisecond integration time.

As shown in FIG. 9A, 9 capture oligonucleotides were designed for detection of the target oligonucleotide hsa-let-7a-5 p. These nine capture oligonucleotides contain decreasing numbers of nucleotides after the miRNA complementary insertion site (in underlined font) at the 3' end of their sequences. Figure 9B shows that the optimal activity of the TET-miRNA assay was achieved when 5-8 nucleotides were added to the 3' of the target oligonucleotide complementary insert.

Example 7-TET-miRNA assay sensitive to mutations in the MIR-340 target oligonucleotide sequence

Mu.l ATP sulfurylase (NEB, M0394S, 300U/ml), 0.25. mu.l Klenow (NEB, Lg fragment, M0210S, 5000U/ml), 5. mu.l His-Si-luciferase (prepared in the laboratory), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl. RTM.) were mixed together₂) Mu.l APS (30mM), 1. mu.l capture oligonucleotide (T2, 100. mu.M) and 1.8. mu.l dNTP mix (33 mM each) to prepare a total volume of 100. mu.l reaction mix. The reaction mixture was added to individual wells of a white 96-well plate containing 1. mu.l of each target oligonucleotide (MIR-340#1-MIR-340#7(SEQ ID NO:27-33), 100. mu.M) and immediately placed in a TECAN plate reader to be in the chamberLuminescence signal was read at 400 msec integration time for 1200 sec at room temperature.

As shown in FIG. 10A, MIR340 target oligonucleotide (MIR-340#1(SEQ ID NO:27)) and various mutant sequences (MIR-340#2- #7) (SEQ ID NO:28-33) were used in this experiment, as well as 2 controls (former-unmutated MIR-340#2, and Ctrl-reaction mixtures that do not include any target oligonucleotide) (mutated nucleotides are shown in underlined font). Fig. 10B to 10C show the luminescence signals and kinetics measured for various mutations as shown in fig. 10A. The data in fig. 10A show that all tested mutations induced detectable differences in the measured signals.

Example 8 TET-miRNA assay naturally occurring miRNAs in human serum and human plasma can be detected

Mu.l ATP sulfurylase (NEB, M0394S, 300U/ml), 0.25. mu.l Klenow (NEB, Lg fragment, M0210S, 5000U/ml), 5. mu.l His-Si-luciferase (prepared in the laboratory), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl. RTM.) were mixed together₂) Mu.l APS (30mM), 1. mu.l capture oligonucleotide (100. mu.M) targeting a naturally occurring miRNA as listed in FIG. 10A, and 1.8. mu.l dNTP mix (33 mM each) to prepare a 40. mu.l total volume reaction mixture. The reaction mixture was added to individual wells of a white 96-well plate containing 60 μ l of human serum or plasma and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 500 seconds with a 400 millisecond integration time.

As shown in fig. 11A, 6 capture oligonucleotides were designed to detect 6 different naturally occurring mirnas in commercially obtained human serum. Real-time kinetics of the TET-miRNA reaction are shown. Fig. 11B is a summary of fig. 11A, where the bioluminescent signals were integrated for 500 seconds to show the relative amounts of the various mirnas in the test serum samples.

As shown in figure 12, a set of 6 naturally occurring mirnas from plasma samples from 3 human donors (collected in Guthrie Medical Center, Sayre PA) were tested with TET-miRNA.

Example 9 Elimination of TET-miRNA Activity by ribonuclease (RNase) treatment

By mixing 0.05. mu.l ATP sulfurylase (N)EB, M0394S, 300U/ml), 0.25. mu.l Klenow (NEB, Lg fragment, M0210S, 5000U/ml), 5. mu.l His-Si-luciferase (laboratory preparations), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl)₂) 2 μ l APS (30mM), 1 μ l capture oligonucleotide (100 μ M) targeting a naturally occurring miRNA as described in FIGS. 12A-12B, and 1.8 μ l dNTP mix (33 mM each) to prepare a 40 μ l total volume reaction mixture. The reaction mixture was added to individual wells of a white 96-well plate containing 60 μ l of human plasma and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 500 seconds with a 400 millisecond integration time.

Figure 13A shows a set of 4 naturally occurring mirnas tested with TET-miRNA on plasma samples from 3 subjects collected from Guthrie (Sayre, PA). Figure 13B shows a significant decrease in signal observed after 30 minutes of treatment with rnase-a at room temperature.

Example 10 assay for detection of target oligonucleotides reacted in solution using NP-stationary phase

Biotinylated enzyme was immobilized to streptavidin-coated microspheres (500nm SiO. RTM.) according to the manufacturer's instructions₂Bangs Laboratory, IN, USA), and then spin-washed away unbound protein 3 times. Equal amounts of NP-tethered or untethered enzyme were added to a reaction mixture (100 μ l total volume per reaction) containing: mu.l His-Si-luciferase (prepared in the laboratory), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl)₂) Mu.l APS (30mM), 1. mu.l capture oligonucleotide (Cap-HAS-MIR-451a, 1. mu.M) and 1.8. mu.l dCTP/dTTP/dGTP mixture (33 mM each). The reaction mixture was added to individual wells of a white 96-well plate containing the target oligonucleotide (HAS-MIR-451a, 1. mu.M) and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 2000 seconds with a 400 millisecond integration time.

Commercially available human serum samples were spiked with equal amounts of the target oligonucleotide HSA-MIR-451a and added to TET-miRNA reaction mixtures including the soluble enzyme luciferase/ATP-sulfurylase/Klenow (fig. 14A, Sol) or tethered to NPs (fig. 14B, NP). Note that DNA-polymerase and ATP-sulfurylase were tethered to the NP using non-directed immobilization by biotinylation. ATP sulfurylase (NEB, M0394S, 300U/ml) and Klenow (NEB, Lg fragment, M0210S, 5000U/ml) were biotinylated using the EZ-Link Sulfo-NHS-LC-biotinylation kit (Thermo Scientific, USA) according to the manufacturer's instructions.

Example 11-immobilization of commercial BST2.0 to NP-inhibited TET-miRNA reaction assay by Biotin-streptavidin binding

BST2.0(NEB, M0537S, 8000U/ml) was biotinylated using the EZ-Link Sulfo-NHS-LC-biotinylation kit (Thermo Scientific, USA) according to the manufacturer's instructions. Biotinylated enzyme was immobilized to streptavidin-coated microspheres (500nm SiO. RTM.) according to the manufacturer's instructions₂Bangs Laboratory, IN, USA), and then spin-washed away unbound protein 3 times. Equal amounts of NP-tethered or unbounded BST2.0 were added to a reaction mixture (100 μ l total volume per reaction) containing: 0.05 μ l ATP sulfurylase (NEB, M0394S, 300U/ml), 5 μ l His-Si-luciferase (laboratory prepared), 5 μ l luciferin (200mM), 5 μ l20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl)₂) Mu.l APS (30mM), 1. mu.l capture oligonucleotide (Cap-HSA-MIR-451a, 1. mu.M) and 1.8. mu.l dCTP/dTTP/dGTP mixture (33 mM each). The reaction mixture was added to individual wells of a white 96-well plate containing the target oligonucleotide (HSA-MIR-451a, 1. mu.M) and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 2000 seconds with a 400 millisecond integration time.

Commercially obtained human serum samples were spiked with equal amounts of HSA-MIR-451a and added to TET-miRNA reaction mixtures including the soluble enzyme luciferase/ATP-sulfurylase/bst 2.0 (fig. 15A, Sol), or when bst2.0 was immobilized to NPs by biotinylation (fig. 15B, NP). The data show that biotinylation has a negative effect on its activity, and even more so when bst2.0 is tethered to NP by non-directed immobilization.

Example 12 TET-miRNA assay is only minimally affected by temperature

By mixing 0.05. mu.l ATP sulfurylase (NEB, M0394S, 300U/ml), 0.25. mu.l Klenow (NEB, Lg fragment, M0210S, 5000U/ml)) 5. mu.l His-Si-luciferase (prepared in the laboratory), 5. mu.l luciferin (200mM), 5. mu.l 20 XTluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl)₂) Mu.l APS (30mM), 1. mu.l capture oligonucleotide targeting the Has-let-7a-5p miRNA (100. mu.M), 1.8. mu.l dNTP mix (33 mM each) and Has-let-7a-5p oligonucleotide to prepare a total volume of 100. mu.l reaction mix. The reaction mixture was added to individual wells of a white 96-well plate and immediately placed in a TECAN plate reader to read the luminescence signal at the different temperatures indicated. Luminescence was measured for 1000 seconds with a 400 millisecond integration time.

As shown in fig. 16A, commercially obtained human serum samples were spiked with equal amounts of target oligonucleotide and added to the TET-miRNA reaction mixture at different temperatures (25 ℃ -40 ℃). Only minor differences were observed in the initial parameters of reaction kinetics (as measured from slope, fig. 16B) and efficiency (as measured from integrated signal, fig. 16C).

Example 13-analysis of two different capture oligonucleotide designs for detection of miRNA in human serum

Mu.l ATP sulfurylase (NEB, M0394S, 300U/ml), 0.25. mu.l Klenow (NEB, Lg fragment, M0210S, 5000U/ml), 5. mu.l His-Si-luciferase (prepared in the laboratory), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl. RTM.) were mixed together₂) Mu.l of APS (30mM), 1. mu.l of CAP1(SEQ ID NO:34) or CAP2(SEQ ID NO:35) capture oligonucleotide (100. mu.M), 1.8. mu.l of dNTP mix (33 mM each) and test oligonucleotide to prepare a total volume of 100. mu.l reaction mixture. The reaction mixture was added to individual wells of a white 96-well plate and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature. Luminescence was measured for 1000 seconds with a 400 millisecond integration time.

Figure 17A depicts the design of 2 capture oligonucleotides tested to detect 6 different naturally occurring mirnas in human plasma. CAP1 is a random nucleotide sequence, and in CAP2, the 5 'and 3' tails contain only adenosine. Figure 17B shows the real-time kinetics of TET-miRNA reactions to detect naturally occurring mirnas and DNA-based target oligonucleotides (test oligonucleotides) with similar sequences. Fig. 17C is an enlarged portion of the data presented in fig. 17B to show the luminescent signals of the various miRNA molecules. Fig. 17D is a summary of fig. 17A, where the bioluminescent signals were integrated for 500 seconds to show the relative amounts of the various mirnas. Figure 17E is a summary of the results of measurements of only naturally occurring mirnas (as shown in figure 17C).

Example 14 comparison of various DNA polymerase activities in the TET-miRNA assay

Mu.l ATP sulfurylase (NEB, M0394S, 300U/ml), DNA polymerase (as shown in FIG. 17A), 5. mu.l His-Si-luciferase (prepared in the laboratory), 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl, etc.) were mixed₂) Mu.l APS (30mM), 1. mu.l capture oligonucleotide targeting hsa-let-7a-5p miRNA (100. mu.M), 1.8. mu.l dNTP mix (33 mM each) and hsa-let-7a-5p oligonucleotide to prepare a total volume of 100. mu.l reaction mix. The reaction mixture was added to individual wells of a white 96-well plate and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 500 seconds with a 400 millisecond integration time.

Figure 18A is a list of DNA polymerases used in this experiment and some of their key features. As shown in fig. 18B, commercially obtained human serum samples were spiked with equal amounts of miRNA oligonucleotides and added to the TET-miRNA reaction mixture (at room temperature) in the presence of various DNA polymerases. The bar graph of the reaction kinetics (calculated from the initial slope of the reaction) shows that Bst, Klenow and Terminator DNA polymerase provide the fastest reaction kinetics under these experimental conditions. Control 1(CTRL1) reactions lack capture oligonucleotides, whereas control 2(CTRL2) does not include DNA polymerase. The data bars for the Bst and Klenow variants used to obtain the data in the previous figures are highlighted.

Example 15 target oligonucleotide detection Using TET-miRNA when tethered versus in solution

Preparation of an equivalent amount of NP-tethered or untethered enzyme, 5. mu.l His-Si-luciferase, 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl)₂) A reaction mixture consisting of 2. mu.l of APS (30mM), 1. mu.l of capture oligonucleotide (T2(SEQ ID NO:7), 100. mu.M) and 1.8. mu.l of a dCTP/dTTP/dGTP mixture (33 mM each). Adding the reaction mixtureIndividual wells of a white 96-well plate containing increasing amounts of target oligonucleotide (R6(SEQ ID NO:6), 200nM, 500nM and 1uM) were immediately placed in a TECAN reader to read the luminescence signal for 1400 seconds at room temperature with 400 msec integration time.

FIG. 19A shows a schematic of the His-Si-enzyme design (ATPS: ATP-sulfurylase, DNA-pol: DNA polymerase, Luc: luciferase). Genes encoding ATP-sulfurylase (MET3, sulfate adenylyltransferase, Saccharomyces cerevisiae), BST (Bacillus stearothermophilus) DNA polymerase I (pol) gene), and Klenow (Escherichia coli) strain LD93-1 DNA polymerase I) were fused with His-Si tags into pET17b vector for bacterial protein expression. His-Si-protein was purified using Ni-NTA beads and stored in Natural Protein Buffer (NPB) containing sorbitol until use. Fig. 19B shows the results of measurement of the enzyme activity when tethered versus in solution made using the reaction mixture described above. The data show that when TET-miRNA enzyme is immobilized to 500nm SiO₂Kinetics of individual reactions when on NP (NP, left), or when in solution (Sol, right). Figure 19C is a bar graph of reaction kinetics (calculated from the initial slope of the reaction) showing that the coupling reaction occurs with faster kinetics when TET-miRNA enzyme is tethered to NP. Figure 19D is a summary of NP TET-miRNA reactions in detecting increasing concentrations of R6 target oligonucleotide.

Example 16-comparison of His-Si-Klenow versus His-Si-BST Activity in TET-miRNA assay (tethered versus untethered)

Using an equivalent amount of NP-tethered or untethered enzyme, 5. mu.l His-Si-luciferase, 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl)₂) Measurement of enzyme activity when tethered versus in solution was performed with reaction buffer consisting of 2. mu.l APS (30mM), 1. mu.l capture oligonucleotide (T2(SEQ ID NO:7), 100. mu.M) and 1.8. mu.l dCTP/dTTP/dGTP mix (33 mM each). The reaction mixture was added to individual wells of a white 96-well plate containing increasing amounts of target oligonucleotide (R6(SEQ ID NO:6), 1. mu.M) and immediately placed in a TECAN plate reader to read luminescence signals at room temperature with 400 msec integration timeNumber 1200 seconds.

As shown in FIG. 20A, the large Klenow fragment from E.coli (E.coli strain LD93-1 DNA polymerase I) was expressed as a fusion protein downstream of the His-Si affinity tag. FIG. 20B shows the activity of His-Si-Klenow in solution compared to DNA-pol I (His-Si-BST2 (from Bacillus stearothermophilus)). His-Si-BST showed slightly better kinetics of activity in terms of initial reaction rate and overall activity. FIG. 20C shows when the reaction enzyme is tethered to 500nm SiO₂Comparison of the activities of two DNA polymerases in nanoparticles. Also, His-Si-BST showed better activity. In addition, both Klenow and BST show improved activity when the reaction includes tethered enzymes.

Example 17 comparison of target oligonucleotide detection Using varying ratios of His-Si-ATPS/His-Si-BST/NP

The PCR reaction was carried out using 20. mu.l NP-His-Si-BST (enzyme/NP at various ratios, as shown in FIG. 20B), 20. mu.l NP-His-Si-ATPS, 5. mu.l His-Si-luciferase, 5. mu.l luciferin (200mM), 5. mu.l 20 XTluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl)₂) The enzyme activity was measured in a reaction mixture consisting of 2. mu.l of APS (30mM), 1. mu.l of capture oligonucleotide (T2(SEQ ID NO:7), 100. mu.M) and 1.8. mu.l of dCTP/dTTP/dGTP mixture (33 mM each). The reaction mixture was added to individual wells of a white 96-well plate containing 1. mu.l of target oligonucleotide (R6, 100. mu.M) and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 2000 seconds with a 400 millisecond integration time.

FIG. 21A is a summary of experiments in which the ratio of His-Si-BST2(DNA-Pol) to ATPS increased from 0.1:1 to 2.5: 1. FIG. 21B is a summary of experiments in which the ratio of His-Si-BST2(DNA-Pol) to NP increased from 0.1:1 to 10: 1.

Example 18 detection of miRNA (RNA oligonucleotides) Using TET-miRNA (His-Si-enzyme) and DNA Capture oligonucleotides

The reagent was prepared from 20. mu.l NP-His-Si-BST, 20. mu.l NP-His-Si-ATPS, 5. mu.l NP-His-Si-luciferase, 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl)₂) 2. mu.l of APS (30mM), 1. mu.l of capture oligonucleotide (T2(SEQ ID NO:7), 100. mu.M) and 1.8. mu.l of a mixture of dCTP/dTTP/dGTP (33 mM each)Reaction mixture, measurement of enzyme activity was performed. The 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. mu.M) and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 2500 seconds with a 400 millisecond integration time.

FIG. 22A shows the sequences of target oligonucleotides (RNA and DNA) as provided by manufacturing company (IDT, San Diego CA), and the sequence of capture oligonucleotides (T2) designed to detect them. Figure 22B shows TET-miRNA reactions using tethered enzymes to detect RNA versus corresponding DNA target oligonucleotides. Fig. 22C is a summary of the data presented in fig. 22A, showing the calculated initial rate of reaction (slope).

Example 19 analysis of target oligonucleotide mismatch sensitivity of different non-tethered DNA polymerases (His-Si-Klenow vs His-Si-BST)

The buffer solution consisting of 20. mu.l NP-His-Si-BST or NP-His-Si-Klenow, 20. mu.l NP-His-Si-ATPS, 5. mu.l NP-His-Si-luciferase, 5. mu.l luciferin (200mM), 5. mu.l 20X luciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl)₂) The enzyme activity was measured in a reaction mixture consisting of 2. mu.l of APS (30mM), 1. mu.l of capture oligonucleotide (T2(SEQ ID NO:7), 100. mu.M) and 1.8. mu.l of dCTP/dTTP/dGTP mixture (33 mM each). The reaction mixture was added to individual wells of a white 96-well plate containing 1 μ l of each target oligonucleotide (1 μ M) and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 2000 seconds with a 400 millisecond integration time.

As shown in figure 23A, increasing percentages of mismatched nucleotides were introduced into the target oligonucleotides tested herein (in underlined font) at the 3' end of the target oligonucleotide sequence relative to the sequence of the capture oligonucleotide T2. Both the R1(SEQ ID NO:1) and R6(SEQ ID NO:6) oligonucleotides matched the capture oligonucleotide sequence; however, they contain different levels of GC content. Figure 23B is a summary of the initial rates of TET-miRNA reactions for various oligonucleotides and shows significantly faster kinetics for 100% matching sequences of Klenow and BST as calculated from the initial reaction slope. Figure 23C shows that calculating the ratio between reaction rates in the presence of various mismatch oligonucleotides and 100% match oligonucleotides provides a measure of the sensitivity of the two DNA polymerases to the amount of mismatch in the target oligonucleotide, where BST polymerase sensitivity is 2-5 times higher, in terms of percent mismatch.

Example 20 TET-miRNA reaction tethered when capture oligonucleotides were in solution versus immobilized (non-oriented)

The buffer solution was prepared from 20. mu.l NP-His-Si-BST or NP-His-Si-Klenow, 20. mu.l NP-His-Si-ATPS, 5. mu.l NP-His-Si-luciferase, 5. mu.l luciferin (200mM), 5. mu.l 20 XTluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl)₂) The enzyme activity was measured in a reaction mixture consisting of 2. mu.l of APS (30mM), 1. mu.l of capture oligonucleotide (T2(SEQ ID NO:7), 100. mu.M) and 1.8. mu.l of dCTP/dTTP/dGTP mixture (33 mM each). The reaction mixture was added to individual wells of a white 96-well plate containing 1 μ l of target oligonucleotide (1 μ M) and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 1500 seconds with a 400 millisecond integration time.

Using immobilization in solution or by non-specific adsorption to SiO₂On NP (capture oligonucleotide with SiO)₂NP was incubated at room temperature for 30 minutes, then spin-washed into native protein buffer) capture oligonucleotides were subjected to TET-miRNA reaction. Figure 24 shows that adsorption of the capture oligonucleotide to the NP significantly reduces its ability to detect the target oligonucleotide.

Example 21-analysis of TET-miRNA reaction when Capture oligonucleotides are in solution versus immobilized to SiO2 NP with Biotin-streptavidin

Mu.l His-Si-BST, 20. mu.l His-Si-ATPS, 5. mu.l His-Si-luciferase, 5. mu.l luciferin (200mM), 5. mu.l 20 Xluciferase buffer (50mM HEPES, 40mM KCL, 200mM MgCl) were used₂) The enzyme activity was measured in a reaction mixture consisting of 2. mu.l of APS (30mM), 1. mu.l of NP-capturing oligonucleotide (T2(SEQ ID NO:7), 100. mu.M) and 1.8. mu.l of dCTP/dTTP/dGTP mixture (33 mM each). The reaction mixture was added to individual wells of a white 96-well plate containing 1. mu.l of target oligonucleotide (R6(SEQ ID NO:6), 1. mu.M) and immediately placed in a TECAN plate reader to read the luminescence signal at room temperature for 2000 seconds with a 400 millisecond integration time.

FIG. 25A identifies the 2 forms of immobilizable capture oligonucleotides generated, in which a biotin tag is attached to the 5 'or 3' end (biotinylated oligonucleotides were purchased from IDT, CA, USA). 3' and 5' biotinylated oligonucleotides were immobilized to streptavidin-coated SiO according to the manufacturer's instructions₂NP (500nm, Bangs Laboratory, IN, USA), and then spin-washed 3 times to remove unbound protein. When enzymes (DNA-Pol, ATP and Luc) are in solution, 500nm SiO tethered to streptavidin-coated is used₂The capture oligonucleotide of NP, the TET-mRNA reaction, indicated low activity. Figure 25B shows that significantly higher activity was obtained when TET-mRNA enzyme was also immobilized to NP (via Si-tag) in the presence of 5' biotinylated capture oligonucleotide; however, the 3' biotinylated capture oligonucleotide showed very low or no activity.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the following claims.

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