CN116710572A

CN116710572A - Ready-to-use nanopore platform for attomole DNA/RNA oligonucleotide detection using osmium tagged complementary probes

Info

Publication number: CN116710572A
Application number: CN202080107691.9A
Authority: CN
Inventors: 阿纳斯塔西娅·卡纳瓦利奥蒂
Original assignee: A NasitaxiyaKanawaliaodi
Current assignee: A NasitaxiyaKanawaliaodi
Priority date: 2020-10-05
Filing date: 2020-10-05
Publication date: 2023-09-05
Also published as: AR123690A1; KR20230080472A; EP4225911A1; TW202229561A; AU2020471747A9; AU2020471747A1; JP2024500005A; EP4225911A4; WO2022075965A1; BR112023006304A2; CA3198227A1

Abstract

Provided herein is a method for detecting the presence of a nucleic acid target molecule in a biological sample. In certain aspects, the methods comprise contacting a test sample comprising (i) a biological sample comprising a nucleic acid target molecule, and (ii) an osmium-forming single-stranded oligonucleotide probe comprising at least one nucleic acid sequence that is complementary to substituted or unsubstituted osmium tetroxide (OsO ₄ ) -pyrimidine residues covalently bonded to 2,2' -bipyridine groups (OsBp groups).

Description

Ready-to-use nanopore platform for attomole DNA/RNA oligonucleotide detection using osmium tagged complementary probes

Electronically submitted reference to sequence Listing

The contents of the electronically submitted sequence listing in the form of an ASCII text file (name 68812_199738_ST25.Txt; size: 6755 bytes; creation date: 10/2/2020) submitted with the present application are incorporated herein by reference in their entirety. Statement regarding federally sponsored research and development

The present application was completed with U.S. government support under the NIH/NHGRI SBIR program awarded foundation number R43HG 010841. The united states government has certain rights in this application.

Background

Blood or other body fluid samples known as liquid biopsies (Bronkhorst, a.j., ungerer, v. and holdenrier, s. (2019); vidal, j., taus, a. And montabout, c. (2020);O.、o., szemes, t, and Nagy, b. (2018); oellerich, m., schutz, e., beck, j. And Walson, p.d. (2019); stewart, c.m. and Tsui, d. (2018); giannopoulou, l., kasimir-Bauer, s. and liannibou, e.s. (2018); saryl, u., srivasta va, a. And Abbosh, p.h. (2019); finotti, a. Et al (2018); valpione, s. And Campana, l. (2019); and Kwapisz d. (2017)) contains relevant information about the health status of the individual, disease progression, whether the individual is disease free after surgery and even whether a certain treatment strategy seems promising (Vidal, j., taus, a. And montabout, c. (2020)). Liquid biopsies are procedures that are far less invasive than surgical/tumor biopsies. The body fluid contains cell free DNA (cfDNA), some of which may originate from a tumor (circulating tumor DNA; ctDNA). In 2016, the U.S. food and drug administration (US Food and Drug Administration) approved the first liquid biopsy test for EGFR-activated mutations in non-small cell lung cancer patients as a concomitant diagnostic test to achieve therapy selection (Kwapisz d. (2017)). It is believed that the cell free DNA of diseased individuals is fragmented and shorter than healthy individuals O., bir, o., szemes, t, and Nagy, b. (2018)). In addition to DNA, body fluids contain transferred RNA derived fragments and non-coding RNA oligonucleotides in the range of 20 to 300 nucleotides (nt) (Kim HK, yeom JH, kay MA. (2020); poller W et al (2018); mitchell, P. Et al (2008); aggarwal, V.; priyanka, K. And Tuli, H.S. (2020); meseme, D.; drak Alsibai, K.; nicolas, A.; bieche, I. And Morellon, (2015)). Wherein there is a group of single stranded (ss) RNAs of 17 to 25nt long, called microRNAsRNA or miRNA. They were found 20 years ago and proved to be tiny modulators that control posttranscriptional expression of proteins (Ambros, v. (2001); bartel, d.p. (2004)). mirnas are highly conserved and unexpectedly stable in body fluids (Mitchell, p. Et al (2008); mall, c., docket, d.m., durbin-Johnson, b. And Weiss, r.h. (2013)). Currently, over 2,300 human mirnas are known (Alles j. Et al (2019)), which are the subject of over 100,000 scientific publications. Upregulation or downregulation of mirnas is associated with a variety of human diseases including cancer, heart disease, kidney disease, obesity, diabetes, etc. (Bartel, d.p. (2004)); mirnas have been proposed as biomarkers (Farazi, t.a., hoell, j.i., morozov, p. and Tuschl, t. (2013); poibriny, i.p. (2017)) and potential therapeutic agents (rupamoole, r. and Slack, f.j. (2017)) in personalized medicine. Body fluids contain traces of cfDNA, ctDNA, fragmented coding RNAs (Kim HK, yom JH, kay MA. (2020)), non-coding RNAs (beller W et al (2018)) and mirnas that require simple, validated and highly sensitive assays to test (Finotti, (2018), valpione, s. And Campana, l. (2019), raabe, c., tang t., brosius j. And Rozhdestvensky, t. (2014)). Current techniques for "small RNA" identification and quantification include microarrays, NGS sequencing (Ion Torrent or Illumina (small RNA-seq)), and qRT-PCR based methods that have been largely successful to date (Ferracin, m. And Negrini, m. (2018); valihrach, l., androvic, p. And Kubista, m. (2020); gines, g., menezes, r., xiao, w., rondelz, y., taly, v. (2020)). However, these techniques require a large amount of infrastructure and skilled personnel, are not suitable for on-the-fly testing, and are never possible for home testing.

Nanopore-based technology using solid or protein nanopores has proliferated over the past 30 years (Kasianowicz, j.j., brandin, e., brandon, d. And Deamer, d.w. (1996), butler, t.z., gundlach, j.h., and Troll, m. (2007), maglia, g., heron, a.j., stoddart, d., japan, japanng, d., and bayey, h. (2010), haque, f., li, j., wu, h.c., liang, x.j., and Guo, P.S (2013), fuller c.w., et al (2016), laszlo, a.h., et al (2014), oxford Nanopore Technologies, nanotech.com, under Resources/Publications). 2014, oxford Nanopore Technologies (ONT)) The first portable nanopore device was proposed and commercialized, and DNA and RNA can be sequenced almost anywhere, as long as there is a computer and internet (Oxford Nanopore Technologies website: nanoporetech.com under Resources/Publications). The ONT technology is based on CsGg protein nanopores (Cao, b. Et al (2014)) with diameters of less than 2nm, inserted into a planar lipid bilayer membrane separating two electrolyte filled compartments (fig. 1A). Applying a voltage across the two compartments results in a constant current of electrolyte ions (I _o ) Recorded as a function of time (i-t). Single molecule passing through the pore I _o Reduced to a lower level of residual ion current (I _r ). This is recorded as having (I) _r ) And "event" of residence time (τ) (fig. 1B). Currently, ONT platforms are exclusively used for DNA/RNA sequencing (Oxford Nanopore Technologies website: nanoporetech. Com under Resources/Publications), while comparable nanopore platforms have been successfully used for single molecule analysis (Chen, X., wang, L. And Lou, J. (2020); chaudhary, V.; janga, S. And Yadav, N.R.; 2018); wanunun M, dadosh T, ray V, jin J, mcReynolds L),M. (2010); gu, l.q. and Wang y. (2013); arata, h., hosokawa, k, and Maeda, m. (2014); henley, r.y., vazquez-Pagan, a.g., johnson, m., kanavarioti, a. And wannu, m. (2015); xi, d. et al (2016); zahid, o.k., wang, f., ruzicka, j.a., taylor, e.w., and Hall, a.r. (2016); ding, Y, and Kanavaloti, A. (2016); tian, k., shi, r., gu, a., pennella, m., and Gu, l.q. (2017); zhang, y., rana, a., stratton, y., czyzyk-Krzeska, m.f., and esfandiiri, l. (2017); huang, g., willems, k, soskine, m., wloka, c, and Maglia, g. (2017); galenkamp, n.s., soskine, m., hermans, j., wloka, c., and Maglia, g. (2018); sultan m., kanovarioti, a. (2019); cao, C.et al (2019); hao, w., haoran t., cheng y, and Yongxin, l. (2019)).

The ONT provides a portable nanopore device carrying two types of flow channels; minion with 512 channels and florgle with 126 channels, all monitored simultaneously (Oxford Nanopore Technologies website: nanoporetech.com under Resources/Publications). ONT has generalized these devices for direct sequencing of DNA and RNA of a minimum length of 200 nucleotides (nt) (Oxford Nanopore Technologies website: nanoporetech.com under Resources/Publications). However, attempts to sequence RNAs shorter than 200nt according to ONT protocols have not been successful to date (Workman, RE et al (2019)), lacking circularization and rolling circle amplification to produce long DNA with 100nt repeats (Wilson, b.d., eisenstein, m.and Soh, h.t. (2019)). Most of the DNA/RNA available in biological fluids is fragmented and the length is estimated to be within 200bp @O., bir, o., szemes, t, and Nagy, b. (2018)) and mirnas are very short (Ambros, v. (2001)), and thus are not suitable for direct ONT sequencing protocols. Although several other experimental nanopore platforms have been successfully used for miRNA profiling, they have not proven suitable for commercial use. Thus, there remains a need for a technology that is readily accessible, readily available, relatively inexpensive, and does not require any special skills or infrastructure.

Disclosure of Invention

Provided herein is a method for detecting the presence of a nucleic acid target molecule in a biological sample. In certain aspects, the method comprises the steps of: (a) Contacting a test sample comprising (i) a biological sample comprising a nucleic acid target molecule, and (ii) an osmium (osmylated) single-stranded oligonucleotide probe comprising at least one pyrimidine residue covalently bonded to a substituted or unsubstituted osmium tetroxide (OsO 4) -2,2' -bipyridyl group (OsBp group), wherein the sequence of the probe is at least partially complementary to the sequence of the nucleic acid target molecule to allow formation of a hybridized probe/target complex; (b) Detecting the number of events in the test sample in which unhybridized osmium probe passes through the nanopore using the nanopore device; and (c) (i) comparing the number of events detected in the test sample with the number of corresponding probe sample events in which non-hybridized osmium probes pass through the nanopore in the absence of nucleic acid targets, wherein a decrease in the number of events detected in the test sample relative to the number of probe sample events is indicative of formation of hybridized probe/target complexes in step (a) and the presence of nucleic acid target molecules in the test sample; (c) (ii) comparing the number of events detected in the test sample with noise of a corresponding baseline sample that does not contain any osmium probes, wherein a lack of an increase in the number of events detected in the test sample relative to noise of the baseline sample is indicative of the formation of hybridized probe/target complexes in step (a) and the presence of nucleic acid molecules in the test sample; and/or (c) (iii) comparing the number of events detected in the test sample with the number of corresponding control sample events in which unhybridized osmium probes pass through the nanopore in the presence of a known amount of nucleic acid target molecules, wherein a decrease in the number of events detected in the test sample relative to the number of control sample events indicates an increase in formation of hybridized probe/target complex in step (a) and a greater amount of nucleic acid target molecules in the test sample than in the control sample, or wherein an increase in the number of events detected in the test sample relative to the number of control sample events using the same amount of probes indicates more unhybridized probes and thus indicates a lower amount of nucleic acid target molecules in the test sample as compared to the control sample. In certain aspects, at least one osmium pyrimidine is a thymine residue (T).

Furthermore, certain aspects provide a kit comprising an osmium probe of the present disclosure and a control nucleic acid target molecule that can hybridize to the probe, and the use of such probe for detecting a nucleic acid target molecule using a nanopore device, wherein the nucleic acid target molecule is optionally ctDNA, cfDNA, miRNA or non-coding RNA, optionally wherein the non-coding RNA is less than about 300 bases in length.

Drawings

FIGS. 1A-D. (A) Schematic of nanopores within a planar bilayer lipid membrane separating two electrolyte-filled compartments. Application of a constant voltage to the flow cell directs ions through the nanopore, thereby producing a measurable ion current. (B) I-t trace obtained from voltage driven ion channel experiments, wherein constant current of electrolyte ions through the pores (I _o ) Interrupted by the passage of molecules. These molecules appear to have a residual ion current I _r And "event" of residence time τ. (C) OsBp labelling reaction: osO (o) ₄ And 2, 2-bipyridine (bipy) are low in association constant, but their mixtures add to the C5-C6 double bond of pyrimidine and form stable conjugates. The addition of OsBp produces chromophores that absorb in the 312nm range that the native nucleic acid does not absorb (see examples). (D) instantiation of the concept behind the proposed diagnostic test. ssDNA and ssRNA pass through the nanopore and exhibit little count because they pass faster than the relatively slow collection rate of the device; the ds nucleic acid is too large to pass through the nanopore. Although larger than ss native nucleic acid, osmium ss nucleic acids pass through the pores, but are slower than the collection rate of the device, and thus produce a large number of events. When an osmium nucleic acid (probe) is added to a sample containing its complementary nucleic acid (target), the probe and target form a hybrid. When the concentration of the target is equal to or higher than the concentration of the probe, the probe is hybridized. The probe is then blocked from passing through the hole and little or no event is observed. The large number of events that the probe generates while freely passing through the well demonstrates the absence of the target in the sample.

Fig. 2A, B. Voltage driven ion channel (nanopore) experiments using a florgle ONT device; the sample is in>In 90% ONT buffer. (A) the same Flingle flow cell was used for 1h at-200 mV: (i) 5. Mu.M probes T8 (RNA) and (ii) T8 (RNA) and d (CT) ₁₀ 5. Mu.M each. Notably, the two molecules are only partially complementary to each other. Counts (counts) of events were obtained using osbp_detect software to analyze and report raw fast-5 file data (kanovarioti, a. And Kang, a. Please see RNA (OsBp) event detection Python package in public coexistence repository: github. Com/kangaroo96/osbp_detect on the world wide web, and step installation instructions, please see github. Com/kangaroo96/osbp_detect/blob/master/instructions on the world wide web; see example). Plotting the count as I _r /I _o The sliding window size (bin size) is 0.05. (B) at-190 mV for 2h, the same Flingle flow cell was used: (i) 5. Mu.M probe dmiR122, (ii) a mixture of dmiR122 and miRNA122, each 5. Mu.M, and (iii) a mixture of miRNA122 and miRNA140 at-180 mV, each 10. Mu.M, usingDifferent flowle flowcells. Notably, dmiR122 carries 4 OsBp moieties and its sequence is fully complementary to miRNA 122. Data acquisition and analysis were as described in (a).

Fig. 3A-D. Alternative methods of testing hybridization between an osmium probe and a target. (A) Enzymatic extension of osmium primers using ssM mp18 DNA and DNA polymerase as templates; time points were obtained at 5, 10 and 20 min. No primer and M13rev (-48) were used as negative controls. All other osmium primers, except BJ1, showed enzymatic extension comparable to positive control M13fwd (6097). BJ1 does not extend due to the presence of T (OsBp) at the 3' end. (B) (C) and (D) from different sample analysis of overlapping HPLC patterns, wherein the sample in about 90% ONT buffer is about 5 u M. The same HPLC method B was used for all samples (see examples). Intact ss and ds oligonucleotides appear as peaks, whereas osmium oligonucleotides and hybrids with one osmium strand appear as broad peaks; the hybrids elute later than ss nucleic acids. Sample composition (B): complete BJ2, complete complement of primer M13for (-41). The HPLC profile of their equimolar mixture was consistent with hybridization. (C) sample composition: miRNA21, probe 21EXT carrying 8 dT (OsBp) moieties, and equimolar mixtures of both. The HPLC profile of the mixture was consistent with no hybridization due to the large number of single OsBp tags, 6 within the 22nt sequence, which could distort the helix of the probe and prevent ds formation. (D) The sample composition was almost the same as according to (B), except that in these samples BJ2 carried 6 dT (OsBp) moieties (having a broad shape and the first peak of absorbance at 312 nm). The HPLC profile of the mixture is consistent with hybridization due to the fact that most OsBp moieties are contiguous, such that the remainder of the sequence can still hybridize to the target.

Fig. 4A-D. The same samples tested by HPLC and nanopores; the samples were in >90% ont buffer. (A) Overlapping HPLC profiles of about 0.2 nanomolar samples in ONT buffer (i) probe BJ1, (ii) target here shown at much higher loadings, and (iii) a mixture of about 0.2 nanomolar probe and target in about 30% excess compared to probe (hybrid). The HPLC profile of the mixture was consistent with hybridization. (C) an overlapping HPLC profile of: (i) About 0.2 nanomolar loading of probe BJ2 TA (OMe) and (ii) an approximately equimolar mixture thereof with the complementary target, complementary primer M13for (-41). The target was identified as a small peak, 5% excess compared to the probe. The HPLC profile of the mixture (hybrid) was consistent with ds formation. HPLC profiles of these two samples are shown at 272 and 312nm (see examples). (B) And (D) a nanopore experiment performed for up to one hour using the same sample as in (a) and (C), respectively, using a min flow cell; (B) The samples in (D) were used as they are, whereas the samples in (D) were used after 1000-fold or 100-fold dilution in ONT buffer. (B) Probe BJ1 was tested at-180 mV (dashed line) and showed little count. Without sample addition, the voltage was raised to-220 mV and additional nanopore experiments were performed at-220 mV (solid line), counting over 100,000. The hybrid samples showed very few counts (solid lines, each near the x-axis, tested at-190 mV for 1 h) compared to the counts obtained using BJ 1. (D) nanopore experiments using the same flow cell: (i) control/buffer test (0.75 h at-180 mV), (ii) probe BJ2 TA (OMe) at a loading of 0.38 picomolar (pmole) (2 h at-210 mV), (iii) equimolar mixture of probe and target, each at 3.8 picomolar loading (1 h at-210 mV). For (B) and (D), the observation that the counts of the hybrid samples were lower compared to the probe samples suggests target identification, which is consistent with HPLC results. Nanopore experiments using BJ2 TA (OMe) probes at 0.38 picomolar loadings showed detectability of the probes at sub-picomolar levels. (d) Experiments using hybrid samples showed that the hybrid persisted under the experimental conditions of duration and applied voltage. Data acquisition and analysis were as described with respect to fig. 2A.

Fig. 5A-D. Nanopore experiments for advanced probe design and probe detection were tested in 15% human serum-85% ONT buffer. (A) Three consecutive nanopore experiments were performed using the same flow cell. (i) buffer test at-180 mV for 0.75h, (ii) probe 2XdmiR122, test at-180 mV for 1.5h (dashed line), and (iii) without adding sample, the voltage was raised to-220 mV and the experiment was run for 1.5h. This set of experiments provided conclusive evidence that, at least, the probe did not pass through the ONT nanopore at-180 mV, even during experiments up to 1.5h. In addition, it illustrates that the probe passes through the well at an applied voltage of-220 mV and produces a high detectable event count. (B) Four consecutive nanopore experiments were performed using the same but previously used flow cell carrying only about half, i.e. 250, of the working nanopores. (i) buffer test at-180 mV, (ii) probe 122EXT at a loading 3-fold less than the conventional loading of 5 μm (corresponding to 0.38 nanomolar), test at-180 mV (dashed line), (iii) no sample added, voltage raised to-220 mV, and (iv) new sample with the same loading of probe 122EXT as in (ii), but prepared in 15% human serum-85% ONT buffer. (C) HPLC profile of: (i) intact miRNA122, (ii) probe 2XdmiR122 at 0.5 μm at a concentration 10-fold lower than typical 5 μm sample concentrations, and (iii) a mixture (hybrid) of probes to targets=1:2 also at a probe concentration of 0.5 μm. All three samples were in >90% ONT buffer and monitored at 260nm using HPLC method B (see examples). The latter two samples were used as received for the nanopore experiment shown in fig. 5D. (D) continuous nanopore experiments on the same flowcell. (i) buffer test, -1 h at 220mV, (ii) probe 2XdmiR122, -3 h at 220mV, and (iii) hybrid sample with excess miRNA122 compared to probe, test 1h at-220 mV. This experiment demonstrates the sensitivity of 2XdmiR122 detection, albeit through up to 3h of the experiment, and demonstrates that hybridization to the target results in a severe reduction in counts, i.e., silencing. Data acquisition and analysis of nanopore experiments are as described with respect to fig. 2A.

Fig. 6A, B. Targeting miRNA21 in complex mixtures. (A) HPLC profile of two samples analyzed using HPLC method B (see examples): (i) Probe dmiR21 (OMe) at a 0.15 nanomolar loading and (ii) a 1:2 mixture of this probe and miRNA21 at a 0.30 nanomolar probe loading. The HPLC profile of the probe shows two peaks, the larger peak eluting after the smaller peak. This profile is consistent with a defined value of an average of 2.85 OsBp moieties per molecule, meaning that the preparation includes molecules with 2 OsBp tags and molecules with 3 OsBp tags. HPLC profile of a 1:2 mixture of probe and target shows a single rather sharp peak, eluting after a broad and complex peak. Spikes have been shown to correspond to excessive miRNA21 targets. We attribute the broad complex peaks to the results of multiple hybridosomes, i.e., one target and multiple probes, all complementary to the target, but each of them carries an OsBp moiety at a different nucleobase. It is reasonable to think that chromatography resolved these hybrids because it resolved the topoisomer with such a short osmium oligonucleotide (Kanavarioti, a. (2016)) (HPLC method B in the examples). The observations of different HPLC profiles between probe and mixture samples were consistent with hybridization. (B) Four nanopore min experiments, two of which used the exact samples analyzed by HPLC in (a): (i) Probe dmiR21 (OMe), tested at-180 mV for 2h, showing more than 100,000 events, (ii) 1:2 mixture of this probe with miRNA21, tested at-180 mV for 1h, showing negligible counts (hybrids). Two additional experiments tested the effect of excess non-target RNA on translocation properties of probe dmiR21 (OMe) and its hybrids with miRNA21. The excess of non-target RNA was 10-fold higher in load than the probe and consisted of equimolar amounts of miRNA140 and 100nt long RNA. Nanopore experiments performed in the presence of excess non-target RNA (iii) hybridized to miRNA21, -180mV for 1h, and (iv) probe dmiR21 (OMe), -200mV for 2h. According to the ONT scheme, a higher voltage is used here to compensate for the number of hours that the flow cell has been operating. No effect, if any, of excess non-target RNA on the hybrid was detected, as the count was too low. The effect of excess non-target RNA on the probe appears as profile shift (profile shift) and the count is reduced by a factor of 2, although this reduction may be due to a reduction in the number of working nanopores by a factor of about 2 as well. Notably, probe dmiR21 (OMe) translocates efficiently at an applied voltage of-180 mV, as it does not contain any adjacent osmium-forming dT. Data acquisition and analysis were as described with respect to fig. 2A.

Fig. 7A-D. Nanopore experiments targeting miRNA140 or miRNA21 at a single digit attomole (attomole) loading. (A) Continuous nanopore experiments using the same flow cell: (i) Buffer test, -1 h at 210mV, (ii) 47 femtomole (fmole) loadingIs set at about 210mV for 1.5h. (B) Continuous nanopore experiments using the same flow cell used with less than 200 working nanopores: (i) buffer test, -1 h at 210mV, (ii) probe 140EXT (mU) at 3.5 attomole (amole) loading, -1.5 h at 210mV, and (iii) equimolar mixture of 140EXT (mU) and miRNA140, each at 3.5 attomole loading, -1.5 h (hybrid) at 210 mV. (C) HPLC profile of sample with equimolar concentrations of probe 21EXT (mU) and target miRNA21 using HPLC method B (example). The appearance of a single peak was consistent with hybridization. The samples were monitored at two different wavelengths to show absorbance at 312nm due to the presence of the osmium probe. (D) continuous nanopore experiments using the same flow cell; the sample was first tested by HPLC and then the probe and hybrid were diluted 10 with ONT buffer, respectively ⁹ Or 3x10 ⁸ Multiple (see examples). (i) buffer testing, (ii) probe 21EX (mU) at 0.9 attomoles, and (iii) 1:1 mixture (hybrid) of probe 21EXT (mU) and miRNA21, each at 2.8 attomoles loading. The hybrids showed significantly fewer counts compared to the probes. Data acquisition and analysis of nanopore experiments are as described with respect to fig. 2A.

Fig. 8. Samples of the tsv file were obtained by running osbp_detect software on the fast-5 file. Left, sample probe T8 (RNA); right, sample d (CT) ₁₀ T8 (RNA) =1:1 mixture, both in about 90% ONT buffer (see fig. 2A for experimental conditions).

Fig. 9. I-t recordings from two nanopore experiments are in the range of 15 to 60 s. Top, probe T8 (RNA), i-T recordings from two different channels. Bottom, d (CT) ₁₀ T8 (RNA) =1:1 mixture, i-T recordings from the same two channels as the top. The vertical line intersecting the x-axis (=0pa) is an instrument-generated line obtained by voltage inversion, not an event. The top record shows multiple depth events and the bottom record shows a small number of shallow events. Shallow events are attributed to molecules that collide at the opening of the pore without passing through the pore, and in selecting "all I _r /I _o <0.6 "they are not counted (see fig. 8 and examples).

Fig. 10A-D. HPLC profile of each component and 1:1 mixture thereof in sample solvent about 90% ONT buffer. All HPLC patterns were shown at 260nm, except for osmium probes and hybrids shown at 272nm and 312nm in the upper right hand corner pattern. The HPLC profile of the mixture demonstrates hybridization, wherein the main peak elutes after each component. (A) Shows hybridization of two intact oligonucleotides BJ1 and their complementary intact oligonucleotides (complement primer M13for (-20)). Comparable results for another pair of intact oligonucleotides are shown in FIG. 3B. (B) Shows hybridization of probe BJ1 with 5 OsBps in 30nt with its complementary intact oligonucleotide (complement primer M13for (-20)). (C) Shows only partial hybridization of intact BJ1 with its complementary osmium complement primer M13for (-20) of 11 OsBps in 35 nt. (D) Shows hybridization of intact BJ2 with its complementary osmium complement primer M13for (-41) of 6 OsBps in 35 nt. Analysis was performed using HPLC method B (see examples).

Fig. 11. Nanopore experiments using probes BJ2 and BJ4 showed few events at-180 mV and a large number of events at-220 mV. Probes BJ2 and BJ4 tested at-180 mV (dashed line) showed negligible count numbers. Without the addition of new samples, the voltage was raised to-220 mV and the probes were tested at-220 mV (solid line) and a number of events were detected. Both probe samples were used at a loading of 0.2 nanomolar. Experiments were performed on the same flow cell in the order BJ 2-180 mV, BJ 2-220 mV, BJ 4-180 mV and BJ 4-220 mV; the duration of each experiment was 1h. Data acquisition and analysis were as described with respect to fig. 2A. The large difference in counts at different applied voltages clearly shows that these and other similarly designed probes do not pass through proprietary CsGg nanopores at lower voltages and require high applied voltages of about-220 mV to translocate.

Fig. 12. miRNA21 or miRNA21-A using probe 21EXT (8T (OsBp), see sequences in Table 1) ₁₅ HPLC profile of the 1:1 mixture (non-hybrid). The sample was in about 90% ONT buffer as sample solvent. HPLC profile (example) was obtained using HPLC method B. The HPLC profile of the mixture sample closely matches the sum of the HPLC profiles of the two components, providing evidence that hybridization was not detected in both cases . The two cases differ in that due to the added A ₁₅ Tail, miRNA21-a compared to miRNA21 ₁₅ Is eluted a few minutes later.

Fig. 13. The effect of applied voltage on counts observed at-180 mV, -200mV, -220mV using 10. Mu.M each of the mixture of intact miRNA122 and miRNA 140. The data shown at-180 mV are identical to those in FIG. 2B (Flingle), but are normalized by multiplying by 10, because there are about 10 times more working channels of MinION compared to Flingle. The increase in applied voltage slightly reduces the count of events, consistent with faster translocation and reduced detectability. 10. Mu.M miRNA21-A at-220 mV ₁₅ Experiments performed showed counts comparable to the combination of miRNA122 and miRNA140, but with no a ₁₅ Tail mirnas have different profiles compared to one another. The effect of voltage on intact RNA is in sharp contrast to the effect of voltage on most probes tested in this study. No detectable count difference was observed between-200 and-220 mV in the control/buffer.

Fig. 14. Good linear correlation of the number of osmium pyrimidine in an oligonucleotide lacking T as a function of the number of U in the sequence was obtained. The correlation does not seem to be severely dependent on whether the sequence is DNA, RNA, or carries a 2' -OMe group on all or part of the bases (see Table 1). The linear correlation can be used to estimate the number of osmium pyrimidine obtained according to scheme c for any given sequence. The linear correlation was due to the observation that deoxyuridine (dU) was osmium-formed 4.7 times faster than deoxycytidine (dC) (Ding, y. And Kanavarioti, a. (2016)), and the conditions of scheme c, which produced only a small fraction of osmium-formed pyrimidine, not nearly 100% of the osmium-formed oligonucleotides.

Fig. 15. HPLC profiles of four complete M13 primers and their corresponding T-osmium derivatives were obtained using HPLC method a (see example). The oligonucleotides were analyzed using water as sample solvent. T-osmium formation of these oligonucleotides was performed using protocol o (see example). The reason that osmium oligonucleotides appear as multiple peaks is because addition of OsBp to the top or bottom of a C5-C6 double bond results in a topoisomerase that is resolved by the chromatography method.

Fig. 16. HPLC profile of four complete BJ1-4 and their corresponding T-osmium derivatives obtained using HPLC method a. The oligonucleotides were analyzed using water as sample solvent. T-osmium formation of these oligonucleotides was performed using protocol o (see example).

Fig. 17. 2 complete complements of primer M13for (-20) and primer M13for (-41) shown at 260nm, and HPLC profiles of their corresponding T-osmium derivatives shown at 272nm and 312nm were obtained using HPLC method B (see example). The oligonucleotides were analyzed using water as sample solvent. Briefly, HPLC method B used a DNA PacPA200 HPLC column from ThermoFisher Scientific, configured as 2x250mm, flow rate of 0.45mL/min and column oven of 15 ℃. The solvent is aqueous solution pH8.0.+ -. 0.2 Mobile Phase A (MPA) and Mobile Phase B (MPB) with 25mM TRIS.HCL buffer; MPB is 1.5M NaCl. Initial conditions were 90% MPA-10% MPB and gradient from 10% to 50% MPB over 20 min. The total analysis time, including column equilibration, was 30min. T-osmium formation of these oligonucleotides was performed using protocol o (see experimental section). The right panel shows atypical but confirmed results, i.e., osmium conjugates eluted later than the parent intact nucleic acid.

Fig. 18A-D. HPLC profiles of BJ2 TA (OMe) (a) and BJ2 AT (OMe) (B) and BJ1EXT (mU) (C) and BJ2EXT (mU) (D) shown AT 260nm and their corresponding T-osmium derivatives shown AT 272nm and 312nm (sequences in table 1). HPLC profile was obtained using HPLC method B (see fig. 17 and examples). The material uses water as a sample solvent. Nanopore experiments with probe BJ2 TA (OMe) showed excellent translocation properties with high numbers at relatively low probe loading (fig. 4D).

Fig. 19A-D. Intact miRNA21, miRNA122, miRNA140 and miRNA21-A shown at 260nm ₁₅ (these are-5 p sequences) and HPLC profiles of the corresponding partially osmium derivatives; different materials were analyzed at different sample loadings. Osmium protocol o for miRNA21-A ₁₅ And scheme c was used for the other 3 mirnas (see table 1 and examples). HPLC method A for analysis of miRNA21-A ₁₅ And HPLC method B was used to analyze the other 3 mirnas (see examples). The material uses water as a sample solvent.

Fig. 20A, B. (A) HPLC profile of intact dmiR21 at 260nm and T-osmium formation at 272nm and 312 nm. (B) HPLC profile of intact 21EXT and its T-osmium product. Osmium formation was performed using protocol o (40 min with 2.63mM OsBp), an early process in which OsO was added ₄ Bipy was then dissolved (see examples). The material in water was used for analysis and analyzed using HPLC method B (see examples).

Fig. 21A-C. (A) HPLC profiles of osmium formation products of dmiR21 (OMe) obtained using scheme b (30 min with 2.63mM OsBp) and scheme c (30 min with 3.94mM OsBp). A third protocol (d) incubated for 30min was used to osmium the probe for nanopore experiments using 5.25mM OsBp (fig. 6B). This is necessary because dmiR21 (OMe) does not contain T and the use of schemes b and c results in very low osmium levels and reduced detectability. (B) Probe integrity 21EXT (mU) and osmium products under two different protocols; prep1, protocol a was used with 2.63mM OsBp for 45min, and prep2, protocol b was used with 2.63mM OsBp for 30min. (C) Fig. 7C was repeated in order to compare the HPLC profile of the hybrid with that of the individual probes (B, above) and to find that they were different. Analysis was performed using HPLC method B (see examples).

Fig. 22A-D. The complete miRNA122 probe shows HPLC profiles at 260nm and its corresponding T-osmium derivative (actual probe) at 272 and 312 nm. Osmium protocol o was used to osmium dmiR122, 2XdmiR122, and 122EXT. dmiR122 (OMe) did not have any T and was osmium-formed using either scheme b or c (see table 1 for sequences and schemes). The material was used for analysis in water and analyzed using HPLC method B (see examples).

Fig. 23A-C. The complete miRNA140 probe is shown at 260nm and its corresponding osmium derivative (actual probe) HPLC profile at 272nm and 312 nm. With respect to HPLC profiles of dmiR140 (A) and 2XdmiR140 (B), osmium protocol o was used, along with 2.63mM OsBp for 40min (early course, at the addition of OsO ₄ Bipy was not previously dissolved). (C) Probe integrity 140EXT (mU) and its osmium products obtained under two different protocols; prep2, protocol a, 45min with 2.63mM OsBp, and prep1, protocol b, and2.63mM OsBp together for 30min. The material in water was used for analysis and analyzed using HPLC method B (see examples).

Fig. 24A, B. (A) HPLC profiles of partially osmium 100nt RNA and 100nt RNA (OMe) obtained using osmium protocol c (see table 1 and examples). (B) T-osmium d (CT) obtained using scheme b ₁₀ Is a HPLC profile of (C). The material uses water as a sample solvent. HPLC method B was used for all samples, and HPLC profiles were shown at 272nm and 312 nm. Osmium protocols and HPLC methods can be found in the examples.

Fig. 25A, B. HPLC profile of partially osmium 22nt RNA. (a) a complementary miRNA21 and (B) a complementary miRNA122. Osmium protocol c was used with 3.94mM OsBp for 30min.

Fig. 26A, B. (A) Fig. 6A is repeated for direct comparison with the HPLC profile on the right. (B) HPLC profile of three samples in 15% serum-85% ONT buffer: miRNA140 was incubated for 2min prior to analysis and 100nt RNA was incubated for 30min prior to analysis. Longer incubation times are the reason why degradation of 100nt RNA appears to be more severe than degradation of miRNA 140. dmiR21 (OMe) (OsBp) in about 5% water-95% ONT buffer was a mixture (a) of mirnas 21=1:2 and the same mixture (B) in 15% serum-85% ONT buffer. HPLC profiles appeared comparable, indicating insignificant degradation of the hybridosome in 15% serum-85% ONT buffer. HPLC method B was used for these analyses (see examples).

Detailed Description

Terms defined directly below are defined in more detail by referring to the specification in its entirety. To the extent necessary to provide descriptive support, the subject matter and/or text of the appended claims is incorporated herein by reference in its entirety.

Definition of the definition

All readers of this written description will understand that the exemplary aspects and embodiments described and claimed herein may be suitably practiced in the absence of any recited features, elements or steps, either specifically disclosed or not specifically disclosed herein.

The term "a" or "an" entity refers to one or more of the entities/one or more; for example, "a probe" is understood to mean one or more/one or more "probes". Thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

As used herein, the term "and/or" should be taken to mean a specific disclosure of each of the specific features or components with or without the other. Thus, "and/or" such as "a and/or B" as used herein in the phrase is intended to include: "A and B"; "A or B"; "A" (alone); and "B" (alone). Similarly, "and/or" such as "A, B and/or C" as used in the phrase is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

It will be understood that whenever an aspect is described herein by the term "comprising," other similar aspects described as "consisting of … …" and/or "consisting essentially of … …" are also provided.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, unless otherwise indicated, "complementary" base pairs refer to A/T, A/U and G/C base pairing.

Numerical ranges include the numbers defining the ranges. Even when not explicitly identified by "and any range therebetween," etc., wherein a series of values, i.e., 1, 2, 3, or 4, are recited, the present disclosure specifically includes any range between the values, i.e., 1 to 3, 1 to 4, 2 to 4, etc.

The headings provided herein are not for ease of reference only and do not limit the various aspects or aspects of the disclosure, which may be obtained by reference to the specification in its entirety.

As used herein, the term "identity" to an amino acid sequence or nucleotide sequence disclosed herein, i.e., "percent identity" refers to a relationship between two or more amino acid sequences or between two or more nucleotide sequences. A sequence is said to be "identical" at a position in a sequence when that position is occupied by the same nucleobase or amino acid in the corresponding position in the compared sequences. The percentage of "sequence identity" is calculated by: the number of positions in the two sequences where the same nucleobase or amino acid occurs is determined to give the number of "identical" positions. The number of "identical" positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of "sequence identity". The percentage of "sequence identity" is determined by comparing the two optimally aligned sequences over a comparison window. For optimal alignment of sequences for comparison, the portion of the nucleotide or amino acid sequence in the comparison window may contain additions or deletions, known as gaps, while the reference sequence remains unchanged. The optimal alignment is such that: even with gaps, as many "identical" positions as possible are created between the reference sequence and the comparison sequence. The percentage of "sequence identity" between two sequences can be determined using, i.e., the program "BLAST" available from the national center for Biotechnology information (National Center for Biotechnology Information), and which combines the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for amino acid sequence comparison), which are based on the algorithms of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90 (12): 5873-5877, 1993).

As used herein, when referring to nucleic acid molecules, the term "complementary" is given its standard definition for complementary Watson-Crick (Watson-Crick) base pairing as understood in the art.

The term "nucleic acid" is a term well known in the art and is used herein to include DNA and RNA. Unless otherwise indicated, a "nucleic acid" molecule and a "polynucleotide" may be used interchangeably. The nucleic acid may comprise conventional phosphodiester bonds or non-conventional bonds (i.e., amide bonds, such as are found in Peptide Nucleic Acids (PNAs)). By "isolated" nucleic acid is meant a nucleic acid molecule that has been removed from its natural environment, such as a sample of genomic DNA obtained from a subject. Isolated polynucleotides or nucleic acids also include synthetically produced such molecules.

As used herein, when referring to an oligonucleotide, the term "intact" or "native" means that the oligonucleotide is not osmium-ized.

As used herein, a "biological sample" is a biological sample derived from a subject, such as a human, animal, plant, bacterium, virus, fungus, or other type of multicellular or unicellular life form. In certain aspects, the biological sample may be obtained directly from the subject, such as by drawing blood, collecting a urine sample, or tissue or liquid biopsy. In certain aspects, the biological sample may be obtained indirectly, such as from biological evidence collected at a crime scene. In certain aspects, the biological sample is a bodily fluid, such as blood, plasma, lymph, saliva, urine, amniotic fluid, spinal fluid, or the like. In certain aspects, the biological sample is a fluid sample having components derived from tissue or cells suspended, lysed (in solution), reconstituted, etc. in the fluid sample. "Complex mixture" refers to a sample that contains various components such as nucleic acids, proteins, carbohydrates, etc., and/or different nucleic acid molecules.

As used herein, an "event" or "count" is detected by: applying a voltage across the two compartments of the nanopore device results in a constant current of electrolyte ions (I _o ) It is recorded as a function of time (i-t). Single molecule passing through the pore I _o Reduced to a lower level of residual ion current (I _r ). This is recorded as having (I) _r ) And "event" of residence time (τ) (fig. 1B).

It has been found that portable nanopore devices from Oxford Nanopore Technologies (ONT) can be reused to detect DNA/RNA polynucleotides (targets) in complex mixtures by performing voltage-driven ion channel measurements. Detection and quantification of targets is achieved by using the unique complementary probes of the present disclosure. The probes were labeled with a bulky osmium tag (osmium tetroxide 2,2' -bipyridine) using a validated labeling technique in a manner that maintained strong hybridization between the probes and the targets. Untagged oligonucleotides pass through the nanopore relatively quickly compared to the collection rate of the device and exhibit event counts comparable to baseline. Python packets are detected, for example, by publicly available software OsBp_detect report counts (Kanavaloti, A. And Kang, A.. Please see RNA (OsBp) events in public coexistence store: https:// gitub. Com/kangaroo96/osbp_detect, and stepwise installation instructions, see herein: https:// gitub. Com/kangaroo 96/osbp_detect/blob/master/instructs. Md). Due to the presence of the bulky osmium tag, the osmium-tagged probe passes more slowly, resulting in multiple counts above baseline, and can even be detected in the single digit attomole (attomole) range. However, in the presence of the target, the probe is "silenced". Silencing is due to double-stranded complexes that are considered not to pass through the nanopore under the applied conditions for practical purposes of this disclosure. Thus, the disclosed ready-to-use platform can be tailored to diagnostic tests to meet the requirements of, for example, the on-the-fly detection and quantification of circulating tumor DNA (ctDNA), cell free DNA (cfDNA) fragmented RNA, and micrornas (mirnas) in body fluids.

Aspects of the present disclosure utilize selective labeling (also referred to herein as tagging) of nucleic acids in an attempt to enhance base-to-base discrimination using osmium tetroxide 2,2' -bipyridine (OsBp) as a label/tag (Ding, y. And kanovarioti, a. (2016); sultan m., kanovarioti, a. (2019); kanovarioti, a. (2015)). OsBp is not reactive with purines and does not cleave phosphodiester bonds in DNA or RNA. OsBp adds to the C5-C6 double bond of pyrimidine and forms two strong C-O bonds without cleavage of the pyrimidine ring (fig. 1C) (Chang, c.h., beer, m. and Marzilli, l.g. (1977); payek e. (1992); reske t.; surkus, a-e.; duwenbee, h. And Flechsig g. —u. (2009); kanavarioti, a. Et al (2012); kanavarioti, a.; 2016); debnath, t.k. And Okamoto, a.; (2018)). OsBp is 28-fold and 7.5-fold more reactive to thymidine (T) than deoxycytidine (dC) and deoxyuridine (dU), respectively (Ding, y. And Kanavarioti, a. (2016)). Labeling conditional protocols have been developed to selectively label T in the presence of other pyrimidines (Kanavarioti, a. Et al (2012)). Furthermore, the present inventors developed Capillary Electrophoresis (CE) and High Performance Liquid Chromatography (HPLC) methods to measure the degree of labeling in short and long DNA and RNA (Kan avarioti, a. Et al (2012); kanovarioti, a. (2016); see examples). Voltage-driven ion channel measurements were performed using SiN solid-state nanopores (Henley, R.Y., vazquez-Pagan, A.G., johnson, M., kanavaloti, A. And Wanuu, M. (2015)), alpha-hemolysin nanopores (Ding, Y. And Kanavaloti, A. (2016)) and CsGg nanopores in Minion (Sultan M., kanavaloti, A. (2019)), and demonstrated that all three platforms allowed translocation of osmium nucleic acids and clearly distinguished them from native nucleic acids. The distinction is shown to have a significantly lower I _r And longer events of τ, and may be increased, for example, by increasing the number and/or position of OsBp moieties of the labeled oligonucleotides. These features are used to pick, detect and count OsBp tagged oligonucleotides in a complex mixture of native DNA and RNA.

FIG. 1D illustrates the concept behind nanopore-based identification and quantification of target oligonucleotides in complex mixtures. Certain aspects of the disclosure are realized by custom-designed OsBp tagged oligonucleotides (probes) as described in detail elsewhere herein that are at least partially complementary to a nucleic acid target molecule sufficient to produce hybridized double-stranded complexes. The use of complementary oligonucleotides as probes has been demonstrated in several experimental nanopore platforms (Wanununu M, dadosh T, ray V, jin J, mcReynolds L, M. (2010); xi, d. et al (2016); zahid, o.k., wang, f., ruzicka, j.a., taylor, e.w., and Hall, a.r. (2016); tian, k., shi, r., gu, a., pennella, m., and Gu, l.q. (2017); hao, w., haoran t., cheng y, and Yongxin, l. (2019)). However, these platforms were based on the conjugation of probes to proteins (Wanunu M, dadosh T, ray V, jin J, mcReynolds L),M. (2010)), nanoparticles (Hao, w., haora t., cheng y. and Yongxin, l. (2019)), homopolymers (Xi, d. et al (2016)), or polypeptides (Tian, k., shi, r., gu, a., pennella, m. and Gu, l.q. (2017)). These areDetection in the platform relies on counting long blocks generated by collision and near "seizing" of the double-stranded hybrid complex at the entrance of the nanopore. None of these methods have achieved commercial availability, which has prevented their widespread use. In contrast to earlier methods based on hybrid detection (Wanunu M, dadosh T, ray V, jin J, mcReynolds L,)>M. (2010); xi, d. et al (2016); zahid, o.k., wang, f., ruzicka, j.a., taylor, e.w., and Hall, a.r. (2016); tian, k., shi, r., gu, a., pennella, m., and Gu, l.q. (2017); hao, w., haora t., cheng y, and Yongxin, l. (2019)), aspects of the disclosure detect translocation of the osmium probe facilitated by a relatively slow acquisition rate (e.g., without limitation, by a mineral (3.012 kHz sampling rate, equivalent to reporting 3 data points every 1 millisecond)) (Oxford NanoporeTechnologies website: nanoporetech.com under Resources/Publications). Although the slow sampling rate misses many, most, or all translocation events of the native DNA/RNA oligonucleotide, events corresponding to translocation of OsBp tagged probes are detected. In the absence of a nucleic acid target molecule (e.g., a complementary probe binding partner), the osmium probe passes through the nanopore and produces a detectable event. In the presence of the nucleic acid target molecule, the probe forms a hybridization complex (e.g., a 1:1 double-stranded hybrid) with the target. The hybridized molecules cannot pass through and not pass through the nanopore (fig. 1D). Thus, hybridized OsBp-tagged probes are "silenced". However, in certain aspects, the hybridization complex does not "plug" and prevent the passage of non-hybridized single stranded nucleic acid through the pore. This may be achieved, for example, by incorporating automatic reversal of voltage to protect the nanopore from such non-productive "blocking" events. Thus, to detect, test, and/or determine the presence or absence of a nucleic acid target molecule in a sample, an osmium probe is added to a particular sample that can be run on a nanopore platform or device by performing a voltage driven experiment. In certain aspects, the absence of a nucleic acid target molecule in the sample may be detected from detection of large size due to translocation of the labeled probe through the nanopore The quantitative events are interpreted. In certain aspects, the presence of a nucleic acid target molecule in a sample can be explained by the absence of events due to the formation of hybrids between the labeled probe and the target. Quantification of the target may be based on known concentrations of labeled probes and 1:1 hybrid formation.

The presence of a T (OsBp) moiety in the middle of the sequence is not a feature common to many potential ctDNA, miRNA or other such targets. Thus, in certain aspects of the probes of the present disclosure, one, some, or all T in the sequence is replaced with uridine (U), deoxyuridine (dU), or 2' -OMe-uridine (mU). Furthermore, in certain aspects, one, some or all of the bases are modified to 2' -OMe. In certain aspects, one or more adjacent T (OsBp) is added at the 3 'end or 5' end of the probe oligonucleotide. Also, in some aspects, one or more additional dabs are added at the 3 'end or 5' end of the probe oligonucleotide. Substitution of T with U, dU or mU reduces or eliminates the presence of OsBp within the complementary sequence that might interfere with hybridization to the nucleic acid target molecule.

RNA/DNA hybrids are known to be more stable than DNA/DNA hybrids. Thus, for example, in certain aspects, RNA-based probes can target DNA after the probes are added to a biological sample to be tested. In certain aspects, the DNA (e.g., dsDNA) is relatively short, e.g., less than 100, 90, 80, 70, 60, 50, 40, 30, 28, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides in length. In certain aspects, the methods comprise a denaturation step prior to hybridization of the RNA-based probes to the nucleic acid target molecules, e.g., to denature dsDNA target molecules and/or to remove any secondary structure of the probes. Compared to DNA-based probes, RNA probes exhibited a different nanopore profile, as shown in fig. 2A (I _r /I _o ) _max Corresponding to those in all other figures representing probes (I _r /I _o ) _max Indicated by the comparison of (a). This feature can lead to probe multiplexing (multiplexing) in order to test more than one target at a time. Similarly, probes lacking 3 adjacent T like dmiR21 (OMe) were observed to translocate at-180 mV, whereas probes having 3 adjacent T required-220 mV. Such can be utilizedDistinction, e.g., translocation differences between probes due to voltage, current, time, etc., and based on the amount and/or location of osmium of the probes, in order to multiplex the probes. Different nanopore profiles will reveal which probe is silenced. Using only one probe, the test can ultimately identify the presence/absence of a target by comparing the total event count of the individual probes to the total event count of the mixture of probes and unknown samples. For multiplexing tests, for example, counts may be plotted as histograms to determine which probe is missing. Thus, certain aspects of the present disclosure provide multiplexing to test more than one nucleic acid target molecule at a time, even within the same test sample.

Certain aspects of the present disclosure provide ion channel single molecule experiments performed using, for example, commercially available portable nanopore devices. The targets tested were DNA and RNA oligonucleotides and, in some aspects, exhibited a detection range of 9 orders of magnitude. This sensitivity is close to that of a single digit attomole target, for example from 11 μl of biological sample. These properties enable the detection and quantification of highly diluted samples such as ctDNA and miRNA present in body fluids.

Provided herein is a method for detecting the presence of a nucleic acid target molecule in a biological sample. One of ordinary skill in the art will recognize that for any of the methods described below, similar methods can also be used to detect/verify the absence of a nucleic acid target molecule in a biological sample if the criteria for detecting a nucleic acid target sample are not met. In certain aspects, the methods comprise contacting a test sample comprising (i) a biological sample comprising a nucleic acid target molecule, and (ii) an osmium-forming single-stranded oligonucleotide probe comprising at least one nucleic acid sequence that is complementary to substituted or unsubstituted osmium tetroxide (OsO ₄ ) -pyrimidine residues covalently bonded to 2,2' -bipyridine groups (OsBp groups). In certain aspects, the substitution occurs on the 2,2' -bipyridine of OsBp. In certain aspects, the test sample comprises a sample buffer in which the biological sample is diluted. The sample buffer composition may vary according to the type of nanopore device/system, the type of biological sample, etc., and may be for each caseAnd (5) determining. The biological sample is typically diluted such that at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% of the test sample volume is sample buffer and the remainder of the volume is the biological sample and/or the solution comprising the probe. However, one of ordinary skill in the art will recognize that the smaller the volume of biological sample used, the smaller the amount of nucleic acid target molecules that may be present, and thus the greater sensitivity is required. The osmium pyrimidine may be thymidine (T), cytidine (C), deoxycytidine (dC), deoxyuridine (dU), uridine (U) or derivatives thereof. In certain aspects, the at least one osmium pyrimidine residue is a thymidine residue (T). In certain aspects, the sequence of the probe is at least partially complementary to the sequence of the nucleic acid target molecule sufficient to allow formation of hybridized probe/target complexes. In certain aspects, at least a portion of the probe sequence is fully complementary to at least a segment of the nucleic acid target molecule sequence. The nanopore device is used to detect the number of events in a test sample in which unhybridized osmium probes pass through the nanopore. The number of events detected in the test sample can then be compared to some other value to determine or indirectly detect the presence or, in some aspect, absence of the nucleic acid target molecule in the test sample. In certain aspects, wherein the only potential source of nucleic acid target molecules in the test sample is due to the source of the biological sample, detecting nucleic acid target molecules in the test sample to detect nucleic acid target molecules in the biological sample. Unlike prior methods of detecting oligonucleotides on nanopore systems, the nanopore device/system of the present disclosure does not require conjugation of probes to proteins, nanoparticles, homopolymers, or polypeptides in order to detect the probes. Thus, in certain aspects, the probe is not conjugated to a protein, nanoparticle, homopolymer, or polypeptide. Furthermore, unlike existing nanopore detection methods, detection is not achieved by counting the long blocks generated by hybridized probe/target complexes that block the nanopore or by melting hybridized probe/target complexes in the nanopore.

(i) In certain aspects, the number of events detected in the test sample may be compared to a "corresponding probe sample event number". The number of corresponding probe sample events are those detected in the probe sample or should be detected theoretically for the probe sample, wherein non-hybridized osmium probes pass through the nanopore in the absence of nucleic acid targets. As explained and shown in more detail in the representative examples elsewhere herein, the presence of a nucleic acid target molecule in a target sample (such as from a biological sample or added as a control) results in hybridization to a complementary osmium probe, thus "silencing" the probe (preventing the probe from passing through the nanopore and resulting in a detectable event). Thus, a decrease in the number of events detected in the test sample relative to the number of events of the probe sample is indicative of the formation of hybridized probe/target complexes and thus the presence of nucleic acid target molecules in the test sample. Because unhybridized probes will pass through the nanopore and result in a detectable event, even in the presence of the nucleic acid target molecule, if not all probes are hybridized, in certain aspects the amount of probes should not greatly exceed or should not exceed the amount of nucleic acid target in the test sample. In certain aspects, the amount of probe should be about equal to or less than the amount of nucleic acid target in the test sample. In certain aspects, the hybridization strength between a target nucleic acid molecule and its complementary probe can also be taken into account. One of ordinary skill in the art can determine the approximate amount of nucleic acid target molecules in a particular type of sample based on available information and routine experimentation, and can further refine the amount of probes for best results. In certain aspects, the number of events is reduced by at least two, at least three, or at least four times, with confidence that the observed reduction is due to the presence of nucleic acid target molecules and probe/target hybrids in the test sample. The confidence level may vary, for example, from probe/target to probe/target, test conditions to test conditions, and nanopore device to nanopore device, and may be determined by routine experimentation for a given system. Thus, in certain aspects, the reduction in the number of events between those detected in the test sample and the corresponding number of probe sample events is at least two, at least three, or at least four times the reduction.

In certain aspects, the corresponding number of probe sample events is the number of events detected simultaneously in one or more probe samples when detecting the number of events in the test sample. Simultaneous detection refers to the detection of an event in a test sample and the detection of an event in a probe sample within the same general period of time, so that one of ordinary skill in the art would consider the detection to be performed as part of the same test or experiment, but not necessarily in parallel. In certain aspects, simultaneous detection of events in the test sample and the probe sample will use a common reagent, such as reagents from the same lot, to reduce variability. In certain aspects, reagents for simultaneous detection are provided together in a kit.

In certain aspects, the number of corresponding probe sample events is a predetermined value for a given amount of probes. The predetermined value of the number of probe sample events for a given amount of probes may be determined empirically by running a test on a probe sample with a known amount of probes and detecting the number of events generated, e.g., on certain types of nanopore devices and under certain conditions, e.g., current, voltage, time, buffer conditions, age and/or amount of use of the nanopore device, etc. This predetermined value may then be used for purposes of comparison with the number of events detected in various test samples under similar conditions. The predetermined value may be used for comparison with a test sample having the same or similar amount of probes, or the value may be extrapolated to a different amount of probes. For a given amount of probe, a predetermined value for the number of events corresponding to the probe sample may also be determined theoretically. It is theoretically determined that confirmation can be, but need not be, made by experimental observation.

In certain aspects, the number of osmium probe sample events in the probe sample is detected by a nanopore device, and then the osmium probe in the probe sample is combined with a biological sample to produce a test sample. The number of events in the test sample can then be detected by the nanopore device to compare with the number of osmium probe sample events detected.

(ii) In certain aspects, the number of events detected in the test sample can be compared to the "noise" of a corresponding baseline sample without any osmium probes. Those of ordinary skill in the art will appreciate that even in the absence of an osmium probe, and even for the sample buffer itself alone in the absence of any biological sample, the nanopore system will be indicative of a certain number of events, referred to herein as "noise. Those of ordinary skill in the art will also appreciate that such noise may be accounted for in various ways that do not limit the methods of the present invention (e.g., such as calibrating an instrument to zero out noise). As explained and shown in more detail in the representative examples elsewhere herein, the presence of a nucleic acid target molecule in a target or control sample (such as from a biological sample or added as a control) results in hybridization to a complementary osmium probe, thus "silencing" the probe (preventing the probe from passing through the nanopore and resulting in a detectable event). Thus, no increase in the number of events detected in the test sample relative to the noise of the corresponding baseline sample is indicative of the formation of hybridized probe/target complexes and thus the presence of nucleic acid molecules in the test sample. Because unhybridized probes will pass through the nanopore and result in a detectable event, even in the presence of the nucleic acid target molecule, if not all probes are hybridized, in certain aspects the amount of probes should not greatly exceed or should not exceed the amount of nucleic acid target in the test sample. In certain aspects, the amount of probe should be about equal to or less than the amount of nucleic acid target in the test sample. In certain aspects, the hybridization strength between a target nucleic acid molecule and its complementary probe can be taken into account. One of ordinary skill in the art can determine the approximate amount of nucleic acid target molecules in a particular type of sample based on available information and routine experimentation, and can further refine the amount of probes for best results. In certain aspects, a lack of increase in the number of events in the test sample relative to the number of events in the baseline sample means a less than two, less than three, or less than four times increase, with confidence that the observed decrease is due to the presence of nucleic acid target molecules and probe/target hybrids in the test sample. The confidence level may vary, for example, from probe/target to probe/target, test conditions to test conditions, and nanopore device to nanopore device, and may be determined by routine experimentation for a given system. Thus, in certain aspects, a lack of an increase in the number of events in the test sample relative to the number of events in the baseline sample means an increase of less than two, less than three, or less than four times.

In certain aspects, the noise of the respective baseline samples is determined simultaneously in one or more baseline samples when detecting the number of events in the test sample. Simultaneous detection refers to the detection of events in a test sample and the determination of noise in a baseline sample occurring as part of the same test or experiment, but not necessarily in parallel, within the same general time period, so that one of ordinary skill in the art would consider such detection to occur. In certain aspects, simultaneous detection of events in a test sample and determination of noise in a baseline sample will use a common reagent, such as a reagent from the same lot, to reduce variability. In certain aspects, reagents for simultaneous detection are provided together in a kit.

In certain aspects, the noise of the corresponding baseline sample is a predetermined value. For example, in certain aspects, it may be predetermined for a particular composition of the sample (e.g., type of sample buffer, type of biological sample, concentration of biological sample in the test sample, etc.). The predetermined value of noise in the baseline sample may be determined empirically by running a test on a sample of known composition and determining the amount of noise, e.g., on certain types of nanopore devices and under certain conditions, e.g., current, voltage, time, buffer conditions, age of the nanopore device, etc. This predetermined value may then be used for purposes of comparison with the number of events detected in various test samples under similar conditions. The predetermined value may be used for comparison with test samples having the same or similar composition, or the value may be extrapolated to different compositions, e.g., higher or lower concentrations of biological samples. A predetermined value of noise for a corresponding baseline sample of a certain composition may also be determined theoretically. It is theoretically determined that confirmation can be, but need not be, made by experimental observation.

In certain aspects, noise of a respective baseline sample of the nanopore device/system is determined, and then an osmium probe is added to the baseline sample, such as a baseline sample comprising a biological sample, to produce a test sample. The number of events in the test sample can then be detected by the nanopore device to compare with the noise amount of the baseline sample.

(iii) In certain instances, it may be useful to use a sample comprising a nucleic acid target molecule and a complementary osmium probe as a control, particularly, for example, where the amount of nucleic acid target molecule in the control sample is known. Thus, in certain aspects, the number of events detected in a test sample can be compared to a corresponding number of control sample events, wherein unhybridized osmium probes pass through the nanopore in the presence of a known amount of nucleic acid target molecules and/or a known amount of osmium probes. Consistent with the use of the probe samples and test samples described above, a decrease in the number of events detected in the test samples relative to the number of events in the corresponding control samples indicates the formation of hybridized probe/target complexes and the presence of a greater amount of nucleic acid target molecules in the test samples than in the control samples. The use of such control samples can be used to explore hybridization between a nucleic acid target molecule and its complementary probe, and can also be used to titrate and/or determine the optimal amount of probe usage for a given amount of nucleic acid target molecule, even in the absence of a biological sample. Such use can be used to develop a quantitative method for detecting the amount of a nucleic acid target molecule in a biological sample.

As noted above, the 2,2' -bipyridine in OsBp attached to the pyrimidine in the oligonucleotide probe may be substituted or unsubstituted. In certain aspects, it is substituted, e.g., with one or more methyl or ethyl groups.

In certain aspects, the nucleic acid target molecule may be a biomarker and/or genotype indicative of health, age, or phenotype associated with a particular disease or disease state. In certain aspects, the nucleic acid target is circulating tumor DNA (ctDNA), cell free DNA (cfDNA), miRNA, fragmented coding RNA, or non-coding RNA. In certain aspects, the non-coding RNA is less than about 300 bases in length. In certain aspects, the nucleic acid target molecule is a single stranded nucleic acid molecule. In certain aspects, the nucleic acid target molecule is present in the biological sample as a single stranded nucleic acid molecule. In certain aspects, as found in biological samples, the nucleic acid target molecule is a strand of a double-stranded nucleic acid molecule. Thus, in certain aspects, the methods comprise denaturing double-stranded nucleic acids and/or nucleic acids having a secondary structure in a test sample (including probes) to form single-stranded nucleic acid strands, such that single-stranded oligonucleotide probes can hybridize to single-stranded target molecules.

As explained in detail elsewhere herein, in certain aspects, the nanopore device allows voltage driven translocation of osmium and non-osmium single stranded nucleic acids, but prevents translocation of double stranded nucleic acids. Although the methods disclosed herein may be performed using commercially available nanopore devices, they are not limited to the type of nanopore device. In certain aspects, the nanopore device utilizes a nanopore having a minimum pore size of about 1.3nM to about 7.1 nM. In certain aspects, the nanopore device utilizes a Phi29, alpha-hemolysin, aerolysin (Aerolysin), mspA, csGg, PA63, clyA, fhuA, or SPP1 protein nanopore, or a bioengineered derivative thereof.

As explained elsewhere herein, the voltage will vary depending on factors such as the nanopore itself, design of the osmium probe, sample composition, age and/or amount of use of the nanopore device, and the like. In addition, different voltages can be applied to specifically drive certain nucleic acids through the nanopore but not others, such as driving non-osmium single-stranded nucleic acids through the pore before applying the voltages required to detect osmium probes or to differentiate between different designs of osmium probes, such as in multiplexed tests. In certain aspects, a voltage of about at least, or at least about-180 mV, -190mV, -200mV, -210mV, -220mV, -230mV, -240mV, or-250 mV is applied to determine the presence of the target. In certain aspects, a voltage of between any or any of about-180 mV, -190mV, -200mV, -210mV, -220mV, -230mV, or-240 mV and any or any of about-190 mV, -200mV, -210mV, -220mV, -230mV, or-240 mV, or-250 mV is applied to determine the presence of the target. In certain aspects, a voltage of less than or less than about-200 mV, -190mV, -180mV, -170mV, -160mV, or-150 mV is applied before the voltage is applied to determine the presence of the target. In certain aspects, a voltage of between any or any of about-200 mV, -190mV, -180mV, -170mV, or-160 mV and any or any of about 190mV, -180mV, -170mV, or-160 mV, -150mV is applied before the voltage is applied to determine the presence of the target. One of ordinary skill in the art will recognize that depending on the particular nanopore device and the characteristics of the nanopore incorporated, the voltage may be positive rather than negative, and the absolute term (absolute terms) may be much higher.

Furthermore, as explained in detail elsewhere herein, in certain aspects the method includes counting events generated by passage of the probe using an algorithm, as reported by a time record, to determine whether the probe is free to translocate through the nanopore. In certain aspects, the nanopore device allows for distinguishing between different osmium probes and multiplexed detection of multiple different nucleic acid targets in a test sample.

Using the methods and probes disclosed herein, in certain aspects, the methods can detect nucleic acid targets in a test sample in an amount of less than or less than about 1pM, 100fM, 10fM, 1fM, 100aM, 10aM, 1aM, or 0.1 aM. In certain aspects, the methods can detect nucleic acid targets in a test sample in an amount of at least or at least about 0.1aM, 1aM, 10aM, 100aM, 1fM, 10fM, 100fM, or 1 pM. Also, in certain aspects, the method can detect a nucleic acid target in a test sample in an amount between any or any of 0.1aM, 1aM, 10aM, 100aM, 1fM, 10fM, or 100fM and about any or any of 1aM, 10aM, 100aM, 1fM, 10fM, 100fM, or 1 pM.

In certain aspects, the methods are quantitative for the amount of nucleic acid target molecules in a test sample and/or biological sample.

Certain aspects of the present disclosure relate to the design of probes for detecting nucleic acid target molecules in any of the methods described herein.

In certain aspects, the probe is DNA. In certain aspects, the DNA scaffold is modified. For example, in certain aspects, at least one of the sugars in the nucleic acid backbone is 2' -OMe-deoxyribose. For example, 1, 2, 3, 4, 5 or more or 5%, 10%, 25%, 50%, 75%, 90%, 95% or more or 100% of the saccharides in the nucleic acid backbone are 2' -OMe-deoxyribose.

In certain aspects, the probe is RNA. In certain aspects, the RNA backbone is modified. For example, in certain aspects, at least one of the sugars in the nucleic acid backbone is 2' -OMe-ribose. For example, 1, 2, 3, 4, 5 or more or 5%, 10%, 25%, 50%, 75%, 90%, 95% or more or 100% of the saccharides in the nucleic acid backbone are 2' -OMe-ribose.

The length of the osmium probes can be adjusted based on the length of the nucleic acid target molecule and considerations such as ease and cost of synthesis, amount of osmium to be generated, and hybridization (e.g., longer probes can have greater specificity and base pairing more complementary to the target than, for example, very short probes). In certain aspects, the osmium probe has a length of any of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 35, 40, 50, 60, or 75 to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 35, 40, 50, 60, 75, or 100. In certain aspects, the osmium probe has a length of about 12 to 50 nucleotides. Because detection of the target nucleic acid molecule is achieved by forming a hybrid probe/target complex with the osmium probe, and because translocation of the probe through the nanopore is in its single stranded form, in certain aspects, it is preferred that the probe not self-hybridize to itself, particularly in the region complementary to the target. Thus, in certain aspects, the portion of the probe that is at least partially complementary to the sequence of the nucleic acid target molecule lacks a contiguous self-complementary sequence of more than 2 nucleotides. In certain aspects, the portion of the probe that is at least partially complementary to the sequence of the nucleic acid target molecule is unstructured. And in certain aspects, the portion of the probe that is at least partially complementary to the sequence of the nucleic acid target molecule does not self-hybridize.

In certain aspects, the osmium single-stranded oligonucleotide probe molecules comprise at least one amino acid sequence that is complementary to substituted or unsubstituted osmium tetroxide (OsO ₄ ) -pyrimidine residues covalently bonded to 2,2' -bipyridine groups (OsBp groups). In certain aspects, the at least one osmium pyrimidine residue is a thymidine residue (T). It has been found that the amount of osmium (the amount of osmium pyrimidine) affects how the probe passes through the nanopore and thus how it can be detected as an event and can beTo distinguish it from non-osmium single-stranded nucleic acids that pass through the nanopore. In certain embodiments, an osmium probe comprises at least two, three, four, five, or six osmium pyrimidine residues. In certain embodiments, an osmium probe comprises two, three, four, five, or six osmium pyrimidine residues. As described elsewhere herein, certain methods prefer osmium thymidine residues (T) over other pyrimidines, thus allowing for even more subtle methods to be employed to osmium the oligonucleotide probe. In certain aspects, an osmium probe comprises at least two, three, four, five, or six osmium thymidine residues (T). In certain aspects, an osmium probe comprises two, three, four, five, or six osmium thymidine residues (T). In addition to the amount/number of osmium residues on the probe, the position of the osmium residues relative to each other can also influence how the probe passes through the nanopore. An OsBp group on one residue can hinder the freedom of movement of an OsBp group on an adjacent residue and thus affect how easily a probe can pass through a nanopore with an OsBp group attached. Three adjacent residues further limit the movement of intermediate residues. Thus, in certain aspects, an osmium probe comprises at least two, three, or four adjacent osmium pyrimidine residues. In certain aspects, an osmium probe comprises two, three, or four adjacent osmium pyrimidine residues. In certain aspects, an osmium probe comprises at least two, three, or four adjacent osmium thymidine residues (T). In certain aspects, an osmium probe comprises two, three, or four adjacent osmium thymidine residues (T). Furthermore, the addition of deoxyadenosine (dA) or adenosine (A) residues at the ends of oligonucleotide probes can facilitate passage through nanopores. In certain aspects, the osmium probe includes or comprises at least one, two, three, four, five, or six adenosine residues (dA or a) at the 5 'end or 3' end of the probe. In certain aspects, the osmium probe includes or comprises at least one, two, three, four, five, or six adenosine residues (dA or a) at the 3' end of the probe. In certain aspects, the osmium probe includes or comprises at least one, two, three, four, five, or six adenosine residues (dA or a) at the 5' end of the probe. In certain aspects, the 5 'terminal or 3' terminal glands One or more of the glycoside residues (dA or A) do not hybridise to the nucleic acid target molecule.

Osmium formation of residues, particularly within the portion of the probe that is complementary or at least partially complementary to the nucleic acid target molecule, can inhibit hybridization to the nucleic acid target molecule. To avoid this and/or enhance hybridization, it may be useful to locate an osmium pyrimidine, such as an osmium thymidine residue, in a non-complementary region of the oligonucleotide probe sequence, such as the 5 'end or the 3' end, and/or to replace the thymidine residue in the probe region complementary to the nucleic acid target with another pyrimidine, and to take advantage of the fact that under certain reaction conditions, the thymidine residue may be osmium preferentially over other pyrimidines.

In certain aspects, an osmium probe does not comprise two or more adjacent osmium pyrimidine residues that are not located at the 5 'end or the 3' end of the probe. Also, in certain aspects, an osmium probe does not comprise two or more adjacent osmium thymidine residues (T) that are not located at the 5 'end or the 3' end of the probe. In certain aspects, an osmium probe comprises at least two, three, or four adjacent osmium pyrimidine residues located at the 5 'or 3' end of the probe. In certain aspects, an osmium probe comprises two, three, or four adjacent osmium pyrimidine residues at the 5 'or 3' end of the probe. In certain aspects, an osmium probe comprises at least two, three, or four adjacent osmium thymidine residues (T) at the 5 'end or 3' end of the probe. In certain aspects, an osmium probe comprises two, three, or four adjacent osmium thymidine residues (T) at the 5 'end or 3' end of the probe. In certain aspects, one or more of the 5 'terminal or 3' terminal adjacent pyrimidine residues is not hybridized to a nucleic acid target molecule. In certain aspects, none of the 5 'terminal or 3' terminal adjacent pyrimidine residues hybridizes to a nucleic acid target molecule. In some of the foregoing aspects, adjacent osmium pyrimidine/thymidine residues are located at the 5' end of the probe. In some of the foregoing aspects, adjacent osmium pyrimidine/thymidine residues are located at the 3' terminus of the probe.

As disclosed herein, in certain aspects, the thymidine residue (T) may be osmium-substituted over other pyrimidines. Thus, in certain aspects, at least about 95%, 96%, 97%, 98%, 99% or 100% of the thymidine residues (T) in the oligonucleotide probe molecule are osmium-substituted, and in certain aspects, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the pyrimidines present in the probe (except for thymidine (T)) are not osmium-substituted. The DNA residues thymidine (T) and the RNA residues uridine (U) are complementary to adenosine (A). In certain aspects, the probe is DNA, but at least one thymidine residue (T) in the probe sequence is replaced by a uridine (U) or deoxyuridine (dU) or 2' -OMe-uridine (mU) residue, except for adjacent thymidine residues (T) at the 5' or 3' end. In certain aspects, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the thymidine (T) residues in the probe sequence are replaced by uridine residues (U, dU or mU) except for adjacent thymidine residues (T) at the 5 'or 3' end. The result is an oligonucleotide probe that contains little or no osmium thymidine residues (T) in the region of the probe that hybridizes to the complementary nucleic acid target molecule by substituting thymidine (T) with uridine (U), but does contain adjacent osmium thymidine at the 3 'or 5' end of the probe.

There are many combinations of features that can be incorporated into the design of osmium oligonucleotide probes for use in the methods of the present disclosure. In one representative design, an osmium probe includes or comprises at least two, three, or four adjacent osmium thymidine residues (T) at the 5 'end of the probe, the probe being DNA, but the thymidine residues (T) in the probe sequence are replaced by uridine residues (U, dU or mU) in addition to adjacent thymidine residues (T) at the 5' end; and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the pyrimidines (other than thymidine (T)) present in the probe are not osmium-esterified. Furthermore, in certain aspects, at least one of the sugars in the nucleic acid backbone is 2' -OMe-deoxyribose. In another representative design, an osmium probe comprises three adjacent osmium thymidine residues (T) at the 5 'end of the probe, the probe being DNA, but the thymidine residues (T) in the probe sequence are replaced by uridine residues (U, dU or mU) except for three adjacent thymidine residues (T) at the 5' end; and all pyrimidines (except thymidine (T)) present in the probe are not osmium-ized. Furthermore, in certain aspects, some or all of the saccharides in the nucleic acid backbone are 2' -OMe-deoxyribose.

As described in more detail elsewhere herein, in certain aspects, a polypeptide may be produced by reacting a polypeptide comprising a substituted or unsubstituted 2,2' -bipyridine and OsO ₄ Aqueous solution of (osmium reagent) is reacted with an oligonucleotide to form 2,2' -bipyridine-OsO ₄ The probes were conjugated to prepare osmium probes. In certain aspects, the conjugated probes are purified from excess osmium reagent. In certain aspects, 2' -bipyridine/OsO in solution ₄ The ratio is equimolar or nearly equimolar. For example, in certain aspects, 2' -bipyridine/OsO in solution ₄ The ratio is about 0.80/1.0, 0.85/1.0, 0.90/1.0, 0.95/1.0, 0.97/1.0, 0.98/1.0, 0.99/1.0, 1.0/0.99, 1.0/0.98, 1.0/0.97, 1.0/0.95, 1.0/0.90, 1.0/0.85 or 1.0/0.80. Alternative methods of preparing osmium probes exist and are contemplated. For example, in some aspects, by combining (dT (OsBp)) _n An oligonucleotide is ligated to the 5 'or 3' end of the probe to prepare an osmium probe, where n is 2, 3, or 4.

Provided herein are kits comprising reagents for performing the methods of the present disclosure. In certain aspects, the kit comprises an osmium probe of the present disclosure and a control nucleic acid target molecule that can hybridize to the probe. In certain aspects, the control nucleic acid target comprises ctDNA, cfDNA, miRNA, a fragmented nucleic acid sequence that encodes RNA or non-encoding RNA. In certain aspects, the non-coding RNA is less than about 300 bases in length.

Also provided herein is the use of the probes of the present disclosure for detecting a nucleic acid target molecule, optionally ctDNA, cfDNA, miRNA, fragmented coding RNA, or non-coding RNA, using a nanopore device. In certain aspects, the non-coding RNA is less than about 300 bases in length.

Examples

Materials and methods

Oligonucleotides and other reagents

Custom RNA oligonucleotides were purchased from Dharmacon (Horizon Discovery Group). Custom deoxyoligonucleotides were purchased from Integrated DNA Technologies (IDT). The sequence and UV/Vis characteristics of their osmium derivatives are described inListed in table 1. The purity of the oligonucleotides was tested by HPLC and was generally found>85%. Oligonucleotides were diluted with Ambion nuclease-free water from Thermo Fisher Scientific without DEPC treatment, typically 100 or 200. Mu.M stock solution, and stored at-20 ℃. HPLC profiles from analysis of intact oligonucleotides and osmium oligonucleotides are included elsewhere herein. Buffer dnase-free and rnase-free tris.hcl 1.0M pH 8.0Ultrapure was purchased from Invitrogen and used to prepare HPLC mobile phases. NaCl crystallized ACS was obtained from Alfa Aesar at a minimum of 99.0%. Distilled water from Alhamra was used to prepare the HPLC mobile phase. 4% osmium tetroxide aqueous solution (0.1575M OsO) ₄ In ampoules, 2mL each) was purchased from Electron Microscopy Sciences.2,2' -bipyridine 99+% (bipy) was purchased from Acros Organics. Human serum and NaOH 1N bioagent from human male AB plasma were purchased from Sigma. ss M13mp18, primer M13 (fwd 6097), NEBuffer 2.1 and Klenow fragment of DNA polymerase I (3' -5 exo) (M0212) were supplied by New England Biolabs (New England Biolabs, ipswich, mass., USA) friends, eppanyi, massachusetts, U.S.A.

Although there are no limitations to the probes of the present disclosure, cost and product quality considerations have led to the selection of DNA rather than RNA oligonucleotides as probes for most exemplary experiments. These experiments were developed around optimizing the design of the probe to make it universally applicable and properly detectable by available nanopore systems (e.g., ONT/CsGg nanopores). In certain aspects, probes that successfully identify miRNA targets at the single digit attomole level have a sequence complementary or at least partially complementary to a nucleic acid target molecule, 2' -ome (mU) that replaces T within the sequence, 3 additional adjacent T residues at the 5' end, and 3 additional dA residues at the 3' end. The oligonucleotides were osmium-formed so that 4 to 5 OsBp tags were added per molecule, 3 of which occupied the 5' end, and the other 1 or 2 were randomly allocated in sequence. Because one end of the probe is very crowded, an applied voltage in the range of-210.+ -.10 mV is required to carry out translocation and detection of this exemplary probe. This feature is advantageous because it allows depletion of non-target material at-180 mV prior to performing diagnostic experiments at-210 mV. Probes can also be multiplexed when using probe designs that exhibit different nanopore profiles. Preliminary experiments in 15% human serum showed that the probe and resulting hybrid are stable in this medium and demonstrate the feasibility of using OsBp to identify short DNA and RNA from body fluid samples using a nanopore platform.

Example 1

(pyrimidine) OsBp is a chromophore

Selective labelling of nucleic acids requires an assay for quality control. Addition of OsBp to the C5-C6 Py double bond and formation of Py (OsBp) generates new chromophores in the wavelength range of 300 to 320nm (kanovarioti, a. Et al (2012)), where the nucleic acids exhibit negligible absorbance. Using this observation using a training set of deoxyoligonucleotides shows that the degree of osmium can be measured using the following equation: r (312/272) =2 x (number of osmium pyrimidine/total nt of nucleotides) (Kanavarioti, a. Et al (2012)). The value R (312/272) is the ratio of the observed peak absorbance at 312nm to the observed peak absorbance at 272nm (the peak shape may be a sharp peak or a broad peak or multiple peaks) (Kanavarioti, a. (2016)). Wavelengths 312nm and 272nm were chosen to maximize the effect and equalize the contributions of the different pyrimidines (Kanavarioti, a. (2016)). The absorbance at 272nm is almost 75% of the absorbance of the intact or osmium nucleic acid at 260 nm. The measurement was normalized using the ratio R instead of absorbance at 312nm and the instrument sampling variation was minimized.

As inferred by experiments using oligonucleotide training sets, when the observed value R (312/272) =2x (number of pyrimidines/total number of nucleotides), osmium was almost 100% complete and all pyrimidines carried one OsBp moiety (Kanavarioti, a. Et al (2012); kanavarioti, a. (2016)). When R (312/272) <2x (number of pyrimidines/total number of nucleotides) is observed, the osmium is partial and the number of osmium pyrimidine or OsBp moieties can be calculated based on the same equation (see table 1). For partial osmium, the number obtained from the equation refers to the OsBp fraction on average. Molecules carry an integer number of OsBp moieties and therefore some molecules will have fewer, while some molecules will have more than calculated, distributed in a statistically unbiased manner. Osmium formation has been shown to occur randomly, but depends on the relative reactivity of OsBp to pyrimidine. The relative reactivity to osmium formation was determined by kinetic measurements in water at 26 ℃ using deoxyribooligonucleotide T/dc=28, dU/dc=3.75 and thus T/du=7.5 (Ding, y. And Kanavarioti, (2016)), and using ribooligonucleotide U/c=4.7 and 5-MeU/c=44; thus 5-MeU/u=9.3, wherein 5-MeU carries the same nucleobase as T (Kanavarioti, a. (2016)). Since OsBp is significantly more reactive towards T than dU or U and dC or C, conditions can be found where all T is almost 100% osmium, while some dU and very few dC become osmium (Kanavarioti, a. Et al (2012)). This significantly higher reactivity is exploited by adding 3T's at the 5' end of the probe, substituting dU or mU for T in the sequence, and optimizing the preparation process, as described in detail below.

Example 2

Preparation of OsBp-nucleic acid

By weighing 15.7mM equivalent of bipy (49.2 mg) in a 20mL scintillation vial, 18mL of water was added and stirred at room temperature until bipy dissolved, then 2mL of 4% OsO provided in ampoule form was transferred ₄ The OsBp reagent was prepared from the entire contents of the solution. And adding Os O ₄ After dissolving bipy (scheme o), in the addition of OsO ₄ Previous dissolution of bipy in water (schemes a, b, c and d) resulted in a more consistent and efficient preparation. Transfer was accomplished using a glass pipette inside the safety shield (MSDS and information were obtained from links of the university California los Angeles division (UCL A) chemical series, see https:// www.chemistry.ucla.edu/sites/default t/files/safety/SOP/SOP_Osmium_tetroxide. Pdf). The resulting solution was an aqueous 20mL 15.75mM OsBp (0.4%) stock solution, osO ₄ And bipy equimolar. The concentration of OsBp stock solution is limited by the solubility of bipy in water, and OsO is added ₄ Does not increase because the complex has a low association constant. OsBp complex accounts for approximately 5% of the total amount as measured by CE (Kanavarioti, a. Et al (2012)). It should be noted that this preparation, as well as any other work using OsBp, was done in a stoppered glass vial in a well ventilated area. OsO (o) ₄ And/or the residual solution of OsBp may be mixed with cornOil blending to neutralize unreacted OsO ₄ And correctly discarded according to specific local regulations (MSDS and information are obtained from links of the los Angeles division school of California university, see https:// www.chemistry.ucla.edu/sites/default/files/safety/SOP/SOP_Osmium_tetroxide. Pdf). Freshly prepared OsBp stock solution was dispensed into HPLC vials and stored at-20 ℃. Each vial can be stored at 4 ℃ and used for one month without failure; typical pipette tips may be used to prepare OsBp labeled nucleic acids. The OsBp stock solution should be validated by running a known reaction prior to first use. For the osmium formation reaction, an 20-fold excess of OsBp over the monomer equivalent of the reactive pyrimidine was used to ensure pseudo-first order kinetics and to ensure successful use of the protocol. The preparation conditions, namely OsBp concentration and labeling duration, vary significantly depending on the presence of T and the desired outcome of osmium formation for all pyrimidines, only T or only a portion of dC and dU in DNA and a portion of C and U in RNA. For the purposes of this study, option b was recommended when T-osmium was required, and option C or option d was recommended when partial osmium of U and C was required. These choices were facilitated by testing additional protocols (o and a), as identified in table 1. Quenching of the osmium formation reaction occurs after purification. Purification from excess OsBp was performed using a spin column (TC-100 FC from TrimGen Corporation) according to manufacturer's instructions. Briefly, the spin column was filled with the manufacturer's proprietary solution and centrifuged at 4,000rpm for 4min; the resulting solution and microcentrifuge tube were discarded. Then 40 to 120. Mu.L of the osmium reaction mixture was transferred to a centrifuge column and centrifuged at 4,000rpm for 4min using a clean microcentrifuge tube. The centrifugation solution was purified osmium oligonucleotide. This purification method retains the volume/concentration of the sample and achieves nearly 100% oligonucleotide recovery.

The recommended protocol b for thymidine (T) -osmium formation is incubation with 2.6mM OsBp in water for 30±2min at room temperature (see table, under b). A recommended protocol for partial U-and C-osmium formation of probes without dT is one in which they are incubated with 3.9mM OsBp for 30.+ -.2 min in water at room temperature (see Table 1, under C) or with 5.2mM OsBp for 30.+ -.2 min (see Table 1, under d). Two additional schemes were tested, but these were foundThe scheme is not ideal: after adding OsO ₄ Bipy was previously undissolved in water and incubated for 40min with 2.6mM OsBp (see Table 1, under o) and 45min with 2.6mM OsBp (see Table 1, under a). The presence of the 2' -OMe group did not significantly affect the degree of osmium, as shown by the comparable degree of osmium formation of 100nt RNA and 100nt RNA (2 ' -OMe) carrying about 50% of the 2' -OMe base. As shown in FIG. 14, the number of U's in the oligonucleotide can affect the degree of osmium formation. This is because OsBp is 3.75 times more reactive to dU than to dC and OsBp is 4.7 times more reactive to U than to C. Because the osmium protocol is incomplete, dU (OsBp) or mU (OsBp) is kinetically superior to dC (OsBp), and U (OsBp) is superior to C (OsBp). Using scheme b, the osmium formation of other pyrimidines was negligible compared to T-osmium formation, but using scheme c, the degree of osmium formation of U, dU, mU, and dC was measurable. FIG. 14 shows a linear correlation between the number of OsBp moieties and the number of U's present in oligonucleotides spanning from 22nt to 100nt oligonucleotides. The slope of the graph=0.43 can be used to estimate the degree of osmium formation using scheme c for any oligonucleotide based on the number of U's in the sequence.

The preparation reaction was performed using a 2mL HPLC vial equipped with a 120 μl glass liner. Removal of the reaction products from these liners and transfer thereof to a purification spin column requires an elongated 20 μl pipette tip. The osmium nucleic acid is as stable as the corresponding nucleic acid and OsBp tag is anergic. They can be stored in 1.5mL microcentrifuge tubes for many years at-20 ℃. Concentrations of 2.6, 3.9 and 5.2mM correspond to 1/6, 1/4 and 1/3 dilutions, respectively, of 15.75mM OsBp stock solution. Deviations from these two schemes are included in the table, identified as schemes o and a. An additional protocol (d) was used to achieve a higher degree of osmium formation with a probe that did not contain any T, such as dmiR21 (OMe), and to make it a detectable probe (see brief description of fig. 21A).

Example 3

Enzymatic extension reactions

The ability of the DNA polymerase to extend the osmium primers was examined in vitro using ssM mp18 annealed to unmodified and osmium oligonucleotides. In NEBuffer 2.1 (New EnglandBiolabs) was mixed with ssM mp18 at a concentration of 42nM with 0.42 μm primer. The sample was heated to 90 ℃ for 30 seconds and cooled to 25 ℃ at 0.1 ℃/sec. The polymerization reaction contained these annealed complexes (8.4 nM ssM13mp 18), 1.25X NEBuffer 2.1, 0.25mM each of dGTP, dATP and dTTP, 0.025mM alpha- [ ³² P]The Klenow fragment (3 '-5' exo) of dCTP and 7.7U/ml DNA polymerase I (NEB, M0212). The reaction was incubated at 37 ℃ and incorporation of the labeled dCMP was monitored by an acid precipitation assay. Time points were taken as 5, 10 and 20 minutes.

As shown in FIG. 3A, no incorporation was noted when no oligonucleotide was added, or if oligonucleotide primer M13rev (-48) was not complementary to ssM mp 18. In contrast, most oligonucleotides predicted to anneal to DNA templates provide robust incorporation, even at osmium, with maximum incorporation corresponding to approximately one round of replication on the M13mp18 DNA template. The incorporation noted for M13 (fwd 6097), BJ2, BJ3 and BJ4 was equivalent, although BJ3 and BJ4 were present with internal single base mismatches (see sequences in Table 1). Although the osmium levels of BJ2, BJ3 and BJ4 were generally equal, it was noted that the incorporation level of BJ1 was significantly lower, most likely due to the presence of OsBp at the terminal 3' -T (OsBp) residue. Control experiments in which BJ1 was mixed with M13 (fwd 6097) showed complete incorporation, thus explaining the soluble inhibitors as the reason for low incorporation in the case of BJ1 (data not shown).

Example 4

HPLC method

HPLC methods for assessing the purity of oligonucleotides were developed and used herein to assess the purity of the oligonucleotides listed in Table 1. The method is optimized and used to evaluate hybridization between two oligonucleotides in a mixture; the method was validated using a 1:1 mixture of known hybridizable complete oligonucleotides (see FIG. 3B and FIG. 10A). The analysis was performed automatically using a thermostated autosampler. HPLC peaks were detected and identified in the uv-visible region of 200-450nm using a Diode Array Detector (DAD). Chromatograms were recorded at 260, 272 and 312nm and selectively reported here. Samples were prepared with RNase-free water, but the buffer was not.

For HPLC analysis, agilent 1100/1200LC HPLC equipped with binary pump, diode Array Detector (DAD), 1290infinit autosampler/thermostat and Chemstation software rev.b.04.01sp1 were used for data acquisition and processing. For sample analysis, IEX HPLC column dnappa 200 from ThermoFisher Scientific (Dionex) was used, configured to be 2X250mm. Instrument and column performance was qualified using standards both before and after each analysis of the study samples. HPLC methods were developed to evaluate hybridization of approximately 90% of the samples in aqueous ONT buffer at pH8 and column thermostats were 35 ℃. This HPLC method (identified as HPLC method B) used a dnappa PA200 column at a flow rate of 0.45mL/min, mobile Phase A (MPA) was an aqueous 25mM TRIS.HCl pH8 buffer, mobile Phase B (MPB) was a 1.5MNaCl aqueous solution in 25mM TRIS.HCl pH8 buffer, gradient from 10% MPB to 50% MPB over 20min, and another 10min for washing and equilibration to the initial condition, 90% MPA. The column temperature was set at 35 ℃ to simulate the flow cell temperature. To include 100nt RNA, the chromatography was modified to HPLC method C. Specifically, the gradient became steeper, from 10% MPB to 75% MPB in 20min, everything else remained intact. RNA of 100nt length eluted as broad peaks resembling a mixture due to approximately neutral pH. This is because aqueous solutions at pH8 do not denature the various conformations of long RNAs, as previously reported (Kanavarioti, a. (2019)). The ONT buffer has a UV-Vis component which elutes in the void volume and does not interfere with the analysis of the sample during this chromatography. The sample loading is typically 5 μl and no more than 10 μl. Hybridization assays may be used in combination with the sample in the ONT buffer or any other medium that facilitates complexing. Some oligonucleotides and their osmium derivatives were analyzed using a method identified as HPLC method a, which is recommended for oligonucleotide purity analysis (Kanavarioti, a. (2019)), but is not suitable for testing hybridization. HPLC method A used DNAPac PA200 column at a flow rate of 0.45mL/min and column temperature of 30 ℃. Mobile Phase A (MPA) was an aqueous solution at pH 12.0±0.2 containing 0.01NNaOH, mobile Phase B (MPB) was an aqueous solution at 1.5M NaCl in pH 12.0±0.2 containing 0.01N NaOH, gradient from 20% MPB to 95% MPB in 12min, and 8min further for washing and equilibration to the initial condition, i.e. 80% MPA.

Example 5

Single molecule ion channel conductance experiments in MinION or Flong (ONT platform) using CsGg nanopores

The ONT instructions were followed to remove air bubbles in the flow cell, flush the storage solution with ONT flush buffer, add sample or store the flow cell as required. Instructions for min (75 μl samples) and florgle (30 μl samples) were obtained from the protocol found on the ONT website. The flow cell requires an adapter but works on the same device as the phase flow cell. The software MINKNOW for running the nanopore experiments was downloaded to the MacBook Pro notebook for these experiments. All functions required to test the flow cell and run the experiment were performed by the MINKNOW software tool. The original data file is obtained in fast-5 format and then analyzed by osbp_detect software. The size of the fast-5 file used for the experiment depends on the flow cell and the duration of the experiment and varies between 1.5GB and 6 GB. Once the experiment is complete, the Fast-5 file can be visualized directly in MatLab (from Mathworks) 2D format. MINKNOW allows real-time monitoring of any selected channel, up to 10 channels, so that i-t recordings can be viewed without waiting for the experiment to complete.

The added sample is an untagged oligonucleotide, an osmium oligonucleotide or a mixture thereof. In not less than 80% of ONT buffer, the concentration of oligonucleotide is usually equal to or lower than 10. Mu.M. No libraries were prepared nor processing enzymes were added, so all translocations reported here were unassisted and voltage driven. The experiment lasted no more than 1.5 hours, but the same experiment could be prolonged by stopping it and restarting it later or the next day without adding a new sample. The same flow cell was avoided from running for more than 4 hours per day and was temporarily stored in ONT buffer. The cleaning of the flow cell with buffer before running a new sample is done just before the next experiment. In most cases, the first experiment was a "buffer test" to evaluate the baseline of the flow cell. The duration of the buffer test was as short as possible, since the nanopores of the flow cell did not last more than 15 hours under our experimental conditions. According to ONT schemeThe applied voltage was increased by about 10mV every 5 operating hours in an attempt to increase the opening ion current (I _o ) Remain unchanged. This is why experiments compared with each other were performed under seemingly different applied voltages. The min flow cell had more than 2000 nanopores, but only 512 were monitored simultaneously. During the first few experiments on the flow cell, the nanopores became inactive, but they were replaced with new working nanopores. Thus, the first 4 to 5 experiments were actually performed with the same number of wells. Thereafter, the number of working nanopores was reduced by 5% to 10% per hour. Notably, while most working nanopores exhibited a comparable number of events ranging from lowest to highest within 5 times, a small fraction deviated and recorded significantly higher event counts. These pores are referred to as "outliers" and it is estimated that about 2.5% of the nanopores are outliers. Analysis of the data provided herein includes all channels. Additional analysis was performed on all experiments by excluding outliers and trends and conclusions were the same as presented herein, even though the actual counts were different.

Example 6

Event detection algorithm-OsBp_detect

Let y be an ordered sequence of real values representing a typical time sequence obtained from a single nanopore. We classify the region of y as one of two states: current I from open channel _o Wherein the nanopore is unoccupied, and a residual current I at the occurrence of a translocation event _r . For high-throughput characterization of single translocation events corresponding to OsBp tagged oligonucleotides, we propose a segmentation algorithm that can determine the start and end positions of y for all events of interest based on user-defined thresholds. The analysis flow (pipeline) is divided into three steps:

1. baseline current estimation

2. Identification of potential candidate events

3. Event filtering based on event features

First, by taking the estimated lower and upper limits { o } of the open circuit current _{Lower limit of} ,o _{Upper limit of} Median of signal values between }Establishing a base line I _o 、b _o ：b _o Median ({ i: i e y, o) _{Lower limit of} <i<o _{Upper limit of} })。

Although the signal noise depends on the nanopore platform used, I from the osmium-tagged oligonucleotide _o And I _r The abrupt transition between states allows for event detection procedures using a single threshold-based resolver. Using this method, an event can be identified if it exceeds a set threshold that is far from the local baseline level. The threshold value is defined by parameter b _all It is defined that it should be low enough to capture as many translocation events as possible. In default, ball=b _o ·(1–(10·σ _o /b _o ) Of which sigma _o Signal noise representing open circuit current. Noise constant sigma _o Is obtained by dividing y into small segments (segment size used, n=100,000) and calculating the global standard deviation σ of the open-circuit current signal value ({ i: i e y, o) _{Lower limit of} <i<o _{Upper limit of} }). The σo value additionally provides a useful quality control indicator to detect holes with unstable baselines and large event rates or holes that have been blocked.

Finally, to quantify effective translocation events, two filter conditions were applied to the identified events. The filter corresponds to the parameter { t } _min ,t _max Minimum and maximum event lengths defined and a range of minimum residual currents { b } _min ,b _max "relative to b _o Is a ratio of (2). The event length threshold { t may be adjusted _min ,t _max To monitor translocation velocity, and { b } _min ,b _max The ability to separate OsBp tagged and untagged oligonucleotide species. Let τ ₁ And τ ₂ Representing the start and end indices of any given event in y. To y _τ1:τ2 Classified as a potential OsBp translocation event, the following two conditions must be met:

＞＞t _min ＜τ ₂ -τ ₄ ＜t _max

the event detection procedure is available as a Python library 'osbp_detect'. A cross-platform graphical user interface has been included to enable direct reporting of translocation events (https:// gitsub. Com/kangaroo 96/osbp_detect) from ONT bulk fast5 files.

Results and discussion

The materials used in this study were all the highest purity synthetic oligonucleotides and are listed in table 1. We developed an osmium protocol. The intact and osmium nucleic acids were further characterized internally by a validated HPLC method (kanovar iota, a. (2019)). Nanopore experiments were performed using ONT devices and ONT supplied flush buffer (ONT buffer or buffers), and further the company's instructions on how to start the flow cell, add samples, select voltages and acquire the original i-t trace. No sample libraries were prepared, nor were enzyme assisted utilized. Samples were prepared in 90-95% ONT buffer unless otherwise indicated. The nanopore experiments reported here were performed at factory preset flow cell temperatures in the range 34-35 ℃. Original i-t traces (fast 5 file) of all channels were captured and analyzed using osbp_detect software (kanovarioti, a. And Kang, a. Please see RNA (OsBp) event detection Python package in public coexistence reservoir https:// gitsub. Com/kangaroo96/osbp_detect, and step-wise installationFor an explanation, please refer to the description herein: https:// github.com/kangaroo 96/osbp_detect/blob/master/instrons). The output, the tsv file, was read using Microsoft Excel. It lists I for each channel ₀ Value, and selected event and I thereof _r Values from which I is calculated _r /I _o . It also lists the start and end times (in the form of data time points) of each event (fig. 8). Osbp_detect allows parameters to be manually set to select events of interest (kanova rioti, a. And Kang, a. Please see RNA (OsBp) event detection Python package in public coexistence store https:// gitsub.com/kangaroo 96/osbp_detect, and step-by-step installation description, see https:// gitsub.com/kangaroo 96/osbp_detect/blob/map/instructs). Here we have chosen a residence time of 4.ltoreq.τ.ltoreq.300 data time points or equivalently 1.3.ltoreq.τ.ltoreq.100 ms and a fractional residual ion current I _r /I _o Events of < 0.55 (FIG. 1B). FIGS. 2, 4, 5, 6 and 7 are given as I using a 0.05 sliding window _r /I _o Event count (abbreviated as count or event) of a function of (a) is provided.

Nanopore experiments using a florgle flow cell

Fig. 2 illustrates the results of using a florgle flow cell. FIG. 2A is a first attempt to observe hybrids. From early work using a min flow cell, T8 (32 nt RNA with 9 pyrimidines and a total of 8 OsBp tags) (see sequence in table 1) required high voltage to pass through, exhibited multiple translocation events, and severely embolized ion current, in (I _r /I _o ) max≡0.1. Repeating the experiment essentially repeated early work on the mineral (Sultan m., kanovarioti, a. (2019)). Although not perfect complement, d (TC) ₁₀ Still used to form ds complexes because of d (CT) ₁₀ 16 of 20nt can be base paired with T8, including 8 GC pairs. Although experiments using probe T8 showed an average of 400 events per channel, probes T8 and d (CT) were used ₁₀ Experiments with 1:1 mixtures of (1) produced less than 50 events per channel in some channels, and the remaining channels reported zero events (fig. 9). FIG. 2B illustratesA test for identifying miRNA122 (Li, X-d. Et al (2017)) was performed. The probe used here was dmiR122, which is the exact complement of miRNA122 and carries 4T (OsBp) (see table 1). Samples with only probe dmiR122 showed a large number of counts, while samples with an approximately equimolar mixture of the probe and miRNA122 showed significantly fewer counts. The third sample, which is 4-fold higher in miRNA loading, consisted of miRNA21 (thumb, t. Et al (2008), lai, j.y. Et al (2017), fulci, v. Et al (2007), wang, y. Et al (2020)) and miRNA140 (Li, X-d. Et al (2017)), also showed fewer counts compared to the probe sample. The latter suggests that it is reasonable to identify targets in a complex mixture of mirnas. These and other experiments demonstrate the feasibility of the concept presented in fig. 1D. They also disclose that both the target and the probe can be RNA or DNA, with the difference that the probe is an osmium oligonucleotide, and the target is not. Further of interest are probes that are DNA oligonucleotides because of their lower cost and higher quality of the synthesized product compared to RNA oligonucleotides.

Alternative method of hybridization between test target and its probe

Independent methods were sought to test hybridization between osmium nucleic acids and their DNA or RNA targets. Enzymatic DNA polymerase extension of unmodified primers using the partial osmium template ssM mp18 was the first attempt to obtain support for hybridization, but no extension of the primers was detected (data not shown). Unmodified ssM mp18 was then tested as template and complementary T (OsBp) primers BJ1, BJ2, BJ3 and BJ4 30nt long were used (see table 1 and fig. 11 and 13). BJ1 carries the same sequence as primer M13for (-20) at the 3 'end and is extended by 13nt at the 5' end. BJ2 carries the same sequence as primer M13for (-41) at the 3 'end and is extended by 6nt at the 5' end. BJ3 and BJ4 have the same sequence as BJ2 except for a mismatch in the sequence. Although BJ2, BJ3 and BJ4 carry 6, 5 and 7T (OsBp) bases, respectively (see Table 1), they all extended successfully (FIG. 3A), indicating that T (OsBp) did not preclude complete 1:1 hybridization between ssM mp18 and probe. In contrast, BJ1 with 6T (OsBp) did not extend, probably due to the presence of T (OsBp) base at the 3' end and the inability of the enzyme to add nucleotides to it (FIG. 3A). The absence of extension of BJ1 does not necessarily mean that hybridization is absent.

These extension experiments were performed assuming much lower salt concentrations than those used for nanopore experiments, they were limited to ssM mp18 and use probes with the same sequence as known primers. To extend the hybridization test to mirnas and any DNA/RNA oligonucleotides, we developed HPLC methods as described elsewhere herein. The HPLC method is based on the HPLC method for testing the purity of oligonucleotides, with the following modifications: it uses (i) ONT buffer as sample solvent and (ii) HPLC column temperature 35 ℃ to simulate ONT working flow cell temperature. It should be noted that HPLC column packing may interfere with hybridization, so all HPLC-based results are purely suggestive. That is, no contradiction between HPLC results and nanopore experiments was observed. To confirm hybridization, HPLC analysis was performed on three separate samples. The three samples contained (i) probes, (ii) nucleic acid target molecules, and (iii) samples with a 1:1 mixture of two components comprising putative hybrids (see FIGS. 3B-3D). The absence of hybridization was consistent with the HPLC profile of the mixture sample, which closely overlapped the "sum" of the HPLC profiles of the two components (fig. 3C). Evidence of hybridization is consistent with the hybrid peaks, which split well from the peaks of the target and probe, and elute typically 1 to 1.5 minutes later than the probe or target. In addition, the probe and hybrid peaks exhibited absorbance at 312nm due to the presence of OsBp tags, but the target peaks were absent. 1:1 mixture samples were intentionally prepared with a slight excess of target to prevent excess probes. That is why in many HPLC chromatograms, analysis of a hybrid sample includes smaller peaks due to the target in addition to large peaks due to the hybrid.

FIG. 3B shows an HPLC chromatogram of a sample in which both oligonucleotides are unmodified nucleic acids. Here, the HPLC profile of the sample with the 1:1 mixture was consistent with strong hybridization based on the features discussed above. FIG. 3D is a repeat of the HPLC analysis in FIG. 3B, except that BJ2 is now osmium-functionalized; it is a probe. The corresponding HPLC profile of the 1:1 mixture in FIG. 3D is also consistent with strong hybridization. The same HPLC method indicated that miRNA21 and probe 21EXT (see table 1) did not hybridize (fig. 3C). The absence of hybridization in FIG. 3C is due to the relatively large number of OsBp moieties present in the sequence of the probe (8 dT total, with 5 dT present in the 21nt sequence). Additional hybridization tests confirm the hypothesis that formation of ds complexes in the presence of a large number of OsBp moieties on the probe is precluded (see fig. 10). In addition to the number of OsBp moieties in the molecule, the actual location is also important. As shown in the BJ1-4 probe, hybridization was strong despite the relatively large number of T (OsBp). This is most likely because these tags occupy a small region of the sequence, leaving two fairly long subsequences available for duplex formation with the target.

Hybridization silences probes in the presence of DNA oligonucleotide targets

The experiments in fig. 2 were performed using probe, target and hybrid concentrations in the 5 μm range. Since the sample volumes of the min and florgle flow cells were 75 μl relative to 30 μl, the 5 μΜ concentrations correspond to a sample loading of about 0.38 relative to 0.15 nanomole, respectively. It is worth mentioning that the sample loading of the nanopore experiment is different from the sample loading of the HPLC analysis, because the HPLC sample loading is usually different from the sample loading of the flow cell. FIG. 4A illustrates an HPLC hybridization test, as described above, using probe BJ1 with 5T (OsBp) and its target, namely, the complementary primer M13for (-20). Samples of probes and hybrids were used as received for nanopore experiments as shown in fig. 4B. We note that probe BJ1 was not enzymatically extended using ssM mp18 as template, which we attribute to the presence of a T (OsBp) base at the 3' end. In fig. 4, both HPLC analysis and nanopore experiments recorded hybridization, as indicated by the large decrease in the number of counts reported for the hybrid sample compared to the counts reported for the probe sample. For (I) _r /I _o ) _max The effect of (c) is significant and for I _r /I _o The effect of the rest of the range is clearly detectable.

The applied voltage being a critical parameter

FIG. 4B illustrates that test probe BJ1 exhibits very low counts at an applied voltage of-180 mV, indicating that probe translocation is efficient at-180 mVLow. Increasing the voltage to-220 mV resulted in significantly more counts than those obtained at-180 mV without adding new samples. Fig. 11 illustrates that the probes BJ2 and BJ4 follow the same pattern as BJ 1. This observation was due to the presence of adjacent dT (OsBp) in the probe, and the lowest observation that dT (OsBp) showed in all the tested pyrimidines (I _r /I _o ) _max (this is a strong indication of severe congestion). Early studies using RNA and minon/CsGg (Sultan m., kanovarioti, a. (2019)) and DNA and a-hemolysin nanopores (Ding, y. And kanovarioti, a (2016)) led to the conclusion that there was severe crowding in adjacent OsBp fractions. Steric hindrance is complex because the probe is a deoxyoligonucleotide and includes several T bases in the sequence. The labeled nucleic acid may be brought close to the CsGg well under the direction of an applied voltage drop, but a certain minimum voltage is required to pass through the well. Experiments have shown that the applied voltage to effectively translocate most of our probes through proprietary CsGg nanopores is in the range of-210±10 mV. In contrast to observations with probes, intact DNA oligonucleotides showed meaningless counts (Ding, y. And kanovarioti, a. (2016); sultan m., kanovarioti, a. (2019)) and target/unmodified mirnas showed measurable counts with slightly decreasing trend as a function of increasing voltage (fig. 13). This is consistent with the expectation that increasing the voltage results in faster translocation and faster translocation in turn results in missed events, as the acquisition rate of the device remains constant at 3 data points per millisecond. In order to make the counts detectable when using native RNA, the total loading was 4 times higher than the highest probe loading. No experiments were performed with voltages above-220 mV to protect protein wells that, according to our experience, did not last as long at-220 mV as at-180 mV. Our observation that the probe hardly passes through the CsGg well at-180 mV is an advantage for diagnostic testing. It provides the opportunity to deplete excess non-target nucleic acid in the sample at-180 mV and then, without adding any new sample, to raise the voltage to-220 mV so that the presence of uncomplexed probes is detected or undetectable.

Universal design of highly detectable probes

The presence of a T (OsBp) moiety in the middle of the sequence is not a feature common to many potential ctDNA or miRNA targets. Thus, advanced probes are designed by: substitution of all dT in the sequence with dU, modification of some or all bases to 2' -OMe, addition of 3 adjacent dT (OsBp) at the 5' end, and in some cases 3 additional dA at the 3' end. The addition of dA at the 3' end is commonly used to facilitate well entry (Kasianowicz, J.J., brandin, E., branton, D.and Deamer, D.W. (1996); butler, T.Z., gundlach, J.H., and Troll, M. (2007); maglia, G., heron, A.J., stoddart, D., japrung, D.and Bayley, H. (2010)). Substitution of the DNA base with a 2' -OMe base has been reported to result in stronger hybridization (Majlessi, m., nelson n.c. and Becker, m.m. (1998)). Substitution of dU for all dT ensures that OsBp is present at a minimum in the sequence. This results in as few OsBp moieties within the sequence as possible and minimal obstruction of hybridization to the target. The addition of 3 adjacent dT's at the 5' end makes the probe undetectable at the applied voltage of-180 mV, and highly detectable at the applied voltage of-220 mV, as shown in earlier experiments using BJ1-4 probes. This probe design was then utilized in nanopore experiments with ultra-low target loadings.

BJ2 TA (OMe) is a probe designed to have the above characteristics (sequences in Table 1). Hybridization between probe BJ2 TA (OMe) and the complementary primer M13for (-41) was tested by HPLC at a concentration range of 5. Mu.M (FIG. 4C). FIG. 4C shows HPLC analysis of probe and hybrid samples. The target peak in the hybrid sample is easily identified because it is about 10% excess compared to the probe. HPLC profiles at two wavelengths, 272nm and 312nm, are shown. Careful examination of these spectra showed that the hybrid peak had approximately half of the contribution at 312nm compared to the corresponding contribution of the probe. This is consistent with the expectation that the hybrids are half of the probes and half of the unmodified targets. The actual sample tested by HPLC was diluted with ONT buffer to produce a sample tested by nanopore. All dilutions following the protocol and in the present study were done by serial 1:10 dilution with ONT buffer in 0.5mL microcentrifuge tubes. The nanopore experiment using probe BJ2 TA (OMe) was performed with a dilution of 1000 fold compared to the HPLC sample, i.e. at a probe loading of 0.38 picomolar. The corresponding experiment using hybrids was performed at 3.75 picomoles, i.e., at 10 times higher hybrid loading. High hybrid loading was chosen to test the stability of hybrids under experimental parameters, especially under the influence of an applied voltage of-220 mV. Although the concentration of the hybrid sample was 10 times higher than the probe, the counts obtained from the hybrid experiments were significantly less than those obtained from the probe experiments, indicating that dissociation of the hybrid under the tested conditions was not significant (fig. 4D).

Hybridization silences probes in the presence of RNA oligonucleotide targets several probe designs were explored during development efforts. Experiments performed using two of those designs are illustrated in fig. 5. Probe 2XdmiR122 is a 44nt oligonucleotide with 8T (OsBp) and consists of two fused dmiR122 (sequences in Table 1). Although probe dmiR122 exhibited a large number of counts at-190 mV (fig. 2B), probe 2XdmiR122 required-220 mV (fig. 5A). The higher voltage was most likely the result of severe congestion in the wells, as 2XdmiR122 was incorporated into two 4nt groups each with 3 OsBp in the 26nt subsequence. Efficient hybridization between miRNA122 and 2XdmiR122 was shown by HPLC (fig. 5C) and confirmed by nanopores, as shown by the large decrease in counts of hybrid samples compared to probe samples (fig. 5D). The advantage of such fusion sequence design is that it takes advantage of the fact that the same base sequence is present in longer RNAs in addition to miRNA targets. The use of probes with a design such as fused 2XdmiR122 may be advantageous for hybridization to miRNA targets rather than long RNA targets, because 2XdmiR122 can form 44nt ds complexes with miRNA122, but only 22nt ds complexes with longer RNAs.

FIG. 5B shows another probe design, exemplified by probe 122EXT (sequences in Table 1). Probe 122EXT has the same sequence as dmiR122, except that 3 adjacent T's are added at the 5' end. The probe required-220 mV to show a large count (solid line), as shown by comparison with the nanopore experiment at-180 mV (dashed line). Nanopore experiments using probe 122EXT in samples prepared in 15% human serum and 85% ONT buffer showed reduced counts compared to samples prepared in above 95% ONT buffer. The decrease in counts may be due to lower ionic strength due to the presence of serum, and/or aging of the flow cell and/or serum interference. Despite the lower counts, probe 122EXT is easily distinguished from control/buffer tests, indicating that detection of the probe is not hindered in unknown samples containing bodily fluids (such as human serum).

miRNA21 is an important biomarker for many diseases (thumb, t. Et al (2008); kao, h. Et al (2017); fulci, v. Et al (2007)), and thus its identification was tested. HPLC tests with miRNA21 and probe dmiR21 (not shown) or 21EXT indicated no hybridization to miRNA21 was detectable (fig. 12). The inability to form hybrids is due to the fact that there are more than 6T (OsBp) moieties, which are spread over 22nt sequences. Advanced probe design results in probes that hybridize efficiently to miRNA 21. Probe dmiR21 (OMe) is a 22nt oligonucleotide complementary to miRNA21, where all bases are 2' -OMe, T is replaced by dU, and osmium leads to an average addition of 2.85 OsBp moieties per molecule (osmium protocol d, see table 1). Osmium products are mixtures of molecules comprising predominantly OsBp moieties having 2 or 3 OsBp moieties, and molecules comprising OsBp moieties at different bases (referred to herein as topoisomers) (Sultan m., kanovarioti, a. (2019); kanovarioti, a. Et al (2012)). Chromatography split molecules carrying one, two or three OsBp moieties and usually also topoisomers (kanovarioti, a. (2016). This is why the HPLC profile of the probe consisted of two separate peaks due to 2-tagged molecules and 3-tagged molecules (fig. 6A). Similarly, the HPLC profile of the hybrid exhibited multiple peaks (fig. 6A). HPLC was also used to test the stability of RNA (see below) and of the hybrid of dmiR21 (OMe) with miRNA21 in sample solvents containing 15% human serum and 85% ont buffer. Fig. 26B shows that the tested RNAs, i.e. miRNA140 and 100nt RNAs, all degrade within minutes while the hybrid peaks remained almost unchanged, indicating that the hybrid formed between our probe and its RNA target was expected to be stable for the duration of the experiment performed in human serum.

Hybridization was consistent with the different HPLC profiles observed with the probe and hybrid samples (fig. 6A). Since dmiR21 (OMe) does not contain 3 adjacent T (OsBp), -an applied voltage of 180mV is sufficient to pass the probe through the well. Many events were reported using the 0.75 nanomolar probe sample, whereas significantly fewer events were reported using the 1.5 nanomolar hybrid sample (fig. 6B, compare solid and dashed lines). The counts using the hybrids appeared to be comparable to those obtained using buffer (not shown). Additional nanopore experiments were performed using the same probe and hybrid loadings, but in the presence of other RNA components. These components are non-target nucleic acids, miRNA140 and 100nt RNA, with a total loading 10-fold higher than the probe. The nanopore profile of probe samples with or without excess non-target RNA is different, indicating influence from excess material and/or aged flowcell. In addition, two experiments performed in the presence of non-target RNA indicated effective identification of the target by comparing the large count of probe samples with the small count of hybrid samples (the second dashed line, almost indistinguishable from the first dashed line (hybrid)). This distinction suggests that the presence of non-target mirnas and longer RNAs in complex mixtures does not hinder target identification.

Because of the importance of mirnas as biomarkers (Li, X-d. Et al (2017), thum, t. Et al (2008), lai, j.y. Et al (2015), kao, h. Et al (2017), fulci, v. Et al (2007), wang, y. Et al (2020)), experiments were performed at ultra-low loadings with probes that were widely applicable to any target sequence and that had optimal translocation properties. Probes 140EXTmU and 21EXTmU targeting miRNA140 and miRNA21, respectively, were selected. These probes have a sequence complementary to their target, with mU replacing the T within the sequence, 3 additional adjacent dts at the 5 'end, and 2 or 3 additional dabs at the 3' end. These oligonucleotides were osmium-tagged using a validated labeling method, adding an average of 4 to 5 OsBp tags per molecule, 3 of which occupied the 5' end, and 1 or 2 additional randomly distributed in sequence (see table 1). Since one end of the probe is severely crowded, an applied voltage in the range of-210.+ -.10 mV is required to achieve effective translocation and detection of the probe (as discussed above). FIG. 7A shows the flight at 47Detection with excellent sensitivity at the molar level with 140EXT (mU), and figure 7B shows detection of this probe at an even higher sensitivity of 3.5 attomoles of probe loading. The identification of the target miRNA140 is also evident by comparing the probe to the hybrid (with the hybrid loading also at 3.5 attomoles). FIG. 7C shows an HPLC profile of a hybrid sample between probe 21EXT (mU) and miRNA21, which is then diluted 3X10 ⁸ Fold was used for nanopore experiments to test hybrids at 2.7 attomoles level (fig. 7D). Diluting a21 EXT (mU) probe sample (HPLC profile not shown) by 1X10 ⁹ Multiple to perform the nanopore experiment at 0.9 attomole level (fig. 7D), which is the lowest loading tested in this study. The identification of miRNA21 is evident in fig. 7D by visual comparison of probe counts to hybrid counts. The reason for the lower count (top, up to 6,000) observed with the 140EXT (mU) probe compared to the count (bottom, up to 24,000) observed with the 21EXT (mU) probe is at least in part due to aging of the flow cell, with only about 30% of the working wells. The proportionality of the probe count for a particular probe could be obtained from the ONT/OsBp platform was not tested here, mainly because the probe concentration is a known quantity. Most importantly, the identification of targets was based on nanopore profiles, which showed meaningless numbers of counts comparable to those of control/buffer experiments. The main reason we test each probe through the nanopore is because we want to compare different probe designs and confirm high detectability and high sensitivity.

Potential of ONT/OsBp nanopore platform

The probe loading in the above experiments ranged from 0.38 nanomolar to 0.9 attomolar, spanning almost 9 orders of magnitude. Within this range, evidence for probe detection is provided, and clearly distinguishes between samples containing the target and samples not containing the target. The lower detectability limit was proposed as a 3-fold higher event count than the count of hybridosomes with the probe alone. The proportionality between event counts and probe concentration was not tested here, as the duration of the experiment remained in the range of 1 to 3h and did not reflect the change in sample loading. Since our test does not detect hybridosomes, the hybrid molecules are not measured, and thus quantification of the target depends on the known probe loading. The test requires an estimate of target loading and, depending on the results of the first experiment, probe loading can be changed by a factor of 5 reduction or elevation in the next experiment. It is estimated that in certain aspects, target quantification can be achieved with an accuracy of about 30%. Many representative probe designs were explored and a design was proposed that could match almost any ssDNA or ssRNA oligonucleotide target. Probes of this type, such as 140EXT (mU) and 21EXT (mU), exhibit high sensitivity at the level of attomolar loading. No attempt was made to test the specificity. Discrimination between one target and another target with similar sequences would require specific problem-specific analysis. The HPLC method developed here can evaluate hybridization between a target and a tentative probe (tentative probe), and it can also evaluate the degree of probe hybridization and tentative differentiation between two targets with similar sequences. Here, the flow cell temperature is a factory preset temperature, but the nanopore protein is known to be stable in a specific temperature range. The flow cell temperature, if at the discretion of the user, can provide a means of improving specificity. We also did not replace proprietary ONT wash buffer. The latter was developed for sequencing and different buffers may be developed to be more suitable for other applications including the present invention.

Preliminary experiments showed that the hybrids of miRNA21 and dmiR21 (OMe) were relatively stable in 15% -85% serum-ONT buffer and probe 122EXT was detectable through the nanopore in 15% -85% serum-ONT buffer. These experiments demonstrate that it is feasible to use the ONT/OsBp nanopore platform with blood serum samples. Considering that the MinION flow cell uses a sample volume of 75. Mu.L and 15% is likely serum, then approximately 11. Mu.L of human serum sample can be tested directly in the nanopore experiment. Assuming that 11 μl of human serum sample contains about 3 attomoles of miRNA21, miRNA140 or experimentally any other miRNA, a probe designed according to the present invention should be able to detect it and as a corollary to detect the presence/absence of the target. In this case, aspects of the invention meet the requirements of a follow-up, point-of-care diagnostic test.

*****

The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

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Sequence listing

<110> Ananasta Kana-Kana tile Li Aodi (Kanavaloti, anastassia)

<120> nanopore platform for DNA/RNA oligonucleotide detection using osmium-labeled probes

<130> 68812-199738

<160> 35

<170> PatentIn version 3.5

<210> 1

<211> 17

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 1

gtaaaacgac ggccagt 17

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 2

cgccagggtt ttcccagtca cgac 24

<210> 3

<211> 17

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 3

caggaaacag ctatgac 17

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 4

agcggataac aatttcacac agg 23

<210> 5

<211> 35

<212> DNA

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<220>

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<400> 5

ttggcactgg ccgtcgtttt acaacgtcgt gactg 35

<210> 6

<211> 30

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<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 6

cagtcacgac gttgtaaaac gacggccagt 30

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 7

tttguaaaac gacggccagu aaa 23

<210> 8

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 8

acaacgtcgt gactgggaaa accctggcgt taccc 35

<210> 9

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 9

gggtaacgcc agggttttcc cagtcacgac 30

<210> 10

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 10

gggtaacgcc agggtttccc cagtcacgac 30

<210> 11

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 11

gggtaacgcc agggtttttc cagtcacgac 30

<210> 12

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 12

tttcgccagg guuuucccag ucacgacaaa 30

<210> 13

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 13

aaacgccagg guuuucccag ucacgacttt 30

<210> 14

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 14

tttcgccagg guuuucccag ucacgacaaa 30

<210> 15

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 15

uagcuuauca gacugauguu ga 22

<210> 16

<211> 37

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 16

uagcuuauca gacugauguu gaaaaaaaaa aaaaaaa 37

<210> 17

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 17

ucaacaucag ucugauaagc ua 22

<210> 18

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 18

tcaacatcag tctgataagc ta 22

<210> 19

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 19

tttcaacatc agtctgataa gcta 24

<210> 20

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 20

ucaacaucag ucugauaagc ua 22

<210> 21

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 21

tttcaacauc agucugauaa gcuaaa 26

<210> 22

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 22

uggaguguga caaugguguu ug 22

<210> 23

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 23

caaacaccau ugucacacuc ca 22

<210> 24

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 24

caaacaccat tgtcacactc ca 22

<210> 25

<211> 44

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 25

caaacaccat tgtcacactc cacaaacacc attgtcacac tcca 44

<210> 26

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 26

tttcaaacac cattgtcaca ctcca 25

<210> 27

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 27

caaacaccau ugucacacuc ca 22

<210> 28

<211> 22

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 28

cagugguuuu acccuauggu ag 22

<210> 29

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 29

ctaccatagg gtaaaaccac tg 22

<210> 30

<211> 44

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 30

ctaccatagg gtaaaaccac tgctaccata gggtaaaacc actg 44

<210> 31

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 31

tttcuaccau aggguaaaac cacugaa 27

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 32

ctctctctct ctctctctct 20

<210> 33

<211> 32

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 33

agagagagcc agagagagcc agagagccuu ca 32

<210> 34

<211> 100

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 34

uuacagccac gucuacagca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 35

<211> 100

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 35

uuacagccac gucuacagca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

Claims

1. A method for detecting the presence of a nucleic acid target molecule in a biological sample, the method comprising the steps of:

(a) Contacting a test sample comprising (i) a biological sample comprising a nucleic acid target molecule, and (ii) an osmium-forming single-stranded oligonucleotide probe comprising at least one compound that is conjugated to substituted or unsubstituted osmium tetroxide (OsO) ₄ ) A pyrimidine residue to which a 2,2' -bipyridine group (OsBp group) is covalently bonded, wherein the sequence of the probe is at least partially complementary to the sequence of the nucleic acid target molecule to allow formation of a hybridized probe/target complex,

optionally, wherein at least one osmium pyrimidine residue is a thymidine residue (T);

(b) Detecting a number of events in the test sample in which unhybridized osmium probes pass through a nanopore using a nanopore device; and

(c) (i) comparing the number of events detected in the test sample with the number of corresponding probe sample events in which non-hybridized osmium probes pass through the nanopore in the absence of the nucleic acid target, wherein a decrease in the number of events detected in the test sample relative to the number of probe sample events is indicative of the formation of the hybridized probe/target complex in step (a) and the presence of the nucleic acid target molecule in the test sample;

(c) (ii) comparing the number of events detected in the test sample to noise of a corresponding baseline sample without any osmium probes, wherein a lack of an increase in the number of events detected in the test sample relative to noise of the baseline sample is indicative of the formation of the hybridized probe/target complex in step (a) and the presence of the nucleic acid molecule in the test sample; and/or

(c) (iii) comparing the number of events detected in the test sample with the number of corresponding control sample events in which unhybridized osmium probe passes through the nanopore in the presence of a known amount of the nucleic acid target molecule, wherein a decrease in the number of events detected in the test sample relative to the number of control sample events indicates an increase in the formation of the hybridized probe/target complex in step (a) and a greater amount of the nucleic acid target molecule is present in the test sample than in the control sample, or wherein an increase in the number of events detected in the test sample relative to the number of control sample events using the same amount of probes indicates more unhybridized probe and thus a lower amount of the nucleic acid target molecule in the test sample than in the control sample;

optionally:

wherein the nanopore device does not require conjugation of the probe to a protein, nanoparticle, homopolymer or polypeptide in order to detect the probe, or wherein the probe is not conjugated to a protein, nanoparticle, homopolymer or polypeptide;

and/or the number of the groups of groups,

wherein detection is not achieved by counting the long-blocking by the hybridized probe/target complex blocking the nanopore or by melting the hybridized probe/target complex in the nanopore.

2. The method according to claim 1, comprising the steps of:

(c) (i) comparing the number of events detected in the test sample with the number of corresponding probe sample events in which non-hybridized osmium probes pass through the nanopore in the absence of the nucleic acid target, wherein a decrease in the number of events detected in the test sample relative to the number of probe sample events is indicative of the formation of the hybridized probe/target complex in step (a) and the presence of the nucleic acid target molecule in the test sample; and/or

(c) (ii) comparing the number of events detected in the test sample with noise of a corresponding baseline sample without any osmium probes, wherein a lack of an increase in the number of events detected in the test sample relative to noise of the baseline sample is indicative of the formation of the hybridized probe/target complex in step (a) and the presence of the nucleic acid molecule in the test sample.

3. The method according to claim 1, comprising the steps of: (c) (i) comparing the number of events detected in the test sample with the number of corresponding probe sample events in which unhybridised probes pass through the nanopore in the absence of the nucleic acid target, wherein a decrease in the number of events detected in the test sample relative to the number of probe sample events is indicative of the formation of the hybridised probe/target complex in step (a) and the presence of the nucleic acid target molecule in the test sample.

4. A method according to any one of claims 1 to 3, wherein the respective probe sample event number is the number of events detected simultaneously in one or more probe samples when detecting the event number in the test sample and/or is a predetermined value for a given amount of probes;

optionally, wherein the predetermined value of the number of probe sample events for a given amount of probe has been empirically or has been determined theoretically;

further optionally, wherein after detecting the number of probe sample events, the osmium probes in the probe sample are combined with a biological sample to produce a test sample, and then the number of events in the test sample is detected for comparison with the number of probe sample events.

5. The method according to claim 1, comprising the steps of: (c) (ii) comparing the number of events detected in the test sample with noise of a corresponding baseline sample without any osmium probes, wherein a lack of an increase in the number of events detected in the test sample relative to noise of the baseline sample is indicative of the formation of the hybridized probe/target complex in step (a) and the presence of the nucleic acid molecule in the test sample.

6. The method of any one of claims 1, 2 or 5, wherein noise of a respective baseline sample is a noise value determined simultaneously in one or more baseline samples and/or predetermined for a baseline sample of a particular composition when detecting the number of events in the test sample;

optionally, wherein the predetermined noise value of the baseline sample has been empirically or has been determined theoretically;

further optionally, wherein after determining noise of a baseline sample, the osmium probe is added to the baseline sample to produce the test sample, and then the number of events of the test sample is detected to be compared to noise of the baseline sample.

7. The method according to claim 1, comprising the steps of: (c) (iii) comparing the number of events detected in the test sample with the number of corresponding control sample events in which unhybridised osmium probe passes through the nanopore in the presence of a known amount of the nucleic acid target molecule, wherein a decrease in the number of events detected in the test sample relative to the number of control sample events indicates an increase in the formation of the hybridised probe/target complex in step (a) and a greater amount of the nucleic acid target molecule in the test sample than in the control sample, or wherein an increase in the number of events detected in the test sample relative to the number of control sample events using the same amount of probes indicates more unhybridised probe and thus a lower amount of the nucleic acid target molecule in the test sample than in the control sample.

8. The method of claim 1 or 7, wherein the respective control sample event number is the number of events detected simultaneously in one or more control samples when detecting the event number in the test sample and/or a predetermined value for a given amount of probe mixed with a given amount of nucleic acid target molecule;

optionally, wherein said predetermined value for the number of control sample events for a given amount of probe mixed with a given amount of nucleic acid target molecule has been determined empirically or has been determined theoretically.

9. The method of any one of claims 1 to 8, wherein the amount of the probe in the test sample is about equal to or less than the amount of target nucleic acid molecule in the test sample.

10. The method of any one of claims 1 to 9, wherein the 2,2' -bipyridine in the OsBp group is substituted;

optionally, wherein the 2,2' -bipyridine in the OsBp group is substituted with one or more methyl or ethyl groups.

11. The method of any one of claims 1 to 10, wherein:

(i) The probe is DNA, and optionally wherein at least one of the sugars in the nucleic acid backbone is 2' -OMe-deoxyribose; or alternatively

(ii) The probe is RNA, and optionally wherein at least one of the sugars in the nucleic acid backbone is 2' -OMe-ribose.

12. The method of any one of claims 1 to 11, wherein the osmium probe has a length of about 12 to 50 nucleotides;

optionally, the portion of the probe that is at least partially complementary to the sequence of the target nucleic acid molecule lacks a continuous self-complementary sequence of more than 2 nucleotides and/or is unstructured/non-self-hybridizing.

13. The method of any one of claims 1 to 12, wherein the nucleic acid target is circulating tumor DNA (ctDNA), cell free DNA (cfDNA), miRNA, fragmented coding RNA, or non-coding RNA, optionally wherein the non-coding RNA is less than about 300 bases in length;

optionally, the composition may be used in combination with,

wherein the nucleic acid target molecule is a single stranded nucleic acid molecule; and/or

Wherein the method further comprises denaturing the double-stranded nucleic acids in the sample in step (a) to form single-stranded nucleic acid strands such that the single-stranded oligonucleotide probes are capable of hybridizing to single-stranded target molecules.

14. The method of any one of claims 1 to 13, wherein:

(i) The osmium probe comprises at least two, three, four, five or six osmium pyrimidine residues, optionally wherein the osmium probe comprises at least two, three, four, five or six osmium thymidine residues (T);

(ii) The osmium probe comprises at least two, three, or four adjacent osmium pyrimidine residues, optionally wherein the osmium probe comprises at least two, three, or four adjacent osmium thymidine residues (T);

and/or the number of the groups of groups,

(iii) The osmium probe comprises one, two, three, four, five, or six adenosine residues (dA or a) at the 5 'end or 3' end of the probe, optionally wherein one or more of the 5 'end or 3' end adenosine residues do not hybridize to the target nucleic acid molecule, optionally wherein none of the 5 'end or 3' end adenosine residues (dA or a) hybridize to the target nucleic acid molecule.

15. The method of any one of claims 1 to 14, wherein:

(i) The osmium probe comprises at least two, three, or four adjacent osmium pyrimidine residues located at the 5 'end or the 3' end of the probe, optionally wherein one or more of the 5 'end or the 3' end pyrimidine residues is not hybridized to the target nucleic acid molecule, optionally wherein none of the 5 'end or the 3' end pyrimidine residues is hybridized to the target nucleic acid molecule;

optionally, wherein the osmium probe comprises at least two, three, or four adjacent osmium thymidine residues (T) located at the 5 'end or 3' end of the probe, optionally, wherein one or more of the 5 'end or 3' end thymidine residues (T) do not hybridize to the target nucleic acid molecule, optionally, wherein none of the 5 'end or 3' end thymidine residues (T) hybridize to the target nucleic acid molecule;

And/or the number of the groups of groups,

(ii) The osmium probe does not contain two or more adjacent osmium pyrimidine residues that are not located at the 5 'end or the 3' end of the probe,

optionally, wherein the osmium probe does not comprise two or more adjacent osmium thymidine residues (T) not located at the 5 'end or 3' end of the probe.

16. The method of any one of claims 1 to 15, wherein at least about 95%, 96%, 97%, 98%, 99% or 100% of pyrimidine residues within the sequence of the oligonucleotide probe molecule that is complementary to the target are not osmium-esterified.

17. The method according to any one of claim 1 to 16,

wherein at least about 95%, 96%, 97%, 98%, 99% or 100% of the thymidine residues (T) in the oligonucleotide probe molecule are osmium,

and/or the number of the groups of groups,

wherein at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the pyrimidines present in the probe are not osmium-esterified with other than thymidine (T).

18. The method of any one of claims 1 to 17, wherein the probe is DNA, but wherein at least one thymidine residue (T) in the probe sequence is replaced by a uridine (U), a 2' -ome (mU) or a deoxyuridine (dU) residue, except for adjacent thymidine residues (T) at the 5' end or the 3' end;

Optionally, wherein at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the thymidine residues (T) in the probe sequence are replaced by uridine (U), 2' -ome (mU) or deoxyuridine (dU) residues, except for adjacent thymidine residues (T) at the 5' or 3' end.

19. The method of any one of claims 1 to 18, wherein the method is performed by reacting a polypeptide comprising a substituted or unsubstituted 2,2' -bipyridine and OsO ₄ The aqueous solution of (osmium reagent) is reacted with an oligonucleotide probe to form 2,2' -bipyridine-OsO ₄ Conjugated probes, and optionally, purifying the conjugated probes from excess osmium reagents to produce the osmium probes;

optionally wherein the 2,2' -bipyridine/OsO in the solution ₄ The ratio is about 0.80/1.0, 0.85/1.0, 0.90/1.0, 0.95/1.0, 0.97/1.0, 0.98/1.0, 0.99/1.0, 1.0/0.99, 1.0/0.98, 1.0/0.97, 1.0/0.95, 1.0/0.90, 1.0/0.85, 1.0/0.80, any range therebetween, nearly equimolar or equimolar.

20. The method of any one of claims 1 to 18, wherein the osmium probe is prepared by combining (dT (OsBp)) _n An oligonucleotide is attached to the 5 'end or the 3' end of the probe, where n is 2, 3 or 4.

21. The method of any one of claims 1 to 20, wherein the nanopore device allows voltage driven translocation of osmium and non-osmium single stranded nucleic acids, but prevents translocation of double stranded nucleic acids; optionally, the composition may be used in combination with,

wherein the nanopore device utilizes nanopores having a minimum pore size of about 1.3 nanometers to about 7.1 nanometers; and/or

Wherein the nanopore device utilizes a Phi29, alpha-hemolysin, aerolysin, mspA, csGg, PA63, clyA, fhuA, or SPP1 protein nanopore, or a bioengineered version of any of the naturally occurring protein nanopores.

22. The method according to claim 21,

wherein a voltage of at least or at least about-180 mV, -190mV, -200mV, -210mV, -220mV, -230mV, -240mV, or-250 mV is applied to detect the presence of the target;

wherein a voltage of between any or any of about-180 mV, -190mV, -200mV, -210mV, -220mV, -230mV, or-240 mV and any or any of about-190 mV, -200mV, -210mV, -220mV, -230mV, or-240 mV, or-250 mV is applied to detect the presence of the target;

wherein a voltage of less than or less than about-200 mV, -190mV, -180mV, -170mV, -160mV, or-150 mV is applied prior to applying the voltage to detect the presence of the target, and/or

Wherein a voltage of between any or any of about-200 mV, -190mV, -180mV, -170mV, or-160 mV and any or any of about 190mV, -180mV, -170mV, or-160 mV, -150mV is applied to the sample prior to applying the voltage to detect the presence of the target in the sample.

23. The method of any one of claims 1 to 22, wherein the method comprises counting events generated by passage of the probe using an algorithm, as reported by a time-of-passage record, to determine whether the probe is free to translocate through the nanopore.

24. The method of any one of claims 1 to 23, wherein the nanopore device allows for distinguishing between different osmium probes and/or multiplexed detection of multiple different nucleic acid targets in a test sample.

25. The method according to any one of claim 1 to 24,

wherein the method is capable of detecting in the test sample less than or less than about 1pM, 100fM, 10fM, 1fM, 100aM, 10aM, 1aM, or 0.1aM of the nucleic acid target;

wherein the method is capable of detecting in the test sample at least or at least about 0.1aM, 1aM, 10aM, 100aM, 1fM, 10fM, 100fM, or 1pM of the nucleic acid target; and/or

Wherein the method is capable of detecting the nucleic acid target in the test sample in an amount between any or any of 0.1aM, 1aM, 10aM, 100aM, 1fM, 10fM, or 100fM and about any or any of 1aM, 10aM, 100aM, 1fM, 10fM, 100fM, or 1 pM.

26. The method of any one of claims 1 to 25, wherein the method is quantitative for the amount of the nucleic acid target molecule in the test sample and/or biological sample.

27. An osmium single-stranded oligonucleotide probe molecule comprising at least one oligonucleotide that is linked to substituted or unsubstituted osmium tetroxide (OsO ₄ ) -a pyrimidine residue covalently bonded to a 2,2' -bipyridine group (OsBp group);

optionally, wherein at least one osmium pyrimidine residue is a thymidine residue (T)

28. The probe of claim 27, wherein the OsBp group is substituted;

optionally wherein the 2,2' -bipyridine in the OsBp group is substituted with one or more methyl or ethyl groups.

29. The probe of claim 27 or 28, wherein:

30. The probe of any one of claims 27 to 29, wherein the osmium probe has a length of about 12 to 50 nucleotides;

optionally, wherein the probe lacks a continuous self-complementary sequence of more than 2 nucleotides and/or is unstructured/non-self-hybridizing.

31. The probe of any one of claims 27 to 30, wherein:

(ii) Wherein the osmium probe comprises at least two, three, or four adjacent osmium pyrimidine residues, optionally wherein the osmium probe comprises at least two, three, or four adjacent osmium thymidine residues (T);

and/or the number of the groups of groups,

(iii) Wherein the osmium probe comprises one, two, three, four, five, or six adenosine residues (dA or a) at the 5 'end or 3' end of the probe, optionally wherein one or more of the 5 'end or 3' end adenosine residues (dA or a) do not hybridize to the target nucleic acid molecule, optionally wherein none of the 5 'end or 3' end adenosine residues (dA or a) hybridize to the target nucleic acid molecule.

32. The probe of any one of claims 27 to 31, wherein:

(i) The osmium probe comprises at least two, three, or four adjacent osmium pyrimidine residues at the 5 'or 3' end of the probe, optionally wherein one or more of the 5 'or 3' end pyrimidine residues does not hybridize to the target nucleic acid molecule,

and/or the number of the groups of groups,

33. The probe of any one of claims 27 to 32, wherein at least about 95%, 96%, 97%, 98%, 99% or 100% of pyrimidine residues in the oligonucleotide probe molecule that are not adjacent pyrimidine residues at the 5 'or 3' terminus are not osmium-esterified.

34. The probe according to any one of claim 27 to 32,

and/or the number of the groups of groups,

35. The probe of any one of claims 27 to 34, wherein the probe is DNA, but wherein at least one thymidine residue (T) in the probe sequence is replaced by a uridine (U), a 2' -OMe-uridine (mU) or a deoxyuridine (dU) residue, except for adjacent thymidine residues (T) at the 5' end or the 3' end;

optionally, wherein at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the thymidine (T) residues in the probe sequence are replaced by uridine (U), 2' -OMe-uridine (mU) or deoxyuridine (dU) residues, except for adjacent thymidine residues (T) at the 5' or 3' end.

36. The probe of any one of claims 27 to 35, wherein:

(i) The osmium probe comprises at least two, three or four adjacent osmium thymidine residues (T) located at the 5' end of the probe;

(ii) The probe is DNA, but wherein the thymidine residue (T) in the probe sequence is replaced by a uridine (U), 2 '-OMe-uridine (mU) or deoxyuridine (dU) residue except for the adjacent thymidine residue (T) at the 5' end; and optionally, the presence of a metal salt,

wherein at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the pyrimidines present in the probe are not osmium-bound except for thymidine (T); and/or

Wherein at least one of the sugars in the nucleic acid backbone is 2' -OMe-deoxyribose.

37. The probe of any one of claims 27 to 36, wherein the probe is prepared by reacting a probe comprising a substituted or unsubstituted 2,2' -bipyridine and OsO ₄ The aqueous solution of (osmium reagent) is reacted with an oligonucleotide probe to form 2,2' -bipyridine-OsO ₄ Conjugated probes, and optionally, purifying the conjugated probes from excess osmium reagents to produce the osmium probes;

optionally wherein the 2,2' -bipyridine/OsO in the solution ₄ The ratio is about 0.80/1.0, 0.85/1.0, 0.90/1.0, 0.95/1.0, 0.97/1.0, 0.98/1.0, 0.99/1.0, 1.0/0.99, 1.0/0.98, 1.0/0.97, 1.0/0.95, 1.0/0.90, 1.0/0.85, 1.0/0.80, nearly equimolar or equimolar.

38. The probe according to any one of claims 27 to 36, wherein the probe is prepared by combining (dT (OsBp)) _n An oligonucleotide is ligated to the 5 'or 3' end of the probe to prepare the osmium probe, where n is 2, 3, or 4.

39. A kit comprising the probe of any one of claims 27 to 38 and a control nucleic acid target molecule capable of hybridizing to the probe.

40. The kit of claim 39, wherein the control nucleic acid target comprises ctDNA, cfDNA, miRNA, fragmented nucleic acid sequences encoding RNA or non-encoding RNA, optionally wherein the non-encoding RNA is less than about 300 bases in length.

41. Use of the probe of any one of claims 27 to 38 for detecting a nucleic acid target molecule using a nanopore device, wherein the nucleic acid target molecule is optionally ctDNA, cfDNA, miRNA or a non-coding RNA, optionally wherein the non-coding RNA is less than about 300 bases in length.