Use of tethered enzymes to detect nucleic acids

Abstract

The present application relates to a method for detecting a target nucleic acid molecule in a sample. The method includes providing a sample containing the target nucleic acid molecule and a capture oligonucleotide molecule. In one embodiment, the capture oligonucleotide molecule (i) has a length of 30 to 60 base pairs, (ii) a 4 to 8 base pair overhang 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 at the 3' end and the 5' tail that is 40 to 50% of the ATP content of the capture oligonucleotide molecule. In another aspect of the detection method, a specific reagent is bound to a solid support. The present application also relates to compositions and kits useful for carrying out the method of the present application. TIFF2022521772000005.tif144154

Description

[Technical Field]

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

[0002] Field This application relates to the use of tethered enzymes to detect nucleic acids. [Background technology]

[0003] 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 the template nucleic acid in the sample. Conversely, if no amplification product is produced, it indicates the absence of the template nucleic acid in the sample. Such techniques are very important for diagnostic applications, such as determining whether a pathogen is present in a sample.

[0004] Nucleic acids can be amplified by various thermocycling 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 have been developed that utilize DNA polymerases with strand displacement activity. Similarly, for amplification reactions that involve an RNA synthesis step, isothermal techniques have been developed that use reverse transcriptase, RNase H, and DNA-dependent RNA polymerase.

[0005] Nevertheless, 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, distinguishing genetic changes associated with cancer, studying genetic susceptibility to disease, and measuring responses to various types of treatment. Such procedures are also useful for detecting and quantitating microorganisms in food, water, industrial and environmental samples, species, and other types of materials where the presence of specific microorganisms may need to be monitored. 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 aid in the classification of various life forms.

[0006] Advances in molecular biology over the past two decades have enabled the detection of specific nucleic acid sequences in test samples obtained from patients and other subjects. Such test samples include serum, urine, stool, saliva, amniotic fluid, and other bodily fluids. Accordingly, many methods for detecting and / or quantifying nucleic acid sequences are well known in the art. However, an inherent consequence of highly sensitive nucleic acid amplification systems is the generation of by-products. By-products include molecules that can interfere with the amplification reaction in some systems, thereby reducing specificity. This occurs because limited amplification resources, including primers and enzymes necessary for primer extension and transcript formation, are diverted toward by-product formation. In some cases, the appearance of by-products can complicate the analysis of amplicon production by various molecular techniques. Furthermore, in many cases of interest, specific nucleic acid sequences are present at very low concentrations in samples being tested for the desired nucleic acid sequence. In such cases, the presence of the desired molecule cannot be detected unless assay sensitivity is increased.

[0007] The present application is directed to overcoming these and other deficiencies in the art. Summary of the Invention

[0008] overview 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 providing a sample containing the target nucleic acid molecule and contacting the sample with a capture oligonucleotide molecule complementary to at least a portion of the target nucleic acid molecule, thereby hybridizing the capture oligonucleotide molecule to the complementary portion of the target nucleic acid molecule to form a double-stranded nucleic acid molecule. The capture oligonucleotide molecule (i) has a length of 30-60 base pairs, (ii) has a 4-8 base pair overhang at its 3' end, (iii) has a 5' tail, (iv) has a target-specific portion between the 3' end and the 5' tail, (v) has a deoxyadenosine diphosphate content of 40-50%, (vi) has no deoxythymidine phosphate at the 3' end or 5' tail, and (vii) has an ATP content at the 3' end and 5' tail that is 40-50% of the ATP content of the capture oligonucleotide molecule. The double-stranded nucleic acid molecule, polymerase, and dNTP mixture are contacted to form a polymerase extension mixture, and the polymerase extension mixture is subjected to conditions under which the target nucleic acid molecule is extended and releases free phosphate. The released free phosphate is then converted to adenosine triphosphate, which is then metabolized by luciferase to generate a bioluminescent readout signal indicative of 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 providing a sample containing a target nucleic acid molecule and contacting the sample with a capture oligonucleotide molecule complementary to at least a portion of the target nucleic acid molecule, thereby hybridizing the capture oligonucleotide molecule to the complementary portion of the target nucleic acid molecule and forming 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 and a free phosphate is released. Adenosine triphosphate is then enzymatically generated from the released free phosphate, and the adenosine triphosphate generated from the free phosphate is metabolized by luciferase to generate a bioluminescent readout signal indicating the presence of the target nucleic acid molecule in the sample. The DNA polymerase, the luciferase, and the adenosine triphosphate-generating enzyme 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, comprising a capture oligonucleotide molecule 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, 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 bioluminescent 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, thereby hybridizing to the complementary portion of the target nucleic acid molecule and forming a double-stranded nucleic acid molecule. The capture oligonucleotide molecule (i) has a length of 30 to 60 base pairs (bp), (ii) a 4 to 8 base pair overhang 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 5' tail, and (vii) the 3' end and 5' tail have an ATP content that is 40 to 50% of the ATP content of the capture oligonucleotide molecule. The kit also contains 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.

[0012] Another final aspect of the present application relates to 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.

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

[0014] The present application offers several advantages over other detection methods and systems, including: 1) speed—enzymatic assays can be performed quickly, providing a readout within minutes; 2) a luminescence-based readout is used, allowing for stand-alone, highly portable systems and devices without the need for large excitation components (such as those required for fluorescence); 3) sensitivity—due to the release of multiple free phosphates with each hybridization event, and the enzymatic assay reaction facilitates signal amplification in both the detection and readout steps; 4) low manufacturing cost—potential components of such systems, including nanoparticles, can be fabricated from inexpensive materials and easily mass-produced; 5) multiplexing capabilities—the combination of biochemical reactions may allow for the detection of multiple nucleic acid molecules in a single system in certain embodiments of the present application; 6) the use of tethered enzymes facilitates maximum enzyme stability and activity; and 7) the use of tethered enzymes allows for the reaction and readout to be localized to specific regions of the system (e.g., specific regions of a card). 9) the use of a tethered enzyme limits the reaction and readout, thereby reducing the size of the photodetector in the reader; 10) the immobilization of capture oligonucleotides improves the ability to detect multiple target oligonucleotides in specific regions of the system (e.g., specific regions of a card); 11) the use of isothermal amplification allows detection at ambient temperature without the need for temperature cycling; and 12) the design of the capture oligonucleotide allows for a single-step reaction without interfering / inhibiting by-products, and the ability to introduce a bioluminescent enzyme into the single-step reaction allows for an additional level of signal amplification resulting from the free phosphate released from metabolized AP molecules (which is returned to the ATP-generating reaction). [The present invention 1001] 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, thereby hybridizing the capture oligonucleotide molecule to the complementary portion of the target nucleic acid molecule and forming a double-stranded nucleic acid molecule, wherein the capture oligonucleotide molecule (i) has a length of 30-60 base pairs, (ii) has a 4-8 base pair overhang at its 3' end, (iii) has a 5' tail, (iv) has a target-specific portion between the 3' end and the 5' tail, (v) has a deoxyadenosine diphosphate content of 40-50%, (vi) has no deoxythymidine phosphate at the 3' end or the 5' tail, and (vii) has 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: [The present invention 1002] 1001. The method of claim 1001, wherein the DNA polymerase is bound to a solid support. [The present invention 1003] 1002. The method of claim 1002, 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. [The present invention 1004] 1001. The method of claim 1001, wherein said luciferase is bound to a solid support. [The present invention 1005] 1004. The method of claim 10, wherein said luciferase is attached to said 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. [The present invention 1006] The method of claim 1001, wherein the step of generating adenosine triphosphate comprises subjecting the released free phosphate to an enzymatic reaction of bound glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase to generate adenosine triphosphate. [The present invention 1007] The method of claim 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] 1007. The method of claim 1007, wherein said glyceraldehyde 3-phosphate dehydrogenase and said phosphoglycerate kinase are bound to a solid support. [The present invention 1009] The method of claim 1001, 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. [The present invention 1010] 1009. The method of claim 10, wherein said adenosine triphosphate sulfurylase is bound to a solid support. [The present invention 1011] The target nucleic acid molecule is present in the sample at a concentration of 10 per liter. -5 The method of claim 1001, wherein the compound is present in a submolar concentration. [The present invention 1012] 1001. The method of claim 1001, wherein said target nucleic acid molecule is a microRNA. [The present invention 1013] 1001. The method of claim 10, wherein the subjecting step is carried out at a temperature of 0 to 100°C. [The present invention 1014] The method of claim 1013, wherein the subjecting step is carried out at a temperature of 25 to 40°C. [The present invention 1015] 1001. The method of claim 1001, wherein said 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] 1001. The method of claim 1001, further comprising the step of quantifying said bioluminescent readout signal to determine the presence or concentration of said target nucleic acid molecule in said sample. [The present invention 1017] The method of claim 1016, wherein the presence of said target nucleic acid molecule in said sample is determined. [The present invention 1018] The presence of the target nucleic acid molecule in the sample is determined by: 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 the present invention 1017, wherein the method is determined by a procedure including: [The present invention 1019] The method of claim 1016, wherein the concentration of said target nucleic acid molecule in said sample is determined. [The present invention 1020] 1001. The method of claim 1001, wherein deoxyadenosine triphosphate is omitted from said polymerase extension mixture. [The present invention 1021] 1001. The method of claim 1001, wherein said sample is selected from the group consisting of blood, urine, cerebrospinal fluid, saliva, tissue, and synthetic material. [The present invention 1022] 1001. The method of claim 1001, which is carried out in solution. [The present invention 1023] 1001. The method of claim 1001, wherein a plurality of capture oligonucleotide molecules are provided for detecting a plurality of target nucleic acid molecules. [The present invention 1024] 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, luciferase, and adenosine triphosphate-generating enzyme are each bound to a solid support; The method comprising: [The present invention 1025] 1024. The method of claim 1024, wherein the capture oligonucleotide molecule has a length of 30 to 60 base pairs. [The present invention 1026] 1024. The method of claim 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 claim 1026, wherein said capture oligonucleotide molecule has a 5' tail. [The present invention 1028] 1028. The method of claim 1027, wherein said capture oligonucleotide molecule has a target-specific portion between said 3' end and said 5' tail. [The present invention 1029] 1024. The method of claim 1024, wherein said capture oligonucleotide molecule has a deoxyadenosine diphosphate content of 40-50%. [The present invention 1030] 1028. The method of claim 1027, wherein said capture oligonucleotide molecule does not have a deoxythymidine phosphate at said 3' end or said 5' tail. [The present invention 1031] 1024. The method of claim 1024, wherein said 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 said capture oligonucleotide molecule. [The present invention 1032] The method of claim 1024, wherein a plurality of capture oligonucleotide molecules are provided for detecting a plurality of target nucleic acid molecules. [The present invention 1033] 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 mixture, 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: [The present invention 1034] A kit according to the present invention 1033, comprising a plurality of capture oligonucleotide molecules for detecting a plurality of target nucleic acid molecules. [This invention 1035] 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, 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 mixture, an enzyme for generating adenosine triphosphate from the released free phosphate; Luciferase to generate a bioluminescent readout signal The kit comprising: [The present invention 1036] A kit according to the present invention 1035, comprising a plurality of capture oligonucleotide molecules for detecting a plurality of target nucleic acid molecules. [This invention 1037] 1. A composition comprising a capture oligonucleotide molecule, wherein the capture oligonucleotide molecule (i) has a length of 30-60 base pairs, (ii) has a 4-8 base pair overhang at its 3' end, (iii) has a 5' tail, (iv) has a target-specific portion between the 3' end and the 5' tail, (v) has a deoxyadenosine diphosphate content of 40-50%, (vi) has no deoxythymidine phosphate at the 3' end or the 5' tail, and (vii) has an ATP content at the 3' end and the 5' tail that is 40-50% of the ATP content of the capture oligonucleotide molecule. [Brief explanation of the drawings]

[0015] [Figure 1]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 insert sequence within the capture oligonucleotide, generating a double strand. This allows DNA polymerase to bind and initiate a polymerization reaction, which releases free phosphate (PPi) through nucleotide incorporation (isothermal replication). In the presence of ADP, NAD+, and GAP, these PPi groups are then used by the enzymatic reaction of the bound GAPDH-PGK to generate ATP. Finally, luciferase hydrolyzes the ATP, generating a bioluminescent signal. [Figure 2] Figure 1 shows another embodiment of the TET-miRNA reaction. The miRNA (or other single-stranded nucleic acid polymer) anneals to a complementary insert sequence within the capture oligonucleotide, generating a double strand. This allows DNA polymerase to bind and initiate a polymerization reaction that releases free phosphate (PPi) through nucleotide incorporation (isothermal replication). In the presence of APS, these PPi groups are then used by the enzyme ATP-sulfurylase to generate ATP. Finally, luciferase hydrolyzes the ATP, generating a bioluminescent signal. [Figure 3] FIG. 1 shows the design and characteristics of capture oligonucleotides (Cap oligos). [Figure 4] FIG. 1 shows that omission of dATP from the reaction mixture improves the kinetics and sensitivity of the assay. [Figure 5A] FIG. 1 shows that the TET-miRNA assay can detect mismatched (mm) nucleotides at the 3′ end of target oligonucleotides, as well as various analyses or quantifications of the data. [Figure 5B] FIG. 1 shows that the TET-miRNA assay can detect mismatched nucleotides at the 3′ end of target oligonucleotides, as well as various analyses or quantifications of the data. [Figure 5C]FIG. 1 shows that the TET-miRNA assay can detect mismatched nucleotides at the 3′ end of target oligonucleotides, as well as various analyses or quantifications of the data. [Figure 5D] FIG. 1 shows that the TET-miRNA assay can detect mismatched nucleotides at the 3′ end of target oligonucleotides, as well as various analyses or quantifications of the data. [Figure 6] A and B show an examination of the range of sensitivity of the TET-miRNA assay for the detection of target oligonucleotides. [Figure 7] 1A-C show that the design of the 5' and 3' tails of the capture oligonucleotide affects TET-miRNA kinetics. [Figure 8] A and B show that the dATP content of the capture oligonucleotide affects TET-miRNA kinetics. [Figure 9] A and B show that the 3' tail length of the capture oligonucleotide affects the activity and kinetics of the TET-miRNA reaction. [Figure 10] 1A to 1C show that the TET-miRNA assay is sensitive to variations in the sequence of the MIR-340 target oligonucleotide (lung cancer-associated microRNA). [Figure 11] A and B show that the TET-miRNA assay can detect naturally occurring miRNAs in human serum. [Figure 12] FIG. 1 shows that the TET-miRNA assay can detect naturally occurring miRNAs in human plasma. [Figure 13] A and B show that TET-miRNA activity is abolished by ribonuclease (RNase) treatment. [Figure 14] A and B show a comparison of target oligonucleotide detection using reactions with (unoriented) NP immobilization and reactions in solution. [Figure 15]Figures A and B show that immobilization of commercially available BST2.0 onto NPs via biotin-streptavidin binding inhibits the TET-miRNA reaction assay. [Figure 16] Figures A-C show that the TET-miRNA assay is only slightly affected by temperature. [Figure 17-1] AC show testing (untethered) of two different capture oligonucleotide designs for detecting miRNA in human serum. [Figure 17-2] D and E show testing (untethered) of two different capture oligonucleotide designs for detecting miRNA in human serum. [Figure 18A-1] FIG. 1 shows a comparison of the activity of different DNA polymerases in a TET-miRNA assay. [Figure 18A-2] This is a continuation of Figure 18A-1. [Figure 18B] FIG. 1 shows a comparison of the activity of different DNA polymerases in a TET-miRNA assay. [Figure 19-1] A and B show a comparison of target oligonucleotide detection using TET-miRNAs when tethered and in solution. [Figure 19-2] C and D show a comparison of target oligonucleotide detection using TET-miRNAs when tethered and in solution. [Figure 20] Figures A-C show a comparison of the activity of His-Si-Klenow and His-Si-BST in the TET-miRNA assay when tethered and in solution. [Figure 21] A and B show a comparison of target oligonucleotide detection using various ratios of His-Si-ATPS / His-Si-BST / NP. [Figure 22] 1A to 1C show the detection of miRNA (RNA oligonucleotide) using TET-miRNA (His-Si-enzyme) and DNA capture oligonucleotide. [Figure 23]1A-C show a comparison of the sensitivity of different untethered DNA polymerases (His-Si-Klenow and His-Si-BST) to target oligonucleotide mismatches. [Figure 24] FIG. 1 shows a comparison of testing TET-miRNA reactions when the capture oligonucleotides are in solution and when they are immobilized (non-oriented). [Figure 25A] FIG. 10 shows a comparison of testing TET-miRNA responses when capture oligonucleotides are in solution and when immobilized on SiO 2 NPs via biotin-streptavidin. [Figure 25B] FIG. 10 shows a comparison of testing TET-miRNA responses when capture oligonucleotides are in solution and when immobilized on SiO 2 NPs via biotin-streptavidin. DETAILED DESCRIPTION OF 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 providing a sample containing the target nucleic acid molecule and contacting the sample with a capture oligonucleotide molecule complementary to at least a portion of the target nucleic acid molecule, thereby hybridizing the capture oligonucleotide molecule to the complementary portion of the target nucleic acid molecule to form a double-stranded nucleic acid molecule. The capture oligonucleotide molecule (i) has a length of 30-60 base pairs, (ii) has a 4-8 base pair overhang at its 3' end, (iii) has a 5' tail, (iv) has a target-specific portion between the 3' end and the 5' tail, (v) has a deoxyadenosine diphosphate content of 40-50%, (vi) has no deoxythymidine phosphate at the 3' end or 5' tail, and (vii) has an ATP content at the 3' end and 5' tail that is 40-50% of the ATP content of the capture oligonucleotide molecule. The double-stranded nucleic acid molecule, polymerase, and dNTP mixture are contacted to form a polymerase extension mixture, and the polymerase extension mixture is subjected to conditions under which the target nucleic acid molecule is extended and releases free phosphate. The released free phosphate is then converted to adenosine triphosphate, which is then metabolized by luciferase to generate a bioluminescent readout signal indicative of 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 providing a sample containing a target nucleic acid molecule and contacting the sample with a capture oligonucleotide molecule complementary to at least a portion of the target nucleic acid molecule, thereby hybridizing the capture oligonucleotide molecule to the complementary portion of the target nucleic acid molecule and forming 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 and a free phosphate is released. Adenosine triphosphate is then enzymatically generated from the released free phosphate, and the adenosine triphosphate generated from the free phosphate is metabolized by luciferase to generate a bioluminescent readout signal indicating the presence of the target nucleic acid molecule in the sample. The DNA polymerase, the luciferase, and the adenosine triphosphate-generating enzyme are each bound to a solid support.

[0018] As shown in Figure 1, DNA polymerase is tethered to one nanoparticle, and GAPDH, PGK, and luciferase ("Luc") are tethered to another. The miRNA target anneals to a complementary insert sequence within either the provided, tethered, or solution-based capture oligonucleotide, generating a duplex. This allows DNA polymerase to bind and initiate a polymerization reaction, which 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 the tethered GAPDH and PGK to generate ATP. This ATP, along with luciferin, is then used by the tethered Luc to generate a bioluminescent signal. The amount of light emitted is directly proportional to the amount of ATP in the system, which in turn corresponds to the amount of target miRNA in the system. In one embodiment, the emitted light can be read quantitatively and / or qualitatively by a photodetector positioned to capture the emitted signal.

[0019] Alternatively, as shown in Figure 2, DNA polymerase is tethered to one nanoparticle, and ATP sulfurylase (ATP-sul) and luciferase ("Luc") are tethered to another. The miRNA target anneals to a complementary insert sequence within either a prepared, tethered, or solution-based capture oligonucleotide, generating a duplex. This allows DNA polymerase to bind and initiate a polymerization reaction, which releases a 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, along with luciferin, is then used by the tethered Luc to generate a bioluminescent signal. The amount of light emitted is directly proportional to the amount of ATP in the system, which in turn corresponds to the amount of target miRNA in the system. In one embodiment, the emitted light can be read quantitatively and / or qualitatively by a photodetector positioned to capture the emitted signal.

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

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

[0022] As used herein, "target nucleic acid" or "target" refers to a portion of a nucleic acid sequence in a sample that is the control for detection or analysis. The term "target" includes all variants of the target sequence, for example, variants with 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 its target sequence, or a copy thereof, to form a stable capture oligonucleotide:target hybrid, while minimizing the formation of stable capture oligonucleotide:non-target hybrids. Thus, the capture oligonucleotide hybridizes to the target sequence, or a copy thereof, to a significantly 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 can be determined by routine testing methods (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2002). nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, which is hereby incorporated by reference in its entirety.

[0025] Figure 3 shows one embodiment of the design of a capture oligonucleotide. As shown in Figure 3, the capture oligonucleotide can be 30 to 60 nucleotides in length, including the 5' extension tail, the internal complementary insertion sequence, and the 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 total oligonucleotide. Optimal capture oligonucleotide sequences include 4 to 8 additional nucleotides in the 3' tail. The capture oligonucleotide is preferably designed so that it does not hybridize to itself to form a hairpin structure that would interfere with hybridization to the target nucleic acid.

[0026] As used herein, the term "luciferase" refers to an oxygenase that catalyzes the light-emitting reaction as follows: TIFF0007719721000001.tif12154

[0027] Thus, luciferase refers to an enzyme or photoprotein that catalyzes a bioluminescent reaction (a reaction that produces bioluminescence), and unless otherwise specified, it can be a naturally occurring luciferase, a recombinant luciferase, or a mutant luciferase. Naturally occurring luciferases can be easily obtained from organisms by those skilled in the art. When the 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. Luciferases can be easily obtained from cultures of bacteria, yeast, mammalian cells, insect cells, plant cells, and the like. Furthermore, recombinant or mutant luciferases can be easily obtained from in vitro cell-free systems using nucleic acids encoding luciferases. Luciferases are available from Promega Corporation, Madison, WI. Several luciferases that are modified mutants or variants of luciferase are also known in the art, and 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 incorporated herein by reference in its entirety.

[0028] The "polymerase extension" reaction of the present application includes all forms of template-directed polymerase-catalyzed nucleic acid synthesis reactions. Primer extension reaction conditions and reagents are known in the art, and any standard method, reagent, enzyme, etc. can be used at this stage (see, e.g., Sambrook et al., (editors), 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 a primer, deoxynucleotides (dNTPs), and a suitable polymerase enzyme, such as Klenow, or indeed any available and suitable 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' to 5' exo), and DNA polymerase I (large Klenow fragment). The above conditions can be selected by tradeoffs according to procedures known in the art.

[0029] Polymerase extension techniques for use in the methods of the present application are isothermal techniques (i.e., techniques performed at a single temperature, or in which the major aspects of the amplification process are carried out at a single temperature). Such techniques rely on the ability of a polymerase to copy the template strand to be amplified and form a linked duplex. The isothermal techniques rely on a strand-displacing polymerase to separate / displace the two strands of the duplex and recopy the template. This well-known property has been the subject of numerous 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 entireties).

[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 the complementary DNA strand based on the capture oligonucleotide sequence. The extension reaction occurs using 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 omitted from the polymerase extension mixture.

[0031] As noted above, polymerase extension techniques for use in the methods of the present application are isothermal techniques (i.e., techniques performed at a single temperature, or in which major aspects of the amplification process are carried out at a single temperature). Thus, in certain embodiments, the polymerase extension reaction is performed at a temperature of 0-100°C, 5-100°C, 10-100°C, 15-100°C, 20-100°C, 25-100°C, 30-100°C, 35-100°C, 40-100°C, 45-100°C, 50-100°C, 55-100°C, 60-100°C, 65-100°C, 70-100°C, 75-100°C, 80-100°C, 85-100°C, 90-100°C, or 95-100°C. In one embodiment, the polymerase extension reaction is performed at a temperature of 25-40°C.

[0032] The polymerase extension reaction releases two phosphate groups (PPi) for each 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 target nucleic acid molecules in a sample. Specifically, the presence or absence of target nucleic acid molecules can be detected by the enzymatic conversion of PPi to ATP, followed by bioluminescent detection of ATP using the signaling molecule luciferase.

[0033] Luciferase and luciferin are used in combination to identify target nucleic acids, since the amount of light produced is substantially proportional to the amount of ATP produced, which in turn is directly proportional to the amount of nucleotide incorporated and target nucleic acid present. Thus, the method includes providing luciferin and O2, which are added to a reaction mixture.

[0034] As described above, the method described herein includes subjecting a polymerase extension mixture to conditions under which a target nucleic acid molecule is extended and releases free phosphate. The released free phosphate is then generated into adenosine triphosphate (ATP) through an enzymatic reaction, and the ATP is then metabolized by luciferase to generate a bioluminescent readout signal. According to this aspect, in one embodiment, generating adenosine triphosphate includes subjecting the released free phosphate to an enzymatic reaction of a 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 produce ATP. The ATP then reacts with luciferase to produce a measurable signal. The reaction scheme is shown below. TIFF0007719721000002.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. TIFF0007719721000003.tif26128

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

[0038] The amount of light produced can be easily determined using a device that is sensitive in the appropriate light, such as a luminometer, and thus luminometric methods have the advantage of being quantifiable.

[0039] In one embodiment, the bioluminescent readout signal is quantified to determine the presence or concentration of target nucleic acid molecules in the sample. The amount of target nucleic acid can be determined from the peak magnitude of the luminescent signal, and / or the time it takes for the signal to reach its peak magnitude, and / or the integrated amount of signal emitted over a period of time, and / or the rate at which luminescence is produced.

[0040] In one embodiment, the target nucleic acid molecules are present at 10 per liter -5 Present in the sample in submolar concentrations.

[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 bioluminescent signal generation, calculating the time required to reach peak bioluminescence, and calculating the magnitude of the peak bioluminescent signal or the integrated bioluminescent signal from time zero to the peak bioluminescence.

[0042] These procedures can be used individually, or can be used as part of an analytical method that incorporates two or more procedures to achieve better quantification.For each procedure or combination of procedures, a threshold value can be determined in advance and calibrated to a known amount of target nucleic acid target.Predetermined values can also be useful for identifying sequences that are similar but not identical to the desired target oligonucleotide (i.e., mutations).Furthermore, the values obtained by these analytical procedures can be evaluated against a cutoff value to determine presence / absence, or can be evaluated as a scale to enable quantitative reading.

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

[0044] The method contemplated according to this embodiment is a multiplex assay that utilizes multiple capture oligonucleotides to determine the presence or absence of one or more of multiple predetermined nucleic acid target sequences in a sample. One area where such a multiplex assay is particularly useful is in screening assays where the normal analytical output indicates the absence of the nucleic acid of interest.

[0045] In multiplexed embodiments of the above methods, the sample is mixed with multiple different capture oligonucleotides, in which the analytical output for a particular result with one capture oligonucleotide is distinguishable from the analytical output for the opposite result with all capture oligonucleotides.

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

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

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

[0049] Suitable supports include organic or inorganic materials and can be of any suitable size or shape (e.g., scaffold sheets, platforms, and / or nanoparticles). Anchoring or immobilizing components of the assays of the present application can serve, for example, to spatially confine the components, as well as to improve the stability and / or function of the components, for example, when performing stepwise or sequential reactions as part of a particular assay. In certain embodiments, the support material can include, for example, a nucleotide sequence or a gel. In certain embodiments, enzymes or components of the assays of the present application can be immobilized on or tethered to the luminal surface of a channel (e.g., a microfluidic channel) of a support material, such as, for example, a nanoparticle or platform.

[0050] Several techniques can be used to immobilize components (e.g., enzymes) of the assays of the present application onto a surface. For example, components can be nonspecifically attached or attached via specific but non-directed chemical reactions (e.g., carboxy-amide bonds). Oriented enzyme immobilization can also be used in accordance with the methods of the present application. Oriented enzyme immobilization offers several advantages, including, for example, positioning the binding tag (e.g., affinity tag) to optimize the activity and stability of the tethered enzyme (see Mukai et al., "Sequential Reactions of Surface-Tethered Glycolytic Enzymes," Chem. Biol. 16(9):1013-20 (2009), which is incorporated herein by reference in its entirety).

[0051] One method for anchoring enzymes involved in nucleic acid detection to surfaces is the use of oriented immobilization. In certain embodiments of the assays described herein, recombinant enzymes or assay components involved in the assay reactions are engineered with affinity tags, allowing the enzymes to bind to surfaces such as silica or nickel, or to surface components such as nickel-nitrilotriacetic acid. For example, affinity tags can 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 anchoring domain will depend on the nature and location of the enzyme's catalytic domain(s), substrate-binding domain(s), and any conformational changes that need to occur in the enzyme.

[0052] The use of affinity-tagged proteins is particularly convenient because 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 solid supports containing the corresponding capture binding moieties. Suitable capture moiety 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 binding oligonucleotides to solid supports are well known in the art, see, e.g., 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), which are incorporated herein by reference in their entireties.

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

[0054] According to this aspect of the application, the DNA polymerase and / or the luciferase can be attached 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, an antibody, and an epitope tag.

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

[0056] In certain embodiments of the present application, the support is a nanoparticle. As used herein, the term "nanoparticle" refers to any particle whose average diameter is in the nanometer range, i.e., whose average diameter is at most 1 μm. The nanoparticles used can be made of any suitable organic or inorganic material 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 nanoparticles are magnetic nanoparticles. In another embodiment, the nanoparticles are magnetic silica-coated nanoparticles ("MSPs").

[0057] In addition to nanoparticles (NPs), supports or scaffolds of various materials can take the form of rods, flat surfaces, graphene sheets, nanotubes, DNA scaffolds, gels, microspheres, or the inner channel walls of microchannels in larger supports. Quantum dots are also contemplated for use as supports in this application. Enzyme immobilization can be achieved by nonspecific binding, chemical modification, affinity tags, or other conjugation techniques.

[0058] In one embodiment of the present application, the method further comprises 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 to the control amount. The control amount of the target nucleic acid can be any amount or range of amounts to be compared with the test amount of the target nucleic acid. The control amount can be the amount of the target nucleic acid in a positive control or negative control sample performed as part of the assay of the present application. The control amount can be either an absolute amount (e.g., μg / ml) or a relative amount (e.g., relative intensity of signal).

[0059] Exemplary negative controls for use in the methods of the present application include a blocking oligonucleotide (with a sequence fully or partially complementary to the target oligonucleotide) targeting the target oligonucleotide, a blocking oligonucleotide (modified at the 3' / 5' ends to inhibit extension) targeting the capture oligonucleotide, 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 methods of the present application include various concentrations (including saturating amounts) of target oligonucleotide mimics (DNA oligonucleotides with sequences identical to those of the target oligonucleotides from the sample), and pre-annealed double-stranded DNA with overhanging single-stranded sequences that can be extended by the polymerase.

[0060] In various related aspects, the present application also relates to devices and kits for carrying out the methods described herein. Such kits include monitors, reagents, and procedures that can be utilized in a clinical or research setting, or that can be adapted for either clinical point-of-care testing or field use. In particular, kits containing the disclosed reagents for use in carrying out the methods described herein will include any of several means for detecting captured target nucleic acid molecules and measuring the bioluminescent signal generated after target capture, along with appropriate instructions. Suitable kits will include sufficient reagents to carry out an assay for detecting target nucleic acid molecules.

[0061] It is understood that such kits are useful for any of the methods of the present application. The selection of specific components will depend on the particular method that the kit is designed to perform. Additional components may be added to detect the analytical output, as measured by the release of ATP and the detection of a bioluminescent signal.

[0062] As noted above, the kits optionally further include instructions for detecting target nucleic acids by the methods described herein. The instructions present in such kits instruct the user on how to use the components of the kit to practice 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.

[0063] Therefore, another aspect of the present application relates to a kit for detecting a target nucleic acid molecule in a sample, said kit 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 mixture, 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; Includes:

[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, thereby hybridizing to the complementary portion of the target nucleic acid molecule and forming a double-stranded nucleic acid molecule. The capture oligonucleotide molecule (i) has a length of 30 to 60 base pairs, (ii) has a 4 to 8 base pair overhang at its 3' end, (iii) has a 5' tail, (iv) has a target-specific portion 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 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. The kit also contains 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.

[0065] The kit may also contain multiple capture oligonucleotide molecules for detecting multiple target nucleic acid molecules. In kits intended for multiplexed capture oligonucleotide-mediated specific nucleic acid detection, the kit contains multiple capture oligonucleotides for multiple nucleic acid targets of interest. When the kit contains multiple capture oligonucleotides, each of the capture oligonucleotides is preferably designed to interrogate a different target nucleic acid sequence.

[0066] A final aspect of the present application relates to 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. [Example]

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

[0068] Example 1: Omitting dATP from the reaction mixture improves assay kinetics and sensitivity A reaction mixture (total volume 100 μl) was prepared by mixing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl Klenow (NEB, Lg fragment, M0210S, 5000 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 1 μl capture oligonucleotide (T2 (SEQ ID NO:7), 1 μM), 1.8 μl dNTP mix (33 mM each), and + / - dATP. This reaction mixture was added to each well of a white 96-well plate containing 1 μl of target oligonucleotide (R6 (SEQ ID NO:6), 1 μM). The reaction mixture was then 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 Figure 4, two identical reaction mixtures were tested using the above conditions, except for the nucleotide mixture dATP (deoxyadenosine triphosphate) added to reaction mixture A. Binding of dATP to luciferase and its hydrolysis by luciferase result in a weakened initial luminescent signal (1), a delayed peak response phase (2), and a reduction in overall signal magnitude (3). This demonstrates the relationship between pure sequencing and the tethered detection method used here, where bioluminescence is generated as the readout. As a result of these data, the supplemental oligonucleotides were designed to be completely free of dTPS in their sequences, eliminating the need for dATP in the reaction mixture.

[0070] Example 2 TET-miRNA assay allows detection of mismatched nucleotides at the 3' end of target oligonucleotides A reaction mixture was prepared in a total volume of 100 μl by mixing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl Klenow (NEB, Lg fragment, M0210S, 5000 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 1 μl 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 to R6 (SEQ ID NOs: 1 to 6), 100 μM), and the plate was immediately placed in a TECAN plate reader and the luminescence signal was read for 3500 seconds at room temperature with a 400 ms integration time.

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

[0072] Example 3 Determining the Sensitivity Range for Target Oligonucleotide Detection Using the TET-miRNA Assay A reaction mixture was prepared in a total volume of 100 μl by mixing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl Klenow (NEB, Lg fragment, M0210S, 5000 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 0.5 μl capture oligonucleotide (T2 (SEQ ID NO: 7) (see Figure 7A), 1 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (33 mM each). This reaction mixture was added to individual wells of a white 96-well plate containing decreasing concentrations of target oligonucleotide (R6 (SEQ ID NO: 6), 0 mM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 1 μM), immediately placed in a TECAN plate reader, and the luminescence signal was read for 2000 seconds at room temperature with a 400 ms integration time.

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

[0074] Example 4: The design of the 5' and 3' tails of the capture oligonucleotide affects the TET-miRNA reaction kinetics A reaction mixture was prepared in a total volume of 100 μl by mixing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl Klenow (NEB, Lg fragment, M0210S, 5000 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl 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). 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 NOs: 7-11), 100 μM), and immediately placed in a TECAN plate reader, and the luminescence signal was read for 1000 seconds at room temperature with a 400 ms integration time.

[0075] As shown in Figure 7A, several designs of capture oligonucleotides were generated (T2-T6 (SEQ ID NOS: 7-11)). All designs contain a similar complementary insert sequence (complementary to the R6 target oligonucleotide, indicated by underlined text). Figure 7B shows the luminescence signal measured in the presence of various capture oligonucleotides (shown in Figure 7A) in response to the addition of the target oligonucleotide. Figure 7C summarizes Figure 7B and shows the differences in annealing kinetics, as indicated by the calculated reaction slopes for the various capture oligonucleotides.

[0076] Example 5. Capture oligonucleotide dATP content affects TET-miRNA kinetics A reaction mixture was prepared in a total volume of 100 μl by mixing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl Klenow (NEB, Lg fragment, M0210S, 5000 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl 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). 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), and immediately placed in a TECAN plate reader, and the luminescence signal was read for 1000 seconds at room temperature with a 400 ms integration time.

[0077] As shown in Figure 8A, six capture oligonucleotides containing increasing percentages of dATP (27% to 63%) were designed for detection of the target oligonucleotide, hsa-let-7a-5p (all with similar complementary insert sequences indicated by underlined letters). Figure 8B summarizes the data showing optimal activity with 40% to 50% dATP content in the capture oligonucleotide sequence. This finding was surprising and represents a significant improvement.

[0078] Example 6. The 3' tail length of the capture oligonucleotide affects the activity and kinetics of the TET-miRNA reaction. A reaction mixture was prepared in a total volume of 100 μl by mixing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl Klenow (NEB, Lg fragment, M0210S, 5000 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl 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). 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), and immediately placed in a TECAN plate reader, and the luminescence signal was read for 1000 seconds at room temperature with a 400 ms integration time.

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

[0080] Example 7 The TET-miRNA assay is sensitive to sequence variations in miR-340 target oligonucleotides A reaction mixture was prepared in a total volume of 100 μl by mixing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl Klenow (NEB, Lg fragment, M0210S, 5000 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 1 μl capture oligonucleotide (T2, 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 (MIR-340#1 to MIR-340#7 (SEQ ID NOs: 27 to 33), 100 μM), and the plate was immediately placed in a TECAN plate reader and the luminescence signal was read for 1200 seconds at room temperature with a 400 ms integration time.

[0081] As shown in Figure 10A, the MIR340 target oligonucleotide (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, as well as two controls (one for unmutated MIR-340#2 and one that was a reaction mixture that omitted any of the target oligonucleotides) (mutated nucleotides are underlined). Figure 10B and C show the luminescence signal and kinetics measured for the various mutations shown in A. The data in Figure 10A show that all mutations tested induced detectable differences in the measured signal.

[0082] Example 8 The TET-miRNA assay can detect naturally occurring miRNAs in human serum and plasma A reaction mixture in a total volume of 40 μl was prepared by mixing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl Klenow (NEB, Lg fragment, M0210S, 5000 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 1 μl of capture oligonucleotides (100 μM) targeting naturally occurring miRNAs listed in Figure 10A, 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 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 a 400 millisecond integration time.

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

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

[0085] Example 9: TET-miRNA activity is abolished by ribonuclease (RNase) treatment A reaction mixture was prepared in a total volume of 40 μl by mixing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl Klenow (NEB, Lg fragment, M0210S, 5000 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 1 μl of capture oligonucleotides (100 μM) targeting naturally occurring miRNAs as shown in FIG. 12, 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 60 μl of human plasma and immediately placed in a TECAN plate reader and the luminescence signal was read for 500 seconds at room temperature with a 400 msec integration time.

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

[0087] Example 10 Analysis of target oligonucleotide detection by immobilization on NPs and by reaction in solution Biotinylated enzymes were immobilized on streptavidin-coated microspheres (500 nm SiO2, Bangs Laboratory, IN, USA) according to the manufacturer's instructions, and unbound proteins were then washed three times by centrifugation. Equal amounts of NP-tethered or untethered enzymes were added to a reaction mixture containing 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl2), 2 μl APS (30 mM), 1 μl capture oligonucleotide (Cap-HAS-MIR-451a, 1 μM), and 1.8 μl dCTP / dTTP / dGTP mixture (33 mM each) in 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] Commercially available human serum samples were spiked with an equal amount of target oligonucleotide, HSA-MIR-451a, and then added to a TET-miRNA reaction mixture containing the soluble enzymes luciferase / ATP-sulfurylase / Klenow (Figure 14A, solution) or the enzymes tethered to NPs (Figure 14B, NPs). Note that DNA polymerase and ATP-sulfurylase were tethered to NPs using non-oriented immobilization via biotinylation. ATP sulfurylase (NEB, M0394S, 300 U / ml) and Klenow (NEB, Lg 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 Immobilization of commercial BST2.0 onto NPs via biotin-streptavidin binding inhibits TET-miRNA response assays 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 on streptavidin-coated microspheres (500 nm SiO2, Bangs Laboratory, IN, USA) according to the manufacturer's instructions, and unbound proteins were washed three times by centrifugation. Equal amounts of NP-tethered or untethered BST2.0 were added to a reaction mixture containing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 1 μl capture oligonucleotide (Cap-HSA-MIR-451a, 1 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (33 mM each) in 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] Commercially available human serum samples were spiked with an equal amount of HSA-mIR-451a and added to a TET-miRNA reaction mixture containing either the soluble enzyme luciferase / ATP-sulfurylase / Bst2.0 (Figure 15A, solution) or Bst2.0 immobilized on NPs via biotinylation (Figure 15B, NPs). These data indicate that biotinylation adversely affected activity, even more so when Bst2.0 was tethered to NPs via non-oriented immobilization.

[0091] Example 12 TET-miRNA assay is only slightly affected by temperature A reaction mixture was prepared in a total volume of 100 μl by mixing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl Klenow (NEB, Lg fragment, M0210S, 5000 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 1 μl of a capture oligonucleotide targeting Has-let-7a-5p miRNA (100 μM), 1.8 μl of dNTP Mix (33 mM each), and the Has-let-7a-5p oligonucleotide. 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 various temperatures indicated. Luminescence was measured for 1000 seconds with an integration time of 400 milliseconds.

[0092] As shown in Figure 16A, commercial human serum samples were spiked with equal amounts of target oligonucleotides and added to TET-miRNA reaction mixtures at various temperatures (25°C to 40°C). Only minor differences were observed in the initial kinetic parameters (measured from slopes, Figure 16B) and efficiency (measured from integrated signals, Figure 16C).

[0093] Example 13 Analysis of two different capture oligonucleotide designs for detecting miRNAs in human serum A reaction mixture was prepared in a total volume of 100 μl by mixing 0.05 μl ATP sulfurylase (NEB, M0394S, 300 U / ml), 0.25 μl Klenow (NEB, Lg fragment, M0210S, 5000 U / ml), 5 μl His-Si-luciferase (prepared in-house), 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 1 μl of CAP1 (not column #34) or CAP2 (SEQ ID NO:35) capture oligonucleotide (100 μM), 1.8 μl of dNTP Mix (33 mM each), and the test oligonucleotide. The 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 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 to detect six different naturally occurring miRNAs in human plasma. CAP1 has a random nucleotide sequence, while CAP2 contains only adenosines in the 5' and 3' tails. Figure 17B shows the real-time kinetics of the TET-miRNA reaction detecting both naturally occurring miRNAs and DNA-based target oligonucleotides (test oligonucleotides) of similar sequence. Figure 17C is an expanded portion of the data shown in Figure 17B, showing the luminescence signals of various miRNA molecules. Figure 17D is a summary of Figure 17A, where the luminescence signals were integrated for 500 seconds and show the relative abundance of various miRNAs. Figure 17E is a summary of measurements of only naturally occurring miRNAs (shown in Figure 17C).

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

[0096] Figure 18A lists the DNA polymerases used in this experiment and some of their key characteristics. As shown in Figure 18B, a commercially available human serum sample was spiked with an equal amount of miRNA oligonucleotide and added to the TET-miRNA reaction mixture (at room temperature) in the presence of various DNA polymerases. The reaction kinetics bar graph (calculated from the initial slope of the reaction) shows that Bst, Klenow, and Terminator DNA-Poly gave the fastest reaction kinetics under these experimental conditions. Control 1 (CTRL1) reaction mixture contained no capture oligonucleotide, while Control 2 (CTRL2) contained no DNA polymerase. The bars for the Bst and Klenow mutants used to generate the data in the previous figure are highlighted.

[0097] Example 15 Comparison of detection of target oligonucleotides using tethered and solution TET-miRNAs A reaction mixture consisting of equal amounts of NP-tethered 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 MgCl), 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 (33 mM each) 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). The plate was immediately placed in a TECAN plate reader and the luminescence signal was read for 1400 seconds at room temperature with a 400 ms integration time.

[0098] Figure 19A shows a schematic diagram 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 and inserted into the pET17b vector for bacterial protein expression. The His-Si-proteins were purified using Ni-NTA beads and stored in native protein buffer (NPB) containing sorbitol until use. Figure 19B shows tethered and solution-based enzyme activity measurements performed using the above reaction mixtures. These data show the kinetics of individual reaction mixtures where the TET-miRNA enzyme was immobilized on 500 nm SiO NPs (NP, left panel) or in solution (Sol, right panel). Figure 19C shows a bar graph of the reaction kinetics (calculated from the initial slope of the reaction mixture) showing that the coupling reaction occurred with faster kinetics when the TET-miRNA enzyme was tethered to the NP. Figure 19D summarizes the NP TET-miRNA reaction in detecting increasing concentrations of the R6 target oligonucleotide.

[0099] Example 16 Comparison of the activity of His-Si-Klenow and His-Si-BST in the TET-miRNA assay (tethered vs. untethered) Enzyme activity measurements, both tethered and in solution, were performed using a reaction buffer consisting of equal amounts of NP-tethered 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 MgCl), 2 μl APS (30 mM), 1 μl capture oligonucleotide (T2 (SEQ ID NO: 7), 100 μM), and 1.8 μl of a dCTP / dTTP / dGTP mixture (33 mM each). 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). The plate was immediately placed in a TECAN plate reader, and the luminescence signal was read for 1200 seconds at room temperature with a 400-millisecond integration time.

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

[0101] Example 17 Comparison of Target Oligonucleotide Detection Using Various Ratios of His-Si-ATPS / His-Si-BST / NP Enzyme activity measurements were performed using a reaction mixture consisting of 20 μl NP-His-Si-BST (at various enzyme / NP ratios, as shown in Figure 20B), 20 μl NP-His-Si-ATPS, 5 μl His-Si-luciferase, 5 μl luciferin (200 mM), 5 μl of 20x luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 1 μl capture oligonucleotide (T2 (SEQ ID NO: 7), 100 μM), and 1.8 μl of a dCTP / dTTP / dGTP mixture (33 mM each). 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 a 400 ms integration time.

[0102] Figure 21A summarizes experiments in which the ratio of His-Si-BST2(DNA-Pol):ATPS was gradually increased from 0.1:1 to 2.5:1, and Figure 21B summarizes experiments in which the ratio of His-Si-BST2(DNA-Pol):NP was gradually increased from 0.1:1 to 10:1.

[0103] Example 18: miRNA (RNA oligonucleotide) detection using TET-miRNA (His-Si-enzyme) and DNA capture oligonucleotides Enzyme activity measurements were performed using a reaction mixture consisting of 20 μl NP-His-Si-BST, 20 μl NP-His-Si-ATPS, 5 μl NP-His-Si-luciferase, 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 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 (33 mM each). 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 a 400 ms integration time.

[0104] Figure 22A 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. Figure 22B shows the TET-miRNA reaction using a tethered enzyme to detect RNA and the corresponding DNA target oligonucleotide. Figure 22C summarizes the data shown in Figure 22A and shows the calculated initial velocity (slope) 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 Enzyme activity measurements were performed using a reaction mixture consisting of 20 μl NP-His-Si-BST or NP-His-Si-Klenow, 20 μl NP-His-Si-ATPS, 5 μl NP-His-Si-Luciferase, 5 μl luciferin (200 mM), 5 μl of 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 1 μl of capture oligonucleotide (T2 (SEQ ID NO:7), 100 μM), and 1.8 μl of dCTP / dTTP / dGTP mixture (33 mM each). This reaction mixture was added to individual wells of a white 96-well plate containing 1 μl of each target oligonucleotide (1 μM). The plate was immediately placed in a TECAN plate reader, and the luminescence signal was read for 2000 s at room temperature with a 400 ms integration time.

[0106] As shown in Figure 23A, the target oligonucleotides tested in this example contained increasing proportions of mismatch nucleotides (indicated by underlined letters) at the 3' end of the target oligonucleotide sequence relative to the sequence of capture oligonucleotide T2. Both R1 (SEQ ID NO: 1) and R6 (SEQ ID NO: 6) oligonucleotides match the capture oligonucleotide sequence, but their GC content is at different levels. Figure 23B summarizes the initial velocities of the TET-miRNA reaction with various oligonucleotides. The 100% matched sequence exhibits significantly faster kinetics, calculated from the initial reaction slope, in both the Klenow and BST cases. Figure 23C shows that calculating the ratio of the reaction rates in the presence of various mismatched oligonucleotides to the 100% matched oligonucleotide provides a measure of how sensitive the two DNA polymerases are to the mismatch content in the target oligonucleotide, with BST polymerase being 2- to 5-fold more sensitive depending on the mismatch percentage.

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

[0108] The TET-miRNA reactions were performed using capture oligonucleotides either in solution or immobilized on SiO2NPs by nonspecific adsorption (the capture oligonucleotides were incubated with SiO2NPs at room temperature for 30 minutes, then washed by centrifugation and stored in a native protein buffer). Figure 24 shows that adsorption of the capture oligonucleotides onto NPs significantly reduces their ability to detect target oligonucleotides.

[0109] Example 21 Analysis comparing TET-miRNA responses when capture oligonucleotides are in solution and when immobilized on SiO2 NPs via biotin-streptavidin Enzyme activity measurements were performed using a reaction mixture consisting of 20 μl His-Si-BST, 20 μl His-Si-ATPS, 5 μl His-Si-luciferase, 5 μl luciferin (200 mM), 5 μl 20× luciferase buffer (50 mM HEPES, 40 mM KCl, 200 mM MgCl), 2 μl APS (30 mM), 1 μl NP-capture oligonucleotide (T2 (SEQ ID NO: 7), 100 μM), and 1.8 μl dCTP / dTTP / dGTP mixture (33 mM each). 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). The plate was immediately placed in a TECAN plate reader and the luminescence signal was read for 2000 seconds at room temperature with a 400 ms integration time.

[0110] Figure 25A identifies two forms of immobilizable capture oligonucleotides produced, in which a biotin tag was attached to either the 5' or 3' end (biotinylated oligonucleotides were purchased from IDT, CA, USA). The 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 unbound proteins were then washed three times by centrifugation. The TET-mRNA reaction showed low activity when the enzymes (DNA-Pol, ATP, and Luc) were in solution and the capture oligonucleotides tethered to streptavidin-coated 500 nm SiO2 NPs were used. Figure 25B shows that in the presence of 5' biotinylated capture oligonucleotides, significantly higher activity was obtained when the TET-mRNA enzyme was also immobilized to NPs (via Si tags), whereas 3' biotinylated capture oligonucleotides showed very low or no activity.

[0111] While preferred embodiments have been illustrated and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like may be made therein without departing from the spirit of the invention, and therefore are deemed to be within the scope of the invention as defined in the appended claims.

Claims

1. 1. A method for detecting a target nucleic acid molecule in a sample, comprising: 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, wherein the capture oligonucleotide molecule comprises: (i) has a length of 30 to 60 bases; (ii) has a 3' tail comprising 4 to 8 nucleotides; (iii) has a 5' tail; (iv) having a target-specific portion between the 3′ tail and the 5′ tail; (v) has a deoxyadenosine phosphate content of 40-50%, and (vi) does not have a deoxythymidine phosphate in the 3' tail or the 5' tail; The process and contacting the double-stranded nucleic acid molecule, a polymerase, and a dNTP mixture to form a polymerase extension mixture, wherein deoxyadenosine triphosphate is excluded from the dNTP mixture; subjecting the polymerase extension mixture to conditions under which the target nucleic acid molecule is isothermally extended and releases 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 polymerase is bound to a solid support.

3. 3. The method of claim 2, wherein the 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 enzymatic reaction of glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase 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 bound glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase comprises contacting adenosine diphosphate, nicotinamide adenine dinucleotide, and glyceraldehyde 3-phosphate to achieve the enzymatic reaction of bound glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase.

8. 8. The method of claim 7, wherein the glyceraldehyde 3-phosphate dehydrogenase and the 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. 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 10. 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. 10. The method of claim 1, wherein the subjecting step is carried out at a temperature of 25 to 40°C.

14. 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).

15. 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.

16. The method of claim 15, wherein the presence of the target nucleic acid molecule in the sample is determined.

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

18. 16. The method of claim 15, wherein the concentration of the target nucleic acid molecule in the sample is determined.

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

20. 10. The method of claim 1 carried out in solution.

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

22. 1. A method for detecting a target nucleic acid molecule in a sample, comprising: 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, wherein the capture oligonucleotide molecule comprises a 3' tail, a 5' tail, a 40-50% deoxyadenosine phosphate content, a target-specific portion between the 3' tail and the 5' tail, and no deoxythymidine phosphate in the 3' tail or the 5' tail; contacting the double-stranded nucleic acid molecule, a polymerase, and a dNTP mixture to form a polymerase extension mixture, wherein deoxyadenosine triphosphate is excluded from the dNTP mixture; subjecting the polymerase extension mixture to conditions under which the target nucleic acid molecule is isothermally extended and releases 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 polymerase, the luciferase, and the adenosine triphosphate-generating enzyme are each bound to a solid support; The method comprising:

23. 23. The method of claim 22, wherein the capture oligonucleotide molecule has a length of 30 to 60 bases.

24. 23. The method of claim 22, wherein the 3' tail comprises 4 to 8 nucleotides.

25. 23. The method of claim 22, wherein multiple types of capture oligonucleotide molecules are provided for detecting multiple types of target nucleic acid molecules.

26. 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; (i) has a length of 30 to 60 bases; (ii) has a 3' tail comprising 4 to 8 nucleotides; (iii) has a 5' tail; (iv) having a target-specific portion between the 3′ tail and the 5′ tail; (v) has a deoxyadenosine phosphate content of 40-50%, and (vi) does not have a deoxythymidine phosphate in the 3' tail or the 5' tail; the capture oligonucleotide molecule; A polymerase and a dNTP mixture in which deoxyadenosine triphosphate has been omitted; an enzyme for generating adenosine triphosphate from the released free phosphate; Luciferase to generate a bioluminescent readout signal The kit comprising:

27. 27. The kit of claim 26, comprising multiple types of capture oligonucleotide molecules for detecting multiple types of target nucleic acid molecules.

28. 26. A composition for use in the method of any one of claims 1 to 25, comprising a capture oligonucleotide molecule, said capture oligonucleotide molecule comprising: (i) has a length of 30 to 60 bases; (ii) has a 3' tail comprising 4 to 8 nucleotides; (iii) has a 5' tail; (iv) having a target-specific portion between the 3′ tail and the 5′ tail; (v) has a deoxyadenosine phosphate content of 40-50%, and (vi) does not have a deoxythymidine phosphate in the 3' tail or the 5' tail; The composition.

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