COMPOSITIONS AND METHODS FOR REVERSE TRANSCRIBING MATURE miRNA FOR qPCR

Abstract

Compositions, kits, systems, and methods are disclosed for detecting small nucleotide target sequences, such as (but not limited to) miRNAs. An oligonucleotide composition includes an internal sequence that forms a hairpin and first and second arms attached to the 5' and 3' ends of the internal sequence, respectively, wherein the first and second arms are complementary to portions of the target nucleotide sequence. Kits, systems, and methods for detecting target nucleotide sequences using the oligonucleotide composition are also disclosed.

Description

COMPOSITIONS AND METHODS FOR REVERSE TRANSCRIBING MATURE miRNA FOR qPCRCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The subject application claims benefit under 35 USC § 119(e) of US Provisional Patent Application No. 63 / 586,810, filed September 29, 2023. The entire contents of the above-referenced patent application(s) are hereby expressly incorporated herein by reference.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT

[0002] Not Applicable.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] The instant application contains, as a separate part of the present disclosure, a Sequence Listing which has been submitted via EFS-Web in computer readable form as an XML file. The Sequence Listing, created September 26, 2024, is named "2023P18408WO Sequence Listing.xml" and is 50,333 bytes in size. The entire contents of the Sequence Listing are hereby incorporated herein by reference.BACKGROUND

[0004] MicroRNAs (miRNAs) are non-coding RNA molecules of approximately 18-28 nucleotides in length that are involved in post-transcriptional gene regulation and modulation of various cellular and molecular processes. Non-coding RNAs are typically classified by size, function (housekeeping vs regulatory), or location (nuclear, nucleolar, ribosomal, etc.). miRNAs, which are approximately 18-28 nucleotides in length, are short compared to other long non-coding RNAs (IncRNAs). The detection of mature miRNAs and its applications have become a prominent focus across numerous research and disease areas. miRNAs represent promising biomarkers for early disease detection and diagnosis, especially where the biomarkers need to traverse the blood-brain barrier (such as, but not limited to, neurodegenerative disorders). miRNAs may function as master regulators of numerous physiological and pathological processes, and changes in their expression patterns are often observed in various diseases. Because of their remarkable stability in biofluids, miRNAs have exciting potential as minimally invasive diagnostic biomarkers (Androvic et al., Nucleic AcidsResearch (2017) 45:el44).

[0005] Given their role as important regulators of biological processes, altered miRNA expression levels have been observed in several diseases. Notably, about 70% of the discovered miRNAs are found in the brain, where they are involved in neuroplasticity and neurodevelopment and are altered in various neurological disorders. One significant trait of miRNAs as biomarkers for neurodegenerative diseases is their ability to cross the blood-brain barrier. This, combined with their high stability in bodily fluids, makes miRNAs ideal candidates for liquid biopsy biomarkers for diagnostic purposes. However, the short length of miRNAs affects their suitability as targets (in particular, but not by way of limitation, as PCR targets), and a wide array of methods have been tested for developing diagnostic assays for specific miRNAs.

[0006] As miRNAs increasingly emerge as potential biomarkers, the need to identify and precisely and reliably measure their total and specific activity has led to the development of numerous tools. This diversity of tools can be partly attributed to the challenges posed by the nature of miRNAs as short targets. Commonly used methods include hybridization-based techniques (e.g., Northern blots), enzyme-based methods (e.g., luciferase assays), and amplification-based approaches (e.g., NGS and PCR). Amplification-based methods, particularly PCR, offer the advantage of widely available equipment and the ability to generate quantitative data with high sensitivity and specificity.

[0007] Numerous amplification-based methods have been documented for the detection of miRNA and, in particular, mature miRNA. The three most common methods include microarrays, next generation sequencing (RNA-Seq), and reverse transcription quantitative PCR (RT-qPCR). However, all of these methods have differing performance levels and their own limitations. As a result, researchers constantly explore new avenues, and new techniques continue to emerge.

[0008] RT-qPCR is the method commonly used for the validation of results from screening experiments and when high accuracy and precision are required. RT-qPCR is useful for diagnostic use, as a small amount of targets can be quantified, even when the amount of material is limited.

[0009] Mature miRNAs have a notably short length of 18-28 nucleotides, making them challenging targets for designing RT-qPCR assays. RT-qPCR assays depend on binding of two primers of similar length, and a conventional PCR primer has approximately the same lengthas a mature miRNA. In addition, the GC content of miRNAs is highly variable. Consequently, a plethora of methods has emerged to cater to the diverse requirements of various applications and research needs. This includes diverse versions of RT-qPCR methods, including stem-loop, PolyA-tailing, and ligation approaches.

[0010] One currently used method is the stem-loop RT primer method, the most common of which is the TaqMan miRNA assay. The stem-loop primers of the TaqMan miRNA assays are composed of a short single stranded sequence at their 3'-ends that anneals to the 3'-end of the targeted miRNA, a double-stranded segment (the stem), and a mini-loop. The stemloop structure shifts the equilibrium to the formation of an RNA / DNA duplex and should prevent binding of the prior to pri- and pre-miRNAs and to any double-stranded DNA (dsDNA) that may be present; however, the presence of genomic and plasmid DNA containing sequences of the corresponding pre-miRNA contribute to significant background signal that can lead to false positive results. In addition, the TaqMan probe does not contribute to the specificity of the reaction, as it binds to a site originating from the sequence of the RT primer.

[0011] Androvic et al. (incorporated supra) disclose a two-tailed RT-qPCR approach that quantifies miRNA expression using specific structured primers for reverse transcription and SYBR-green based qPCR. The two-tailed RT primers include two hemiprobes complementary to separate regions of the target miRNA and of an oligonucleotide tetherfolded into a hairpin structure. While this technique provides a great increase in sensitivity over most other techniques, the methods do not allow for easy use with TaqMan probes for all possible miRNA targets. In addition, the methods require the use of a lower melting temperature (Tm) than desired, thus affecting the versatility, specificity, and sensitivity of the methods.

[0012] Accordingly, there is a need in the art for new and improved reagents and methods for use in the detection and quantification of mature miRNA in patient samples using RT- qPCR.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0013] FIG. 1 provides a schematic description of a two-tailed oligo RT-qPCR design in accordance with the present disclosure.

[0014] FIG. 2 illustrates one non-limiting embodiment of a two-tailed RT-oligo design for use in methods in accordance with the present disclosure. Panel A provides the sequence for an oligo (SEQ ID NO:5). Panel B provides a schematic representation of how the two-tailedoligo of Panel A binds to the miRNA. Panel C illustrates the extension of the oligo during the RT step, resulting in the generation of full-length cDNA. Panel D shows the binding of the primers and probe during the qPCR step.

[0015] FIG. 3 illustrates the design of 5' and 3' arms for two-tailed RT-oligos for detection of six different miRNAs.

[0016] FIG. 4 illustrates structures for three non-limiting embodiments of two-tailed RT- oligos produced for detection of the miRNA miR-9-5p.

[0017] FIG. 5 illustrates structures for three non-limiting embodiments of two-tailed RT- oligos produced for detection of the miRNA miR-30a-5p.

[0018] FIG. 6 illustrates structures for three non-limiting embodiments of two-tailed RT- oligos produced for detection of the miRNA miR-34a-5p (SEQ ID NOS:45-47), and one nonlimiting embodiment of a two-tailed RT-oligo produced for detection of the miRNA miR-125b- 5p (SEQ ID NO:48).

[0019] FIG. 7 illustrates structures for three non-limiting embodiments of two-tailed RT- oligos produced for detection of the miRNA miR-15b-5p.

[0020] FIG. 8 illustrates structures for three non-limiting embodiments of two-tailed RT- oligos produced for detection of the miRNA miR-146-5p.

[0021] FIG. 9 illustrates structures for six non-limiting embodiments of two-tailed RT- oligos produced for detection of the miRNA miR-15b-3p.DETAILED DESCRIPTION

[0022] Before explaining at least one embodiment of the inventive concept(s) in detail by way of exemplary drawings, experimentation, results, and laboratory procedures, it is to be understood that the inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings, experimentation and / or results. The inventive concept(s) is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary - not exhaustive. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0023] Independent of the grammatical term usage, individuals with male, female, or other gender identities are included within the term.

[0024] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0025] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0026] All of the compositions and / or methods disclosed and / or claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present disclosure have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

[0027] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0028] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to 1 or more, 2 or more, 3 or more, 4 or more, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0029] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0030] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0031] As used in this specification and claim(s), the terms "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. For example, unless otherwise noted, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements, but may also include other elements not expressly listed or inherently present therein.

[0032] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0033] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0034] Throughout this application, the terms "about" and "approximately" are used to indicate that a value includes the inherent variation of error for the composition / apparatus / device, the method being employed to determine the value, or the variation that exists among the study subjects. That is, the terms "about" and "approximately" and variations thereof are intended to include not only the exact value qualified by the term, but to also include some slight deviations therefrom, such as deviations caused by measuring error, manufacturing tolerances, wear and tear on components or structures, and combinations thereof, for example. In particular, for example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by ± 20%, or ± 15%, or ± 12%, or ± 11%, or ± 10%, or ± 9%, or ± 8%, or ± 7%, or ± 6%, or ± 5%, or ± 4%, or ± 3%, or ± 2%, or ±1%, or ± 0.5%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0035] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequentlydescribed event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time, or at least 98% of the time. The term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item.

[0036] Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those end points and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the present disclosure. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value.

[0037] As used herein, the phrase "associated with" includes both direct association of two moieties to one another as well as indirect association of two moieties to one another. Non-limiting examples of associations include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety.

[0038] The term "purified" as used herein means at least one order of magnitude of purification is achieved compared to the starting material or of the natural material, for example but not by way of limitation, two, three, four, or five orders of magnitude of purification of the starting material or of the natural material. Thus, the term "purified" as utilized herein does not necessarily mean that the material is 100% purified, and therefore such term does not exclude the presence of other material(s) present in the purified composition.

[0039] The term "sample" as used herein will be understood to include any type of biological sample that may be utilized in accordance with the present disclosure. Examples ofbiological samples that may be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), saliva, sputum, cerebrospinal fluid (CSF), skin, interstitial fluid, tears, mucus, urine, swabs, combinations, and the like. In one non-limiting embodiment, the sample is a volume of whole blood.

[0040] The term "polynucleotide" or "oligonucleotide" as used herein will be understood to refer to a polymer of two or more nucleotides. Nucleotides, as used herein, will be understood to include deoxyribose nucleotides and / or ribose nucleotides, as well as artificial variants thereof. The term polynucleotide also includes single-stranded and double-stranded molecules.

[0041] The term "small non-coding RNA" as used herein refers to RNA molecules of less than about 200 nucleotides and include miRNAs, siRNAs, snRNAs, piRNAs, and circRNAs.

[0042] The term "miRNA" as used herein refers to a small non-coding RNA having a length of less than about 30 nucleotides and that has various regulatory functions.

[0043] Turning now to the inventive concepts, certain non-limiting embodiments thereof are directed to new and improved reagents, compositions, kits, systems, and methods for detection of small target sequences, such as (but not limited to), short DNA target sequences (e.g., SNPs, etc.), short RNA target sequences (e.g., miRNAs, siRNAs, snRNAs, piRNAs, circRNAs, etc.), and short target sequences within a longer DNA or RNA sequence. For example (but not by way of limitation), the detection of mature miRNAs and its applications have become a prominent focus across numerous research and disease areas. miRNAs represent promising biomarkers for early disease detection and diagnosis, especially where the biomarkers need to traverse the blood-brain barrier.

[0044] Certain non-limiting embodiments of the present disclosure are directed to any of the oligonucleotide compositions disclosed or otherwise contemplated herein, including any of the consensus and specific sequences listed in Table 1 and / or shown in FIGS. 2-9 and / or described in detail in the Examples. In specific (but non-limiting) embodiments, the oligonucleotide compositions are two-tailed RT-oligos.

[0045] Certain non-limiting embodiments of the present disclosure include kits and systems that include any of the oligonucleotide compositions disclosed or otherwise contemplated herein, including any of the sequences listed in Table 1 and / or shown in FIGS. 2-9 and / or described in detail in the Examples.

[0046] The compositions / reagents of the kits or systems may be provided in any form that allows them to function in accordance with the present disclosure. For example, but not by way of limitation, each of the reagents may be provided in liquid form and disposed in bulk and / or single aliquot form within the kit or system. Alternatively, in a particular (but nonlimiting) embodiment, one or more of the reagents may be disposed in the kit or system in the form of a single aliquot lyophilized reagent. The use of dried reagents in kits / microfluidics devices is described in detail in US Patent No. 9,244,085 (Samproni), the entire contents of which are hereby expressly incorporated herein by reference.

[0047] In addition to the compositions / reagents described in detail herein above, the kits or systems may further contain other reagent(s) for conducting any of the methods described or otherwise contemplated herein. For example, but not by way of limitation, the kits or systems may include forward and reverse primers and probes for PCR amplification of the at least one target and / or at least one control. The nature of these additional reagent(s) will depend upon the particular assay format, and identification thereof is well within the skill of one of ordinary skill in the art; therefore, no further description thereof is deemed necessary. In one particular (but non-limiting) embodiment, the kits or systems may include a universal probe, such as a TaqMan probe.

[0048] Also, the compositions / reagents present in the kits or systems may each be in separate containers / compartments, or various compositions / reagents can be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the compositions / reagents. For example (but not by way of limitation), the forward and reverse primers for PCR amplification of the at least one target and / or probe may be present in the same container / compartment. In addition, the kit or system may include a device in which one or more of the compositions / reagents are disposed.

[0049] The relative amounts of the various compositions / reagents in the kits or systems can vary widely to provide for concentrations of the compositions / reagents that substantially optimize the reactions that need to occur during the methods and further to optimize substantially the sensitivity and selectivity of an assay. Under appropriate circumstances, one or more of the compositions / reagents in the kit or system can be provided as a dry powder, such as a lyophilized powder, and the kit or system may further include excipient(s) for dissolution of the dried reagents; in this manner, a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present disclosurecan be obtained from these compositions. Positive and / or negative target controls may also be included with the kit or system. In addition, the kit or system can further include a set of written instructions (or access to electronic instructions) explaining how to use the kit or system. A kit or system of this nature can be used in any of the methods described or otherwise contemplated herein.

[0050] Certain non-limiting embodiments of the present disclosure are directed to methods of designing any of the two-tailed RT-oligo compositions disclosed or otherwise contemplated herein. In the method, a 6-9 nucleotide 5' hemiprobe specific for the target sequence (such as, but not limited to, a miRNA) and a 4-6 nucleotide 3' hemiprobe specific for the target sequence (such as, but not limited to, a miRNA) is selected. Then the 5' hemiprobe is attached to a 5' end of any internal consensus sequence disclosed or otherwise contemplated herein to form a 5' arm thereof, and the 3' hemiprobe is attached to a 3' end of the internal sequence to form a 3' arm thereof; the resulting structure forms the two-tailed RT-oligo composition. The method may further include one or more additional steps; nonlimiting examples thereof include examining the sequences of the portions of the RT-oligo that are 5' and 3' to the hairpin / stem loop structure (including the target-specific 5' and 3' arms) to ensure that a palindrome isn't present, and / or examining said sequences to ensure that the arms will remain open and not bind to one another and collapse. Other non-limiting examples of additional steps that may be included in the method include examining the 3' arm of the oligo to ensure that it is sufficient to be extended during the RT step, and / or examining the 5' arm of the oligo to ensure that it will tether to the miRNA to enhance specificity and binding but also be capable of being displaced by the reverse transcriptase enzyme during the RT step.

[0051] Certain non-limiting embodiments of the present disclosure are directed to a method of detecting a target nucleotide sequence in a sample (such as, but not limited to, a biological sample). In the method, any of the oligonucleotide compositions disclosed or otherwise contemplated herein are combined with a sample suspected of containing the target nucleotide sequence to form a mixture, and the mixture is incubated under conditions wherein the first and second arms of the oligonucleotide bind cooperatively to the first and second portions of the target sequence. Reverse transcriptase is added to the mixture, and the reverse transcriptase binds a 3' end of the oligonucleotide and elongates the oligonucleotide based on the target sequence to form a cDNA molecule. The cDNA moleculeis then amplified by qPCR using forward and reverse primers, wherein one of the primers is a target sequence-specific primer, and the other primer is specific for a portion of the internal sequence of the oligonucleotide composition. The presence and / or concentration of the target nucleotide sequence is then detected and / or determined based on the amplification step.

[0052] In certain particular (but non-limiting) embodiments, the target nucleotide sequence to be detected by the method is a short DNA target sequence (e.g., SNPs, etc.), a short RNA target sequence (e.g., miRNAs, siRNAs, snRNAs, piRNAs, circRNAs, etc.), or a short target sequence within a longer DNA or RNA sequence. In a particular (but non-limiting) embodiment, the target nucleotide sequence to be detected by the method is a mature miRNA.

[0053] In certain particular (but non-limiting) embodiments, the method may include one or more additional steps. For example (but not by way of limitation), the method may include a step of performing a heat shock step on the sample before adding the oligonucleotide composition. One non-limiting example of a heat shock step that me be utilized involves incubation of the sample at about 85°C for about 2 to about 5 minutes. In a particular, but non-limiting embodiment, the heat shock step may be followed by exposing the heat shocked sample to a cold shock on ice prior to addition of a reaction mix including the two tailed oligos and performing the RT and qPCT steps.EXAMPLES

[0054] Examples are provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein after. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.

[0055] Standard qPCR techniques are not an option for mature miRNAs due to the exceptionally short nature of their sequences. Consequently, various techniques have been developed to extend miRNAs as a template to make it amendable for qPCR with varying success.

[0056] Androvic et al. (incorporated supra) disclose a two-tailed RT-qPCR approach with SYBR-green detection chemistry. The two-tailed RT primer of Androvic et al. has two hemiprobes connected by a hairpin folding sequence, and the hemiprobes bindcooperatively, one at each end of the target miRNA (i.e., one to the 5' end of the miRNA and another to the 3' end of the miRNA), thereby forming a stable complex. Then reverse transcriptase binds the 3'-end of the hybridized two-tailed RT primer and elongates it to form tailed cDNA, which is amplified by qPCR using two target-specific primers. The sequence of the two-tailed oligo of Androvic et al. is:NNNNNNNNACATGCTCTCcaggtacAGTTGgtacctgTCTCCACTTNNNNN (SEQ ID NO:1), with the bolded portion of the sequence (TCTCCACTT; SEQ ID NO:2) being a critical area for probe design; the GC content of this area of SEQ ID NO:2 is 44%. The two portions of the sequence shown in lower case form the double stranded stem of the hairpin, with a five- nucleotide sequence therebetween that forms the loop of the hairpin.

[0057] H owever, the method of Androvic et al. cannot utilize Taq Man detection chemistry for all possible miRNA targets and instead must rely on SYBR-green detection chemistry, in which individual probes must be produced. In addition, the GC content of the critical area of the RT-oligo of Androvic et al. requires the use of a lower melting point (Tm). These disadvantages limit the versatility, specificity, and sensitivity of the method of Androvic et al.

[0058] Another prior art method of analyzing miRNAs usingtwo-tailed RT-PCR is disclosed in Russian Patent No. RU2783509C1. The two-tailed oligo utilized in this method has a similar sequence to the oligo of Androvic et al. and is shown below:NNNNNNNN CTATGCTCTCcagctaCAGTACACTAGgtagctgTCTCCACTTN N N N N(SEQ ID NO:3).Note that this oligo contains the same critical area of probe design as the oligo of Androvic et al. (TCTCCACTT; SEQ ID NO:2; shown in bold above). As mentioned above, the GC content of the critical area of SEQ ID NO:2 is 44%. In addition, the two portions of the sequence shown in lower case form the double stranded stem of the hairpin, with an eleven (11) nucleotide sequence therebetween that forms the loop of the hairpin. However, the method of Androvic et al. is only effective for certain miRNAs and cannot be applied universally across all miRNAs.

[0059] In the present disclosure, a new and improved, very effective two-tailed RT-qPCR approach is provided that incorporates improvements to widen the applicability of the method across a broader spectrum of miRNAs while also increasing the sensitivity and specificity of the assay. FIG. 1 illustrates one non-limiting embodiment of the basic RT-qPCR method utilized in accordance with the present disclosure. This approach uses an RT primer structured as a hairpin with two open arms that hybridize with the target miRNA. A first (i.e.,5') arm of the two-tailed RT-oligo hybridizes to a first internal portion of the miRNA, while a second (i.e., 3') arm of the two-tailed RT-oligo hybridizes to a second portion of the miRNA. The first and second portions of the miRNA do not overlap so that both the first and second arms of a single oligo can bind at the same time to a single miRNA molecule, as shown in the middle panel of FIG. 1. In a particular (but non-limiting) embodiment, the distance between the first and second portions of the miRNA is about 3 nucleotides, about 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, and the like, as well as a range formed between two of the above values (e.g., a range of from about 3 nucleotides to about 7 nucleotides). In addition, the first portion of the miRNA is 5' to the second portion of the miRNA. The first portion of the miRNA may be an internal or 5' end portion of the miRNA, while the second portion of the miRNA may be an internal portion or a 3' end portion of the miRNA.

[0060] The right panel of FIG. 1 illustrates how reverse transcription occurs. After hybridization of the two-tailed RT-oligo to the miRNA as shown in the middle panel, the reverse transcription (RT) reaction is primed from the second arm of the oligo, and the first arm is eventually displaced by the RT enzyme. The two-tailed RT-oligo is thus elongated to produce a cDNA molecule which includes a sequence that is complementary to the target mature miRNA. Then a qPCR step can be conducted to amplify the entire cDNA molecule, which contains the sequence of both the oligo as well as the miRNA.

[0061] The first (5') arm of the oligo enhances specificity and binding, while only the 3' arm of the oligo is extended during the RT step. The resulting cDNA is then selected with high specificity and extended by the hairpin oligo, allowing the application of standard qPCR assay design strategies in the final step.

[0062] The two-tailed RT-oligo serves at least three functions. First, the two-tailed RT- oligo specifically primes the reverse transcription of the target miRNA template. Second, the two-tailed RT-oligo contributes with additional sequence to the cDNA, thereby providing acDNA that is sufficiently long enough for amplification. Third, the two-tailed RT-oligo contains the sequence to which one of the PCR primers binds.

[0063] One non-limiting embodiment of a two-tailed RT-oligo designed in accordance with the present disclosure has the sequence:N6-9ACATGCTCTCcaggtacXi-5gtacctgCCTATGCTGN4-6 (SEQ ID NO:4)The "N" sequences on the 5' and 3' ends of the above sequences contain the arms that hybridize to portions of the miRNA; the first arm on the 5' end of the oligo is complementary to a first portion of a miRNA to be detected, while the second arm on the 3' end of the oligo is complementary to a second portion of the miRNA. The two portions of the sequence shown in lower case form the double stranded stem of the hairpin structure, with 1-5 nucleotides in between that form the loop of the hairpin. The portion of the sequence shown in bold (CCTATGCTG; SEQ ID NO:8) is considered an important area for probe design and has a GC content of 56%. The higher GC content results in a higher temperature range that can be utilized for PCR, thus making the PCR more specific. Also, the probe area nucleotides were diversified with the addition of Gs.

[0064] FIG. 2 illustrates one non-limiting embodiment of a specific two-tailed RT-oligo designed in accordance with the present disclosure for use in an RT-qPCR method in accordance with the present disclosure. Panel A of FIG. 2 illustrates that the two-tailed RT- oligo has the sequence: / V / V / V / V / V / V / V / VACATGCTCTCcaggtacAGTTGgtacctgCCTATGCTG / V / V / V / V / V (SEQ ID NO:5).

[0065] Panel B of FIG. 2 illustrates how the two-tailed oligo binds to the miRNA, with the first (i.e., 5') arm hybridizing to a first internal portion of the miRNA, and the second (i.e., 3') arm hybridizing to a second portion of the miRNA (which is illustrated as being a 3' end portion of the miRNA). The first and second arms bind at the same time to a single miRNA molecule, as shown in Panel B. As shown in Panel C, reverse transcription is primed from the second arm, the first arm is eventually displaced by the RT enzyme, and the two-tailed RT-oligo is elongated to produce a cDNA molecule shown in Panel D, which includes a sequence complementary to the target mature miRNA. Then a qPCR step is conducted as shown in Panel D, with a forward PCR primer that is specific for the target miRNA and a reverse PCR primer that is specific for a portion of the internal sequence of the oligo (such as, but not limited to, a portion of the sequence that forms the hairpin structure). Based on the novel portion of the internal sequence of the oligo shown in bold in Panel A, a TaqMan probe can also be utilized.

[0066] Other non-limiting embodiments of specific two-tailed RT-oligos designed in accordance with the present disclosure for use in an RT-qPCR method in accordance with the present disclosure include the following:N6-HGCTCTCTCTGAAGTAGTCAGATCTCCN4-IO (SEQ ID NO:58)N6-9TATGCTCTCTCTGAAGTAGTCAGATCTCCACGTN4-6 (SEQ ID NO:59)N6-IIGCTCTCTCTGAAGTAGTCAGATCTCCCAGN4-6 (SEQ ID NQ:60) n6-9TATGCTCTCTCTGAAGTAGTCAGATCTCCACGN4-6 (SEQ ID NO:61)

[0067] In the above sequences, the N nucleotides at the ends of each sequence may include the 5' or 3' arms as well as one or more additional residues, as shown in greater detail herein below.

[0068] In non-limiting examples, the two-tailed RT-oligos constructed in accordance with the present disclosure have a total length of about 40 nucleotides, about 41 nucleotides, about 42 nucleotides, about 43 nucleotides, about 44 nucleotides, about 45 nucleotides, about 46 nucleotides, about 47 nucleotides, about 48 nucleotides, about 49 nucleotides, about 50 nucleotides, about 51 nucleotides, about 52 nucleotides, about 53 nucleotides, about 54 nucleotides, about 55 nucleotides, or the like, as well as a range formed from two of the above values (e.g., a range of from about 40 nucleotides to about 55 nucleotides, a range of from about 44 nucleotides to about 50 nucleotides, a range of from about 45 nucleotides to about 49 nucleotides, etc.).

[0069] Individual optimization of the two-tailed RT-oligo is necessary for each specific miRNA target to achieve optimal detection and quantification. In particular, optimization of the stem and loop of the hairpin structure of the two-tailed RT-oligo may be necessary for each specific miRNA target to achieve optimal detection and quantification. For example (but not by way of limitation), the sequences of the portions of the RT-oligo that are 5' and 3' to the hairpin / stem loop structure (including the miRNA-specific 5' and 3' arms) must be designed so as to ensure that a palindrome isn't present (which could be amplified in either the 5' or 3' direction by the RT, thus resulting in a nonsense product). In addition, the sequences of these sections must also be designed so as to ensure that the arms will remain open and not bind to one another and collapse.

[0070] Also, optimization of the arms of the two-tailed RT-oligo is necessary for each specific miRNA target to achieve optimal detection and quantification. For example (but not by way of limitation), the 3' arm of the oligo will need to be designed to ensure that it issufficient to be extended during the RT step. In addition, the 5' first (5') arm of the oligo will need to be designed to ensure that it will tether to the miRNA and enhance specificity and binding thereto while also allowing for eventual displacement by the reverse transcriptase enzyme during the RT step.

[0071] Specific (but non-limiting) examples of individual optimization of the 5' and 3' arms for six different miRNAs is shown in FIG. 3. Three different 5' arms were selected for each miRNA (each having a length of 6-9 nucleotides); the length of the 3' arm was held constant for each miRNA (with the length of each being 4-6 nucleotides).

[0072] FIGS. 4-9 illustrate optimized structures of full-length two-tailed RT-oligos for detection of various miRNAs. FIG. 4 contains three non-limiting embodiments of two-tailed RT-oligos for detection of miR-9-5p (SEQ ID NO:9), which utilize the 5' arms of SEQ ID NOS:10- 12 and each utilize the 3' arm of SEQ ID NO:13. The two-tailed RT-oligos are based on the consensus sequence of SEQ ID NO:59. The sequences of the two-tailed RT-oligos are represented by SEQ ID NOS: 39-41, respectively.

[0073] FIG. 5 contains three non-limiting embodiments of two-tailed RT-oligos for detection of miR-30a-5p (SEQ ID NO:19), which utilize the 5' arms of SEQ ID NQS:20-22 and each utilize the 3' arm of SEQ ID NO:23. The two-tailed RT-oligos are based on the consensus sequence of SEQ ID NQ:60. The sequences of the two-tailed RT-oligos are represented by SEQ ID NOS: 42-44, respectively.

[0074] FIG. 6 contains three non-limiting embodiments of two-tailed RT-oligos for detection of miR-34a-5p (SEQ ID NO:24), which utilize the 5' arms of SEQ ID NOS:25-27 and each utilize the 3' arm of SEQ ID NO:28. The two-tailed RT-oligos are based on the consensus sequence of SEQ ID NO:61. The sequences of the two-tailed RT-oligos are represented by SEQ ID NOS:45-47, respectively.

[0075] FIG. 6 also contains one non-limiting embodiment of a two-tailed RT-oligo for detection of miR-125b-5p (SEQ ID NO:29), which utilizes the 5' arm of SEQ ID NO: and the 3' arm of SEQ ID NO:32. The sequence of the two-tailed RT-oligo is represented by SEQ ID NO:48.

[0076] FIG. 7 contains three non-limiting embodiments of two-tailed RT-oligos for detection of miR-15b-5p. The two-tailed RT-oligos are based on the consensus sequence of SEQ ID NQ:60. The sequences of the two-tailed RT-oligos are represented by SEQ ID NOS:51-53.Y1

[0077] FIG. 8 contains three non-limiting embodiments of two-tailed RT-oligos for detection of miR-146-5p (SEQ ID NO:34), which utilize the 5' arms of SEQ ID NOS:35-37 and each utilize the 3' arm of SEQ ID NO:38. The sequences of the two-tailed RT-oligos are represented by SEQ ID NOS:62-64, respectively.

[0078] FIG. 9 contains six non-limiting embodiments of two-tailed RT-oligos for detection of miR-15b-3p. The sequences of the two-tailed RT-oligos are represented by SEQ ID NOS:65- 70.

[0079] Table 1 below indicates various sequences disclosed in this application along with the sequence identifiers assigned thereto. For full-length RT-oligos disclosed in Table 1, the miRNA-specific 5' and 3' arms are shown in bold. The accession numbers indicated for the miRNAs are taken from miRBase: the microRNA database (The University of Manchester, UK).TABLE 1

[0080] Reverse transcriptase reactions were performed using the TaqMan MicroRNA Reverse Transcription kit (Thermo Fisher Scientific Inc., Waltham, MA). miRNAs were initially denatured by a heat shock step (i.e., incubation at 85°C for 2-5 minutes) followed by a cold shock on ice; then the reaction mix including the two tailed oligos was added and the experiment finalized following manufacturer's instructions.

[0081] The qPCR step was performed using either of the following 2x master mixes: IDT's Primer Time or Thermofisher's TaqMan FAST Advanced Master Mix with the following thermo cycling protocol:

[0082] Step -> # of Cycles -> Temperature -> Time

[0083] Hold - 1minute ->

[0084] Cycles -> 40 -> 95°C -> 3 seconds ->

[0085] 60°C -> 20 seconds

[0086] While specific reaction conditions are described herein above, it will be understood that any reaction conditions utilized for reverse transcription and / or qPCR known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure. Therefore, the reaction conditions described herein above are for purposes of example only.NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0087] Illustrative embodiment 1. An oligonucleotide composition for detection of a target nucleotide sequence, the oligonucleotide composition comprising: an internal sequence comprising one of SEQ ID NOS:6-7 and 54-57, wherein at least a portion of the internal sequence forms a hairpin, and wherein the internal sequence has a 5' end and a 3' end; a first arm comprising a sequence of 6-11 nucleotides, wherein the first arm is attached to the 5' end of the internal sequence, and wherein at least a portion of the sequence of the first arm is complementary to a first portion of the target nucleotide sequence (such as, but not limited to, about 6-9 nucleotides thereof); and a second arm comprising a sequence of 4- 10 nucleotides, wherein the second arm is attached to the 3' end of the internal sequence, and wherein at least a portion of the sequence of the second arm is complementary to a second portion of the target nucleotide sequence (such as, but not limited to, about 4-6 nucleotides thereof); and wherein the first and second portions of the target nucleotide sequence to which the first and second arms bind do not overlap.

[0088] Illustrative embodiment 1A. The oligonucleotide of any preceding illustrative embodiment, wherein the portion of the first arm that is complementary to the first portion of the target nucleotide sequence has a length of 6-9 nucleotides, and wherein the portion of the second arm that is complementary to the second portion of the target nucleotide sequence has a length of 4-6 nucleotides.

[0089] Illustrative embodiment 2. The oligonucleotide of any preceding illustrative embodiment, wherein the internal sequence comprises SEQ ID NO:6.

[0090] Illustrative embodiment 3. The oligonucleotide of any preceding illustrative embodiment, wherein the internal sequence comprises SEQ ID NO:7.

[0091] Illustrative embodiment 4. The oligonucleotide of any preceding illustrative embodiment, wherein the internal sequence comprises SEQ ID NO:54.

[0092] Illustrative embodiment 5. The oligonucleotide of any preceding illustrative embodiment, wherein the internal sequence comprises SEQ ID NO:55.

[0093] Illustrative embodiment 6. The oligonucleotide of any preceding illustrative embodiment, wherein the internal sequence comprises SEQ ID NO:56.

[0094] Illustrative embodiment 7. The oligonucleotide of any preceding illustrative embodiment, wherein the internal sequence comprises SEQ ID NO:57.

[0095] Illustrative embodiment 8. The oligonucleotide of any preceding illustrative embodiment, wherein the first portion of the target nucleotide sequence is an internal portion of the target nucleotide sequence.

[0096] Illustrative embodiment 9. The oligonucleotide of any preceding illustrative embodiment, wherein the second portion of the target nucleotide sequence contains a S' end of the target nucleotide sequence.

[0097] Illustrative embodiment 10. The oligonucleotide composition of any preceding illustrative embodiment, represented by SEQ ID NO:4.

[0098] Illustrative embodiment 11. The oligonucleotide composition of any preceding illustrative embodiment, represented by SEQ ID NO:5.

[0099] Illustrative embodiment 12. The oligonucleotide composition of any preceding illustrative embodiment, represented by SEQ ID NO:58.

[0100] Illustrative embodiment 13. The oligonucleotide composition of any preceding illustrative embodiment, represented by SEQ ID NO:59.

[0101] Illustrative embodiment 14. The oligonucleotide composition of any preceding illustrative embodiment, represented by SEQ ID NO:60.

[0102] Illustrative embodiment 15. The oligonucleotide composition of any preceding illustrative embodiment, represented by SEQ ID NO:61.

[0103] Illustrative embodiment 16. The oligonucleotide composition of any preceding illustrative embodiment, represented by one of SEQ ID NOS:39-53.

[0104] Illustrative embodiment 16A. The oligonucleotide composition of any preceding illustrative embodiment, wherein the target nucleotide sequence is a short DNA target sequence.

[0105] Illustrative embodiment 16B. The oligonucleotide composition of any preceding illustrative embodiment, wherein the target nucleotide sequence is a short RNA target sequence.

[0106] Illustrative embodiment 16C. The oligonucleotide composition of any preceding illustrative embodiment, wherein the target nucleotide sequence is a short target sequence within a longer DNA or RNA sequence.

[0107] Illustrative embodiment 17. The oligonucleotide composition of any preceding illustrative embodiment, wherein the target nucleotide sequence is a miRNA.

[0108] Illustrative embodiment 17A. The oligonucleotide composition of any preceding illustrative embodiment, wherein the distance between the first and second portions of the miRNA is in a range of from about 3 nucleotides to about 7 nucleotides, whereby the 5' end and 3' end of the oligonucleotide composition are bound to the miRNA at positions that are separated by a distance in a range of from about 3 nucleotides to about 7 nucleotides.

[0109] Illustrative embodiment 18. The oligonucleotide composition of any preceding illustrative embodiment, wherein the miRNA is selected from the group consisting of miR-9- 5p, miR-30a-5p, miR-34a-5p, miR-125b-5p, miR-15b-5p, miR-let7a-5p, miR-15b-3p, and miR- 146a-5p.

[0110] Illustrative embodiment 19. The oligonucleotide of any preceding illustrative embodiment, wherein the miRNA is miR-9-5p, at least a portion of the sequence of the first arm is selected from the group consisting of SEQ ID NQS:10-12, and at least a portion of the sequence of the second arm is represented by SEQ ID NO:13.

[0111] Illustrative embodiment 20. The oligonucleotide of any preceding illustrative embodiment, wherein the miRNA is miR-15b-5p, at least a portion of the sequence of the firstarm is selected from the group consisting of SEQ ID NOS:15-17, and at least a portion of the sequence of the second arm is represented by SEQ ID NO:18.

[0112] Illustrative embodiment 21. The oligonucleotide of any preceding illustrative embodiment, wherein the miRNA is miR-30a-5p, at least a portion of the sequence of the first arm is selected from the group consisting of SEQ ID NQS:20-22, and at least a portion of the sequence of the second arm is represented by SEQ ID NO:23.

[0113] Illustrative embodiment 22. The oligonucleotide of any preceding illustrative embodiment, wherein the miRNA is miR-34a-5p, at least a portion of the sequence of the first arm is selected from the group consisting of SEQ ID NOS:25-27, and at least a portion of the sequence of the second arm is represented by SEQ ID NO:28.

[0114] Illustrative embodiment 23. The oligonucleotide of any preceding illustrative embodiment, wherein the miRNA is miR-125b-5p, at least a portion of the sequence of the first arm is selected from the group consisting of SEQ ID NQS:30-32, and at least a portion of the sequence of the second arm is represented by SEQ ID NO:33.

[0115] Illustrative embodiment 24. A method of detecting a target nucleotide sequence in a sample, the method comprising: combining the sample with the oligonucleotide composition of any preceding Illustrative embodiment to form a mixture and incubating the mixture under conditions wherein the first and second arms of the oligonucleotide bind cooperatively to the first and second portions of the target nucleotide sequence; adding reverse transcriptase to the mixture, wherein the reverse transcriptase binds a 3' end of the oligonucleotide and elongates the oligonucleotide based on the target nucleotide sequence to form a cDNA molecule; amplifying the cDNA molecule by qPCR using forward and reverse primers, wherein one of the primers is a target-specific primer, and the other primer is specific for a portion of the internal sequence of the oligonucleotide composition; and detecting the presence of the target mature miRNA based on the amplification step.

[0116] Illustrative embodiment 25. The method of any preceding illustrative embodiment, wherein the sample is a biological sample.

[0117] Illustrative embodiment 25A. The method of any preceding illustrative embodiment, wherein the target nucleotide sequence is a short DNA target sequence.

[0118] Illustrative embodiment 25B. The method of any preceding illustrative embodiment, wherein the target nucleotide sequence is a short RNA target sequence.

[0119] Illustrative embodiment 25C. The method of any preceding illustrative embodiment, wherein the target nucleotide sequence is a short target sequence within a longer DNA or RNA sequence.

[0120] Illustrative embodiment 25D. The method of any preceding illustrative embodiment, wherein the target nucleotide sequence is a miRNA.

[0121] Illustrative embodiment 25E. The method of illustrative embodiment 25D, wherein the distance between the first and second portions of the miRNA is in a range of from about 3 nucleotides to about 7 nucleotides, whereby the 5' end and 3' end of the oligonucleotide composition are bound to the miRNA at positions that are separated by a distance in a range of from about 3 nucleotides to about 7 nucleotides.

[0122] Illustrative embodiment 25F. The method of any preceding illustrative embodiment, wherein the method further comprises the step of performing a heat shock step on the sample before combining the sample with the oligonucleotide composition.

[0123] Illustrative embodiment 25G. The method of illustrative embodiment 25F, wherein the heat shock step comprises incubation of the sample at about 85°C for about 2 to about 5 minutes.

[0124] Illustrative embodiment 25H. The method of illustrative embodiment 25F or 25G, further comprising the step of exposing the heat shocked sample to a cold shock on ice prior to combining the sample with the oligonucleotide composition.

[0125] Illustrative embodiment 251. The method of any preceding illustrative embodiment, wherein the step of amplifying the cDNA molecule by qPCR comprises the following thermocycling protocol: one cycle of a hold step of incubating for about one minute at about 95°C; and 40 amplification cycles that include incubating for about 3 seconds at about 95°C followed by incubating for about 20 seconds at about 60°C.

[0126] Illustrative embodiment 26. A kit, comprising: the oligonucleotide composition of any preceding Illustrative embodiment; and at least one TaqMan probe.

[0127] Illustrative embodiment 27. A kit for detecting a target nucleic acid, the kit comprising: the oligonucleotide composition of any preceding Illustrative embodiment; a first detection reagent comprising a miRNA-specific forward primer; a second detection reagent comprising a reverse primer specific for a portion of the internal sequence of the oligonucleotide composition; and at least one TaqMan probe.

[0128] Therefore, in accordance with the present disclosure, there have been provided reagents and compositions, as well as kits containing same and methods of use thereof, that fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth herein above, it is evident that many alternatives, modifications, and variations will be apparent to those of ordinary skill in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

What is claimed is:

1. An oligonucleotide composition for detection of a target nucleotide sequence, the oligonucleotide composition comprising: an internal sequence comprising one of SEQ ID NOS:6-7 and 54-57, wherein at least a portion of the internal sequence forms a hairpin, and wherein the internal sequence has a 5' end and a 3' end; a first arm comprising a sequence of 6-11 nucleotides, wherein the first arm is attached to the 5' end of the internal sequence, and wherein at least a portion of the sequence of the first arm is complementary to a first portion of the target nucleotide sequence; and a second arm comprising a sequence of 4-10 nucleotides, wherein the second arm is attached to the 3' end of the internal sequence, and wherein at least a portion of the sequence of the second arm is complementary to a second portion of the target nucleotide sequence; and wherein the first and second portions of the target nucleotide sequence to which the first and second arms bind do not overlap.

2. The oligonucleotide of claim 1, wherein the internal sequence comprises SEQ ID NO:54.

3. The oligonucleotide of claim 1, wherein the internal sequence comprises SEQ ID NO:55.

4. The oligonucleotide of claim 1, wherein the internal sequence comprises SEQ ID NO:56.

5. The oligonucleotide of claim 1, wherein the internal sequence comprises SEQ ID NO:57.The oligonucleotide of claim 1, wherein the first portion of the target nucleotide sequence is an internal portion of the target nucleotide sequence.

7. The oligonucleotide of claim 1, wherein the second portion of the target nucleotide sequence contains a 5' end of the target nucleotide sequence.

8. The oligonucleotide composition of claim 1, represented by SEQ ID NO:58.

9. The oligonucleotide composition of claim 1, represented by SEQ ID NO:59.

10. The oligonucleotide composition of claim 1, represented by SEQ ID NQ:60.

11. The oligonucleotide composition of claim 1, represented by SEQ ID NO:61.

12. The oligonucleotide composition of claim 1, wherein the target nucleotide sequence is a miRNA.

13. The oligonucleotide composition of claim 12, wherein the miRNA is selected from the group consisting of miR-9-5p, miR-30a-5p, miR-34a-5p, miR-125b-5p, miR-15b-5p, miR-let7a- 5p, miR-15b-3p, and miR-146a-5p.

14. The oligonucleotide of claim 13, wherein the miRNA is miR-9-5p, at least a portion of the sequence of the first arm is selected from the group consisting of SEQ ID NQS:10-12, and at least a portion of the sequence of the second arm is represented by SEQ ID NO:13.

15. The oligonucleotide of claim 13, wherein the miRNA is miR-15b-5p, at least a portion of the sequence of the first arm is selected from the group consisting of SEQ ID NOS:15-17, and at least a portion of the sequence of the second arm is represented by SEQ ID NO:18.

16. The oligonucleotide of claim 13, wherein the miRNA is miR-30a-5p, at least a portion of the sequence of the first arm is selected from the group consisting of SEQ ID NQS:20-22, and at least a portion of the sequence of the second arm is represented by SEQ ID NO:23.

17. The oligonucleotide of claim 13, wherein the miRNA is miR-34a-5p, at least a portion of the sequence of the first arm is selected from the group consisting of SEQ ID NOS:25-27, and at least a portion of the sequence of the second arm is represented by SEQ ID NO:28.

18. The oligonucleotide of claim 13, wherein the miRNA is miR-125b-5p, at least a portion of the sequence of the first arm is selected from the group consisting of SEQ ID NQS:30-32, and at least a portion of the sequence of the second arm is represented by SEQ ID NO:33.

19. A method of detecting a target nucleotide sequence in a sample, the method comprising: combining the sample with the oligonucleotide composition of any of claims 1-18 to form a mixture and incubating the mixture under conditions wherein the first and second arms of the oligonucleotide bind cooperatively to the first and second portions of the target nucleotide sequence; adding reverse transcriptase to the mixture, wherein the reverse transcriptase binds a 3' end of the oligonucleotide and elongates the oligonucleotide based on the target nucleotide sequence to form a cDNA molecule; amplifying the cDNA molecule by qPCR using forward and reverse primers, wherein one of the primers is a target sequence-specific primer, and the other primer is specific for a portion of the internal sequence of the oligonucleotide composition; and detecting the presence of the target nucleotide sequence based on the amplification step.

20. The method of claim 19, wherein the sample is a biological sample.

21. The method of claim 19, wherein the target nucleotide sequence is a miRNA.

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