Enrichment or depletion of RNA targets in biological samples

Abstract

The method for enriching or depleting target from sample containing analyte is described.Probe has one of left-handed PNA pair and targeting part, wherein said left-handed PNA pair is a complementary pair of PNA with cyclic backbone modification that induces chiral and left-handed helical structure.Capture surface has the other of said left-handed PNA pair; said left-handed PNA pair is bound to capture surface (can be magnetic beads) to hybridize said probe.

Description

[Technical Field]

[0001] Technical Field The present invention relates to methods and devices for capturing target molecules in biological samples. [Background Art]

[0002] Background Methods for RNA targeting and amplification often employ enzymatic amplification methods, for example, polymerase chain reaction (PCR). Proper storage and handling of reagents are required for these methods. Furthermore, enzyme functionality is critical to ensuring the success of the amplification process. Another challenge is that enzymes are temperature-sensitive and have limited shelf life.

[0003] Sequence-specific RNA enrichment or depletion may be achieved using a biotin-avidin (or generally, streptavidin or neutravidin) affinity reaction, in which a biotinylated probe having a sequence complementary to the sequence of interest is captured on a streptavidin-functionalized surface, for example, microspheres.

[0004] Biotin-avidin is widely used in applications such as enzyme-linked immunosorbent assay (ELISA); immunohistochemistry (IHC); Western blotting, Northern blotting, and Southern blotting; immunoprecipitation; cell surface labeling; affinity purification; fluorescence-activated cell sorting (FACS); and electrophoretic mobility shift assay (EMSA). A notable limitation is that since all biotinylated molecules can bind to any biotin-binding molecule, reagents must be used in combination with other detection-probe systems, for example, primary-secondary antibodies, for multiplex experiments. Importantly, naturally occurring biotin in samples has been found to cause false results in several tests, and has been the subject of a warning by the USFDA requiring contact and discussion with assay developers. Biotin-containing dietary supplements are particularly problematic.

[0005] Furthermore, the storage and transport of streptavidin reagents present logistical challenges due to the need for refrigeration. Sodium azide (potentially an explosive) is commonly used to prevent bacterial growth during long-term storage, and extensive washing processes are required to remove it from the solution before use to prepare the beads and biopharmaceuticals. [Overview of the project] [Means for solving the problem]

[0006] Abstract The present invention provides a method for capturing target molecules in a biological sample. The method of the present invention utilizes peptide nucleic acid (PNA) probes for the capture and delivery of target molecules. In a preferred embodiment, a first chiral PNA probe containing a capture moiety is used to capture a target analyte and is then bound to a complementary second chiral PNA probe bound to a solid support. In a preferred embodiment, the PNA probes are left-handed chiral pairs. The PNA probes of the present invention may be bound to the binding moiety via a linker. The binding moiety may be nucleic acids, glycols, proteins (including antibodies or enzymes), small molecules, carbohydrates or lectins, or any other binding factor that can bind to the target molecule. In one example, a PNA probe bound to a solid support captures another PNA probe via hybridization. That other PNA probe is then bound via a linker to another PNA probe terminated at the binding moiety. The PNA probes preferably have left-handed chirality, and as a result, they hybridize with their PNA complementary binding pairs rather than with native RNA or DNA. The present invention utilizes this approach to specifically capture target molecules in biological samples. The probe may include a linker that connects the left-handed PNA with the targeting portion. In an alternative embodiment, the capture probe may be a right-handed PNA molecule.

[0007] The solid support to which these "keychain" PNA molecules are bound can be any suitable solid support. For example, the solid support may be the surface of beads or magnetic particles. Thus, in certain embodiments, a magnetic field may be used to manipulate (e.g., move or detect) the analyte bound to the capture portion. In some cases, the solid support may be associated with a sensor or sensor surface to sense the bound analyte. Advantageously, this arrangement allows for rapid detection of the analyte by flowing or passing a sample of the analyte, bound to the capture portion of the first chiral PNA molecule, over a sensor surface having a second chiral PNA molecule complementary to the first chiral PNA molecule. The capture of the first chiral PNA molecule by the complementary chiral PNA molecule provides analyte detection. Thus, the method of the present invention is well suited for use with microfluidic systems. Furthermore, since the above chiral PNA molecules do not bind to other analytes (e.g., DNA or RNA), the detection method using the above keychain PNA molecules provides reliable measurement of analytes in a sample.

[0008] The defining characteristics of PNA are its stability and versatility. Therefore, methods using PNA molecules to probe analytes offer several clear advantages over conventional DNA and RNA methods. For example, PNA molecules are resistant to degradation by nucleases and proteases and remain stable even in acidic environments. This enhanced stability allows PNA molecules to capture targets even under suboptimal conditions (in acidic or low-salt solutions). Thus, the method of this invention, by relying on PNA molecules, can capture analytes from samples (e.g., cell lysates) that would be difficult to treat by other methods. Furthermore, PNA molecules have a long shelf life (several months to several years) compared to the six-month shelf life of DNA, RNA, or streptavidin, and because they are resistant to nuclease degradation, refrigeration is not required.

[0009] The method of the present invention does not require enzymes for nucleic acid detection or enrichment. Therefore, the method described herein is not constrained by the same strict operating conditions as similar methods involving enzymes. Furthermore, in most cases, the reagents used in the method described herein do not require refrigeration. Therefore, the method of the present invention can be used anywhere (e.g., in remote locations) and by anyone. For example, the method of the present invention can be carried out over a wide range of temperatures (e.g., in the range of about 18°C ​​to about 80°C). However, in a preferred embodiment, the method of the present invention is carried out at about 37°C.

[0010] The PNA probe of the present invention uses a capture portion to bind an analyte of interest. In some cases, the analyte may be a protein or a cell surface antigen. The capture portion may therefore contain an antibody or antibody fragment. The antibody or antibody fragment of the capture portion may be used to capture an antigen associated with the analyte of interest. The PNA probe having the antibody capture portion is preferably chiral. During use, a second chiral PNA probe complementary to the antibody-conjugated PNA probe may be used to capture the antibody-conjugated PNA probe bound to the target analyte.

[0011] The PNA probe of the present invention may be part of a PNA chain. The PNA chain may contain any number of nucleic acid monomers. Advantageously, since PNA is generally uncharged, it associates with a higher binding strength than similar oligonucleotides of DNA or RNA. Thus, the PNA probe can capture a complementary target with fewer monomers than similar oligonucleotides of DNA or RNA. In some embodiments, the PNA probe of the present invention contains 15 or fewer nucleic acid monomers. Furthermore, in certain embodiments described herein, the method of the present invention aims to modify the charge of the PNA. Preferably, the PNA is chiral. Modifying the charge of the PNA molecule (e.g., negatively charging the PNA molecule) is useful to reduce the binding affinity of the PNA to other negatively charged nucleic acids (e.g., DNA or RNA), thereby increasing the binding specificity to complementary chiral PNA.

[0012] The method of the present invention is particularly useful for multiplexing. Since the left-handed chiral PNA probe of the present invention specifically binds to complementary sequences of other left-handed chiral PNA probes (and not to other nucleic acids), the probe is easily programmable for multiplexing. For example, the length and / or monomer sequence of the left-handed PNA probe may be altered to differentially bind to other complementary probes. Thus, according to several methods, multiple different left-handed chiral PNA probes (bound to different target analytes) can be differentially captured and / or detected by corresponding left-handed PNA probes containing complementary sequences.

[0013] In some embodiments, the PNA probe of the present invention includes a linker. Preferably, the linker connects the PNA probe to the capture portion. The linker may include a cleavable bond. For example, the linker may include a bond that is cleavable by a protease, thereby allowing the captured analyte to be separated from the PNA probe for downstream processing. The linker may be 1 to 120 atoms long and / or may contain one or more of the elements: C, N, O, S, P, and Si. The linker may be in a chain containing one or a combination of the following: single bonds, double bonds, triple bonds, amide bonds, ester bonds, disulfide bonds, imino groups, ether bonds, thioether bonds, and thioester bonds.

[0014] In another aspect, the present invention relates to a targeted enrichment or depletion apparatus for carrying out the method of the present invention. The apparatus includes a chamber for binding an analyte to a probe containing left-handed PNA molecules coupled to a capture portion. The chamber may provide a surface containing a complementary left-handed PNA probe bound to the analyte. The surface may consist of one or more beads. The one or more beads may be releaseable from a compartment of the apparatus. For example, the beads (having the PNA probe) may be packaged in a heat-sensitive substrate (e.g., wax) inside a compartment of the chamber. The beads may be released into the chamber after the probe has bound to the analyte by applying heat. Preferably, the PNA includes a pair of complementary PNAs having a chiral and cyclic skeletal modification that induces a left-handed helical structure. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 shows two modified monomers useful for inducing chirality.

[0016] [Figure 2]Figure 2 shows a process for a multiplexed assay using biotin-streptavidin magnetic beads.

[0017] [Figure 3] Figure 3 shows a multiplexed assay using left-handed PNA.

[0018] [Figure 4] Figure 4 is a schematic diagram showing hybridization of a probe (P) to magnetic beads.

[0019] [Figure 5] Figure 5 shows target capture by hybridization.

[0020] [Figure 6] Figure 6 shows additional steps of an assay for target capture.

[0021] [Figure 7] Figure 7 illustrates the use of right-handed targeting PNA.

[0022] [Figure 8] Figure 8 shows further steps of the method shown in Figure 7.

[0023] [Figure 9] Figure 9 shows an assay involving targeting antibodies.

[0024] [Figure 10] Figure 10 is a continuation of the assay of Figure 9.

[0025] [Figure 11] Figure 11 shows data for capture and tethering of 18s rRNA from total human RNA.

[0026] [Figure 12] Figure 12 shows data for capture and tethering of 18s rRNA from total human isolates.

[0027] [Figure 13] Figure 13 shows data on the detection of 18s RNA from total human RNA.

[0028] [Figure 14] Figure 14 is a plot showing very similar performance of left-handed PNA capture of RNA compared to the previous biotin-streptavidin approach.

[0029] [Figure 15] Figure 15 shows the capture data for left-handed and right-handed PNAs.

[0030] [Figure 16] Figure 16 shows the agarose gel from the 18s ribosomal RNA depletion assay using a whole human RNA sample. [Modes for carrying out the invention]

[0031] Detailed explanation This invention relates to an assay for capturing target molecules from biological samples. More specifically, the invention relates to an assay using a modified peptide nucleic acid (PNA) for capturing a target analyte. Preferably, the PNA is modified by incorporating a specific monomer (described below) to induce chirality. This invention utilizes the induced chirality to manipulate the binding affinity of the PNA to non-PNA molecules. In particular, a preferred method of this invention is to induce chirality in the PNA to reduce the binding affinity of the PNA to DNA and / or RNA in the sample, thereby increasing the selectivity of the PNA to other modified PNA molecules.

[0032] PNAs are oligonucleotide analogs in which the sugar-phosphate backbone is replaced by a pseudopeptide backbone. The PNA backbone typically contains uncharged repeats of N-(2-aminoethyl)glycine units (AEGs) linked by peptide bonds. The synthetic backbone provides PNAs with many beneficial properties, such as low dependence on ionic strength, high chemical stability, high sequence specificity, and resistance to both nucleases and proteases. In some cases, they bind to DNA and / or RNA with high specificity and selectivity, resulting in PNA-RNA and / or PNA-DNA hybrids that are more stable than their corresponding nucleic acid complexes. For further considerations regarding nucleic acid hybridization using PNA, see Ratilainen, 1998, Hybridization of Peptide Nucleic Acid, Biochemistry 1998, 37, 35, 12331-12342, and Weiler, 1997, Hybridisation based DNA screening on peptide nucleic acid (PNA) oligomer arrays, Nucleic Acids Research, Volume 25, Issue 14, 1 July 1997, Pages 2792-2799 (each of these is cited for reference).

[0033] The binding affinity and selectivity of the PNA to nucleic acids (e.g., RNA) can be modified by introducing a chiral stereocenter (e.g., a D-Lys-based unit) into the PNA backbone, and thus, it is a insight of the present invention that modified PNAs are ideal candidates for target capture methods. In a preferred embodiment, the method of the present invention uses a modified PNA synthesized to have left-handed chirality.

[0034] Naturally occurring nucleotides such as DNA and RNA possess right-handed chirality, and among them, achiral and right-handed PNAs can bind to these nucleotides and be used to target them. However, naturally occurring right-handed oligonucleotides generally do not bind to left-handed chiral PNAs due to incompatibility of their helical structures, which prevents conventional Watson-Crick base pairing. Therefore, left-handed PNAs can generally only bind to other left-handed PNAs or achiral PNAs. Consequently, chiral PNAs associate with PNA-DNA double helixes, which are less stable than their achiral analogs. This effect may be more pronounced in the methods of the present invention, which use a modified PNA skeleton containing negatively charged monomers (based on D- or L-aspartic acid and gluaminic acid) that cause repulsive interactions with amino acids or negatively charged phosphate groups of DNA that have bulky nonpolar side chains.

[0035] These properties make left-handed PNA an ideal candidate for use in capture systems, in which case the components of the capture system do not interact with naturally occurring nucleotides. In particular, left-handed PNA is used as a component of a probe according to the method of the present invention. The probe also includes a targeting portion and, preferably, a linker that connects the left-handed PNA to the targeting portion. In certain embodiments, the left-handed PNA is also used as a component of a bead or immobilized on a sensor surface for detection of the target.

[0036] Figure 1 shows two modified monomers useful for inducing chirality. In particular, two enantiomers of cyclic PNA skeleton modified monomers 103 and 105 are shown, which induce chirality of (a) left-handed 103 and (b) right-handed 105. The helical structure of PNA can be influenced by including these monomers within the above PNA sequence.

[0037] The above PNA can be synthesized by methods known in the art, for example, as described in Wu, 2017, Recent advances in peptide nucleic acid for cancer bionanotechnology, Acta Pharmacol ogica Sinica volume 38, pages 798-805 (this is incorporated by reference). Preferably, the above PNA is prepared using one or more of the modified monomers. A PNA sequence without any of the above modified monomers may not have a preferred helical structure and may therefore be achiral. If at least one cyclic skeleton-modified monomer 103, 105 is included, the constrained skeleton structure induces either left-handed (a) or right-handed (b) chirality in the above PNA. Inclusion of one or more monomers may be desired to ensure chirality preference over the entire length of the PNA. The chirality of the above PNA can be evaluated by various techniques known in the art. For example, PNA synthesized to contain one or more of the above monomers can be characterized by nuclear magnetic resonance or by X-ray crystallography.

[0038] The present invention involves incorporating a stereocenter into PNA to form a left-handed PNA pair. The left-handed pair refers to a complementary pair of PNAs having at least one cyclic skeleton-modifying monomer that is chiral and induces a left-handed helical structure. These are sometimes referred to herein as “left-handed PNA pairs.” They should not be confused with γPNA (which, although having a different skeleton chemistry, can also be left-handed).

[0039] The left-handed PNA described above is an enantiomer of the right-handed cyclic modified PNA skeleton, which can be used as the targeting moiety of nucleic acids. However, as described below, the targeting moiety or capture moiety is not limited to PNA.

[0040] Left-handed PNA pairs are used in various ways to provide targeted enrichment of biological entities (e.g., RNA) in assays, where the target molecule in the sample is bound to a surface, for example, beads (e.g., magnetic beads) or a sensor (e.g., a capacitive sensor). The left-handed PNAs described above can also be used to remove relatively abundant targets in depletion-type assays, leaving rarer targets for further detection or analysis.

[0041] For example, in a preferred embodiment, a first chiral PNA molecule linked to a capture moiety is used to capture a target molecule (e.g., RNA). The first chiral PNA molecule, bound to the target molecule via the capture moiety, is then captured by a complementary chiral PNA molecule preferably bound to a solid support. In a preferred embodiment, the PNA molecules are left-handed chiral pairs. The PNA molecules of the present invention may be bound to the capture moiety via a linker. The capture moiety may be any moiety that binds to the target molecule, for example, the capture moiety may be a nucleic acid, glycol, protein (e.g., an antibody or antibody fragment), small molecule, carbohydrate or lectin, or any other molecular binding factor that can bind to the target molecule.

[0042] In one example, a PNA probe bound to a solid support captures another PNA probe via hybridization. This other PNA probe then binds to another PNA probe terminated at the binding site via a linker. The PNA probes preferably have left-handed chirality and therefore specifically hybridize with complementary chiral PNA binding partners but not with native RNA or DNA. The present invention utilizes this approach to specifically capture target molecules in biological samples.

[0043] Advantageously, in some cases, the PNA probe is first hybridized to its target sequence (in one example, 18s ribosomal RNA) and then to the beads using a left-handed PNA pair. This is referred to herein as the “two-step” approach. This approach achieves significantly improved target enrichment compared to the approach in which the probe is first bound to the beads.

[0044] In some cases, the probe is bound to the sensor surface. For example, the probe may be bound to the sensor surface as described in WO 2015 / 086654; WO 2015 / 091139 (these are incorporated herein by reference). The binding generally requires a left-handed PNA containing a capture portion that hybridizes to a left-handed PNA complement bound to the surface. Achiral PNA may replace one or both of the left-handed PNA probes in this application. Achiral PNA exhibits similar hybridization behavior to complementary achiral, right-handed, or left-handed PNAs; however, the removal of the skeletal modification that induces left-handedness in one or both of the complementary pairs of PNAs may eliminate the abiotic nature of probe capture and introduce potential cross-hybridization with endogenous oligonucleotides.

[0045] Figure 2 shows the process for a multiplexing assay using biotin-streptavidin magnetic beads. The above process provides a multiplexing assay using a biotin-streptavidin magnetic bead system for comparison with the present invention. A biotinylated probe (P1) for target 1 is incubated in the sample (I), followed by the addition of streptavidin-coated magnetic beads (II). The beads containing the captured probe and target are removed. The same process of probe incubation and target removal with streptavidin-coated magnetic beads is then performed for the subsequent target (III-VI). The target-probe complex is then removed from the beads (VII). Unlike left-handed PNA systems, due to the fact that the capture portion is biotin-streptavidin in all steps, there may be potential interference from endogenous biotin molecules and cross-contamination from probes / targets not completely removed from the previous steps. Left-handed PNA systems are not affected by endogenous or dietary biotin, as shown in Figure 3.

[0046] Figure 3 shows a multiplexing assay using left-handed PNA. The figure demonstrates the ability to perform the multiplexing assay of the present invention by utilizing the programmability of the left-handed PNA system. In this example, there are three probe-bead sets, each having different targets (P1-3) and different left-handed PNA pairs (L1-3, L1c-3c). All three probes can be incubated simultaneously in the sample (I). Magnetic beads coated with complementary left-handed PNA are then used sequentially to remove the complementary probes and targets without cross-contamination from other probes or native biomolecules (II-IV). The bead-probe-target complexes can be washed, and the probes and targets can be separated from the beads by heating for further analysis (V). Since each target is captured using a specific probe pair, cross-contamination between probes is minimal, and due to the left-handed chirality of the PNA, there is no interference from native DNA / RNA.

[0047] Probes having left-handed PNAs may either have the same left-handed PNA for all probes or each probe may have a different left-handed sequence, and as a result, different sets of beads functionalized with their complements may be used to capture specific targets. Thus, the method of the present invention can be highly customized for various multiplexing applications. The above PNA monomer sequences may be modified to any arrangement of the sequence, and they have no interaction with naturally occurring nucleic acids when removed by heating for stringency, for example, as shown in Figure 15.

[0048] Figure 4 is a schematic diagram showing the hybridization of a probe (P) to a magnetic bead. In particular, two examples, I and II, are shown, where example I shows the components of the system, and example II shows the interactions of those components.

[0049] The left-handed PNA (L) on the probe (P) is hybridized to a magnetic bead (M) functionalized with a left-handed PNA complement (Lc), where the complementary pair of PNAs is chiral and has an annular skeletal modification that induces a left-handed helical structure. In some embodiments, a pair of left-handed PNAs is used for hybridization. A probe (P1) hybridized to a magnetic bead (M) is illustrated, which is common to many embodiments of the present invention. Hybridization is effective and, for example, allows the use of the beads in a sample and the use of a separate probe already bound to the target and / or detection portion, or alternatively, it can be bound after hybridization to the beads.

[0050] In another example, a fixed substrate (i.e., a surface) is functionalized with left-handed PNA(Lc), and a probe (e.g., probe P) hybridizes to the surface or is already bound to the target for subsequent target capture, and capture on the fixed surface enables sensing. The sensing may be capacitive on the sensor, for example, or by radioactive or non-radioactive detection.

[0051] Figure 5 illustrates target capture by hybridization. Specifically, Figure 5 shows (I) hybridization of the first probe P1 to the target by the targeting portion (sometimes referred to herein as the capture portion), and (II) hybridization of the probe to the magnetic bead M by a left-handed PNA pair. In this case, the probe P1 is already bound to the target, and then binding to the bead allows the target to be carried, for example, in a microfluidic flow.

[0052] Figure 6 illustrates a further step in target acquisition. In particular, Figure 6 (which progresses from Figure 5) shows (III) a second probe (P2) binding to the target, and (IV) the hybridization of the second probe to a detection portion by a left-handed PNA pair. This allows for detection by a type of fluorescence sensor well known in the art. The detection portion may be any known type suitable for the application.

[0053] This example demonstrates how a series of entities involved in an assay can be joined together using left-handed PNA pairs, in this case the bead-probe and probe-detection portions, with the linkage between both probes to the target.

[0054] Figure 7 illustrates the use of a right-handed targeted PNA. In particular, Figure 7 shows the use of a probe (P1) having a right-handed targeted PNA, a linker, and a left-handed PNA, where (i) the probe hybridizes to a target nucleic acid sequence by its right-handed targeted PNA, and (II) the probe hybridizes to the beads by a left-handed PNA pair. This illustrates that the probe may have a left-handed PNA pair, a linker, and one of any suitable binding entities (e.g., in this case, a right-handed targeted PNA R1).

[0055] Figure 8 shows a further step of the method shown in Figure 7. In particular, Figure 8 is an extension of the mechanism of Figure 7, where (III) the probe (P2) has a second right-handed targeted PNA (R2) that binds to the target NA, and (IV) the detection portion is coupled by a second left-handed PNA pair.

[0056] Figure 9 shows an assay involving a targeted antibody. Specifically, Figure 9 shows an assay in which (I) probe P1 has a targeted antibody, a linker, and a left-handed PNA, and (II) it hybridizes to a bead (M) by the left-handed PNA.

[0057] Figure 10 is a continuation of the assay in Figure 9. In particular, Figure 10 shows the evolution of the assay from Figure 9, where (III) the above probe (P1) antibody binds to a second probe (P2) having a detection portion or a second left-handed PNA (L2), and (IV) the above second probe binds to a second antibody having a detection portion or a second left-handed PNA complement.

[0058] The enrichment of RNA and tethering with fluorescent achiral PNA are achieved in various examples. The use of left-handed PNA pairs has been shown to efficiently capture long and structurally complex RNA. In the first step described above, many assays benefit from the mechanism by which a PNA probe free in solution hybridizes with the target RNA. In the second step, for example, magnetic beads functionalized with left-handed PNA complementary to the left-handed PNA probe bound to the target RNA are introduced, in the form of microspheres (e.g., "Dynabeads").

[0059] 18s ribosomal RNA is a long and structurally complex RNA, which is used as an endogenous control in many RT-qPCR reactions due to its abundance and consistent expression across cells, even in influenza-infected cells. This makes 18s ideal for normalization at target sequences. Therefore, for the same reasons as its use in PCR, it is important in this assay as well. Using the two-step hybridization described above, 18s ribosomal RNA can be captured from whole human RNA isolates far more efficiently than using capture achiral or right-handed PNA covalently bound to the microspheres described above. Furthermore, 18s ribosomal RNA can be enriched from different biological backgrounds (either blood or saliva) using this method.

[0060] In one aspect, the present invention provides a method for target enrichment. The method comprises the step of introducing a probe containing a left-handed PNA molecule ligated to a capture portion into a sample containing a target analyte. The sample may be any biological sample. For example, the sample may be a fluid sample taken from a subject (e.g., blood, saliva, or urine). The target analyte preferably contains RNA. The RNA may be pathogen RNA. For example, in a preferred embodiment, the target RNA is 18S ribosomal RNA (abbreviated as 18S rRNA). Preferably, the 18S rRNA contains a variable nucleic acid sequence for use in identifying and / or characterizing a biological species, as described, for example, in Hadziavdic, 2014, characterization of the 18S rRNA Gene for Designing Universal Eukaryote Specific Primers, PLoS One 9(2): e87624 (as incorporated by reference).

[0061] The probe of the present invention comprises a left-handed PNA molecule including a capture portion, the capture portion preferably being achiral or having right-handed chirality. The left-handed PNA molecule may include a single-stranded PNA sequence containing a sequence complementary to the partner left-handed PNA probe. The sequence may contain many nucleotide bases (e.g., 2, 5, 10, 15, 20, 25, or more).

[0062] The above method includes the step of capturing the target analyte with the capture portion. The capture step may include binding the capture portion to the target analyte by complementary base pairing. The capture portion may include an oligonucleotide having a sequence complementary to the analyte. The oligonucleotide may include DNA or RNA. The capture portion may include right-handed PNA. The capture portion may be linked to a left-handed PNA molecule by a linker. The linker may contain one or more nucleic acids. The capture step may include contacting a sample containing the target analyte with the PNA probe under conditions that allow the capture portion probe sequence to hybridize with its complementary sequence of the target analyte. The probe may be labeled with a radioactive or chemical tag that allows its binding to be visualized.

[0063] The above method further comprises the step of binding the left-handed PNA molecule with a complementary PNA molecule containing a left-handed chiral structure. The complementary PNA molecule is preferably bound to a surface, thereby enriching the target analyte. The surface may be the surface of a bead. The bead may be a magnetic bead (for example, a magnetic bead sold by ThermoFisher under the trade name Dynabead). Alternatively, the surface may include a surface associated with a sensor for detecting the analyte.

[0064] In one aspect, the present invention relates to a target enrichment or depletion apparatus for carrying out the method of the present invention. The apparatus includes a chamber for binding an analyte to a probe containing left-handed PNA molecules coupled to a capture portion. The chamber may provide a surface containing a complementary left-handed PNA probe to bind to the analyte. The surface may be one or more beads (e.g., magnetic beads). The one or more magnetic beads may be ejectable from a compartment of the apparatus. The compartment may be a compartment in the lid of the apparatus. For example, the beads (having the PNA probe) may be packaged in a heat-sensitive substrate (e.g., wax) inside the compartment of the chamber. The beads may be ejected into the chamber by heating the apparatus after the probe has bound to the analyte by applying heat. Ejecting the beads containing the complementary probe effectively anchors the analyte to the magnetic beads. A magnet associated with the apparatus may be used to draw the beads bound to the analyte into molten wax (which can be re-solidified by cooling). Preferably, the PNA comprises a pair of complementary PNAs having a chiral and left-handed helical cyclic skeletal modification. For example, the apparatus may be a sample apparatus as described in WO / 2015 / 086652 or 2016 / 091868 (each of which is incorporated by reference). [Examples]

[0065] Examples We designed PNA probes using 18s ribosomal RNA (rRNA) as the target sequence. We synthesized one of these designed probes (referred to as 458R, and also called the 458R sequence in this specification) in three versions.

[0066] The first PNA, "458R," was an achiral PNA. The second was "458R RH," a right-handed chiral version with the same sequence as 458R. The third was a PNA probe (referred to as "LH PNA-458R RH") with a 458R RH PNA at one end, coupled to a left-handed chiral PNA. Superparamagnetic Dynabeads (1 μm) were directly functionalized with either the 458R or 458R RH PNA, and in the case of "LH PNA-458R RH," the complementary of the left-handed PNA sequence was functionalized on the beads.

[0067] result Previous data with fluorescently labeled short synthetic RNA sequences (28 nucleotides) showed that, for a complete set of beads functionalized with 5 μM PNA, capture of approximately 80% of the RNA in PBS containing 0.05% Tween® 20 at 57°C. These beads were then tested with actual 18s ribosomal RNA derived from whole human RNA isolates. 1 μg (or 0 μg in the control well) of whole human RNA was used with Dynabeads functionalized with 458R or 458R RH. TM The 18s rRNA was either directly hybridized to the 18s rRNA for 10 minutes, or, alternatively, first hybridized to the "LH PNA-458R RH" PNA probe for 10 minutes, followed by incubation with the left-handed complementary beads for another 10 minutes (all at 57°C in PBS buffer containing 0.05% Tween® 20). To measure the amount of RNA specifically captured by the beads, Atto488 fluorescently labeled achiral PNA was hybridized with the captured 18s rRNA for 10 minutes at 57°C.

[0068] After washing the beads to remove nonspecifically bound RNA, fluorescent PNA was hybridized, and the specific 18s RNA was then transferred to the wells using the magnetic Dynabeads. After tethering the fluorescently labeled PNA to the 18s rRNA, the beads were washed again to remove nonspecifically bound PNA. Finally, the hybridized PNA was eluted from the RNA on the Dynabeads using a 10 mM potassium hydroxide solution and heating at 95°C for 2 minutes. The eluted PNA was fluorescence-measured using a plate reader, and the experimental results are shown in Figure 11. This demonstrates the capture and tethering of 18s rRNA from whole human RNA using different PNA capture systems.

[0069] The data from Figure 11 shows that in all cases, fluorescence was greater than in the control well without RNA, indicating that some 18s rRNA was captured from the total human RNA isolate by each set of beads. For 458R PNA, the difference between the above RNA and the control well without RNA was 0.12, and for 458R RH PNA beads, it was similar, with a fluorescence unit of 0.13. However, the largest difference was 0.41 fluorescence units, observed for the two-step system. Only 458R captured 40% of the amount of RNA captured by the "LH PNA-458R RH" set.

[0070] Tethering using fluorescent achiral PNA was used in all experiments to mimic a second PNA probe that could be used instead in the capture system described above. One advantage of using a left-handed PNA system is that RNA enrichment or depletion can be performed isothermally at a biocompatible temperature. An experiment demonstrating this was performed, referring to Figure 12. In this experiment, hybridization of the probe and the Dynabeads was performed at 37°C.

[0071] Figure 12 shows the capture and tethering of 18s rRNA from whole human isolates at 37°C (N=3), with the trend line representing the mean value (gray dots).

[0072] A linear trend was observed with respect to 18s rRNA tethering at 37°C (normal body temperature), suggesting that capture and tethering do not rely on high temperatures for RNA hybridization. The amounts of PNA eluted for 1 μg and 0 μg of RNA were similar to those previously found, as shown in Figure 11, performed at 57°C, suggesting that high temperatures are not required to access the target sequence or improve specificity using the "LH PNA-458R RH" probe. This consequently means that only minimal hardware and instrumentation are required to perform the above assay and simple heating steps, which simplifies the final instrumentation as thermal cycling is not required for the process.

[0073] Target enrichment from biological matrices was investigated with respect to the use of a left-handed PNA (2-step) system. 10% (v / v) lithium-heparin collected whole blood and 10% saliva (final concentration) were tested with the above assay. These biological samples were treated with a 50% (v / v) solution in PBS containing 20 mM ribonucleoside vanadyl complex (RVC) for RNase inhibition. The above samples were heated at 37°C for 30 minutes, followed by dilution to 10% (v / v) in PBS containing 0.05% Tween® 20, and the addition of either 1 μg of total human RNA (in the blood sample) or 2 picomoles of Cy5-labeled synthetic 28-nucleotide 18s rRNA (in the saliva sample) and the PNA probe. The above PNA probe "LH PNA-RH 458R" was hybridized to the above RNA by heating at 37°C for 10 minutes. 30 μg of LHc-functionalized Dynabeads were added to a solution containing hybridized RNA PNA, followed by a further incubation at 37°C for 10 minutes to hybridize the LH PNA complement on the beads to the LH PNA on the probe. In the case of blood samples, 18s rRNA captured from the total human RNA was washed on the Dynabeads in PBST, and then the beads were placed in a PBST solution containing Atto488-labeled achiral PNA. This fluorescent achiral PNA can be used to bind to a site on the 18s RNA near the 458R position. The fluorescent PNA was hybridized to the 18s on the Dynabeads at 37°C for 10 minutes. The Dynabeads were then washed again, and the RNA was eluted into a solution containing 10 mM potassium hydroxide and heated to 95°C for 2 minutes. The solution eluted from the beads was read using a spectroscopic plate reader at the excitation and emission wavelengths corresponding to the Atto488 dye, which was introduced to the elution plate via hybridization to the 18s rRNA through the assay described above.

[0074] Figure 13 shows data for the detection of 18s RNA from whole human RNA. In particular, Figure 13 shows data from the detection of 18s RNA from a whole human RNA background, as well as from A) whole blood and B) 10% saliva matrix (N=3, and the trend line is the mean (dotted line) to illustrate the use of a left-handed PNA system). The left-handed PNA system provided direct detection of 18s RNA and was observed from the above samples. The data shows the detection of 18s rRNA from a whole human RNA background and A) 10% whole blood matrix, as well as the detection of synthetic 18s RNA from B) 10% saliva background (N=3). The trend line is the mean (dotted line).

[0075] 18s rRNA capture was obtainable from both biological solutions at 10% (v / v) using phosphate-buffered saline buffer containing 0.05% Tween® 20 and 8 mM RNase inhibitor RVC (final concentration). The linear trend in both graphs indicates that RNA capture is proportional to the amount of PNA eluted into solution in the final step of the assay, suggesting that the assay can be applied to different sample types depending on the application at hand. This indicates that the two-step method is a favorable alternative enrichment method to biotin-streptavidin.

[0076] Biotin-Streptavidin Comparison Capture assays were tested to directly measure the performance of biotinylated PNA probes versus left-handed PNA probes in the two-step method described above. For comparison, the "LH PNA-RH 458R" probe was compared to a biotinylated RH 458R probe containing the same right-handed PNA for the RNA target sequence. Cy5-labeled 28-nucleotide synthetic 18s RNA was first incubated with either the biotinylated probe or the "LH PNA-RH 458R" probe, hybridized at 37°C for 10 minutes, and then captured for a further 10 minutes at room temperature with 1 μm Dynabeads functionalized with 30 μg of streptavidin or complementary left-handed PNA. The capture assays were evaluated using fluorescence of residual RNA remaining in solution and measured with a plate reader. The results of the comparative assays are shown in Figure 14. This shows very similar performance of left-handed PNA capture of RNA compared to the standard biotin-streptavidin approach. The above results indicate that similar capture efficiencies were achieved for both sets of RNA capture systems. This demonstrates that the two-step RNA capture method described above is comparable to known biotin-streptavidin systems in terms of target enrichment.

[0077] Specificity of left-handed PNAs for nucleic acid-containing Dynabeads Dynabeads functionalized with left-handed PNA were incubated with both complementary Cy3-labeled synthetic RNA and complementary FAM-labeled left-handed PNA. Due to the right-handed chirality of the RNA, only left-handed PNA should bind to its complement, even though the RNA has the same sequence. The PNA and RNA were incubated together with the beads at 57°C for 10 minutes before removal, and the fluorescence remaining in the solution was measured in relation to the capture efficiency. This experiment was repeated with the same complementary FAM-labeled PNA and random non-complementary Cy3-labeled RNA. Both sets of capture experiments were repeated with bead removal at room temperature. The results of the experiment are shown in Figure 15. Here, the beads were removed from the solution at room temperature (approximately 22°C) or 57°C.

[0078] Figure 15 shows capture data for left-handed and right-handed PNAs. In particular, Figure 15 shows specific capture of left-handed PNAs with complementary left-handed PNAs on the beads, and little capture of complementary RNA due to the RNA's natural right-handed chirality. Less nonspecific RNA capture was observed when the RNA sequence was random compared to the above PNA sequence. Removal of the beads at 57°C prevents RNA binding of the same base to the beads.

[0079] From the above experiments, it can be observed that left-handed PNA Dynabeads specifically capture only the PNA in solution and capture almost no right-handed RNA. This demonstrates the abiotic nature of the left-handed PNA system. In the worst-case scenario, when the beads are removed at room temperature, a small amount of capture is present if the RNA has a precise match to the left-handed PNA sequence. With the use of heat, nonspecific binding is eliminated, as shown in Figure 15, and the left-handed PNA can capture its target sequence, although there is a very slight decrease in capture. If the RNA is not a precise match, it will not be captured by the left-handed PNA, even at room temperature. This demonstrates the abiotic nature of the system.

[0080] Further Examples Whole human RNA samples were run in double-strand electrophoresis on 1% agarose gels. Here, 18s ribosomal RNA was depleted with differently functionalized Dynabeads, as shown in Figure 16. Sample 1 was the whole human RNA standard in PBST. Sample 2 was RNA incubated with 150 μg 458R RH beads. Sample 3 was RNA incubated with 210 μg 458R RH beads. Sample 4 was RNA incubated first with the "LH PNA-458R RH" probe, followed by 150 μg of LH PNA complement beads. Sample 5 was a control containing RNA incubated with 150 μg of streptavidin beads. Sample 6 was RNA incubated first with the "biotinylated 458R RH probe," followed by 150 μg of streptavidin beads.

[0081] All samples were incubated with 0.75 μg of total human RNA for 10 minutes at 68°C with either beads in PBST (lanes 2 and 3) or PNA probes (lanes 4 and 6), and then cooled to 37°C. Subsequently, they were incubated with beads for 10 minutes at 47°C (lanes 4, 5, and 6). The gels were analyzed using Image J, and gray line profiles were obtained vertically for each lane of the gel. Each initial lane was normalized to the RNA concentration in the lane by using the area under the curve of the 28s rRNA band. The area under the curve of the 18s rRNA band was then measured and compared to the RNA standard in lane 1.

[0082] Lane 2 showed approximately 25% reduction in the 18s band, and an even greater reduction was observed in lane 3 at 35%. The most contrasting difference was observed in lane 4 with the LH PNA system, where 70% was removed from the solution. Lane 5 was used as a control for streptavidin beads to evaluate the presence or absence of nonspecifically binding RNA (approximately 20%), and lane 6 was used with a biotinylated 458R RH probe, where 40% of the RNA was removed from the solution.

[0083] The data showed that the above LH PNA two-step system performed better than the biotin-streptavidin two-step system for removing 18s rRNA from total human RNA isolates. It also outperformed Dynabeads directly functionalized with 458R RH PNA, where the two steps appear necessary to bind to larger, more structural RNA targets and successfully move them out of solution. The gel also showed that the PNA probes on the beads were specific to their 18s targets, because the 28s rRNA bands remained generally unchanged in all lanes and were not simultaneously removed from solution.

[0084] advantage Since avidin is a biological protein and naturally occurring in biological samples, the present invention provides abiotic alternatives to avidin for use in enriching or depleting RNA targets. These alternatives may be used to contribute to reducing false-positive results obtained when using biotin-avidin test systems, which are caused by endogenous or dietary biotin. Alternatives to biotin tags provided in DNA synthesis include digoxigenin (DIG), cholesterol, and dinitrophenyl (DNP). These are alternatives to antigen-antibody-based detection. In other applications, protein-based binding systems may be used (e.g., maltose tags in maltose-binding protein systems, chitin tags in chitin-binding proteins). Most alternatives involve biological entities required in labeling or detection processes that necessitate proper storage in a refrigerator or freezer to ensure biological viability. This is not required in left-handed PNA systems, as PNA has been shown to be able to be stored at room temperature for several years.

[0085] The number of available, and especially commercially available, antigen-antibody / protein systems of these types is also limited. Three DNA synthesis companies offer three alternatives to biotin, but only two companies offer DIG or cholesterol tag options for RNA oligo targets. This limits the amount of multiplexing that can be achieved using such systems. When an LH PNA system is used, for example, with an LH PNA containing 12 monomers, it is possible for its user to design 16,777,216 different left-handed PNAs that can be potentially used with corresponding complementary PNAs on beads or surfaces for different targets of interest. These beads may have different physical properties with respect to compositional materials and / or functionalization, if suitable for multiplexing applications. Since DNA and RNA do not bind to these LH PNAs due to chirality, sequences used with an LH PNA system do not interfere even if the target nucleic acid has the same sequence as the LH PNA, as long as the beads are removed at the temperature in the assay for stringency (see Figure 15).

[0086] One other advantage of using the LH PNA probe system is that the LH PNA and its complement can be designed to have a melting temperature within a specific temperature range. Target release via a programmable melting temperature allows for the collection of those targets from the beads or surface without potentially damaging them. This eliminates the need for extremely harsh conditions to dissociate targets from the beads, unlike biotin-streptavidin capture systems which require a boiling step in 95% formamide at 65°C for 5 minutes or 90°C for 2 minutes to remove biotinylated nucleic acids. This step denatures the streptavidin bound to the magnetic beads to release the biotin molecule, and the denaturation of streptavidin means that the beads cannot be reused. Magnetic beads coated with left-handed PNA could be reused for other applications if the probe used in both applications had the same left-handed PNA sequence and the previous probe was sufficiently thermally melted and separated from the beads before being used in its new application.

[0087] In some cases, it is also conceivable that one of the above PNA pairs is left-handed and the other is non-chiral. While the above system may still be viable in such scenarios, the wise design of the achiral PNA must be considered to avoid interaction with naturally occurring right-handed oligonucleotides that the achiral PNA may hybridize with and interfere with the complementary pair of the above PNA.

[0088] The capture of 18s RNA is recognized to be more efficient when using left-handed PNA capture systems compared to achiral or right-handed PNA directly functionalized on beads. Direct detection of 18s RNA is achievable isothermally at 37°C from biological samples (e.g., blood and saliva). The left-handed PNA enrichment system described above is a abiotic alternative to biotin-streptavidin-based extraction systems and shows great potential for future use in RNA and DNA applications. Furthermore, since the sequences of left-handed PNA can be readily altered, the system is "programmable" with each new nucleic acid target captured by paired beads (which may potentially have different physical properties). This suggests that these beads can be used as a versatile tool in many applications such as selection, target depletion, detection or multiplexing of analytes, antigens, nucleic acids, or antibodies in well-established methods such as capture arrays, magnetic separation, or even flow cytometry. This makes the left-handed PNA system a highly versatile, adaptable, and attractive system.

[0089] RNA targeting and amplification have traditionally been performed via enzymatic amplification methods (e.g., polymerase chain reaction (PCR)). Proper storage and handling of the reagents (e.g., enzymes) required for such amplification are crucial to ensuring biological integrity and, therefore, the success of the amplification process. It is shown that these temperature-sensitive and short-lived enzymes can be replaced with abiotic alternatives for RNA enrichment. Direct comparisons showed that a two-step method using left-handed PNA captured nearly four times more 18s RNA from total human RNA isolates than achiral or right-handed PNA directly functionalized on beads. The above method was shown to function isothermally at 37°C and can be used in 10% biological matrix of whole blood or saliva without hindering 18s RNA capture. The above LH system was also capable of successfully removing 18s rRNA from total human RNA samples on agarose gels more successfully than RH 458R-functionalized beads or a biotin-streptavidin two-step system, as visualized. This method presents a promising abiotic alternative to biotin-streptavidin-based targeted enrichment.

[0090] The methods described herein avoid some or all of the shortcomings of previous approaches and provide stable and strong, yet reversible affinity, numerous targets, programmability for binding to specific beads or parts of substrates, and stability within a certain range of temperature conditions with little to no crosstalk with endogenous nucleic acids. Accordingly, the inventors believe that the methods described herein have the potential to be revolutionary in the art.

[0091] Alternative examples The present invention is not limited to the embodiments described and may vary in configuration and details.

[0092] The targeting portion on the first or second probe, including the left-handed PNA and linker, may be, for example, one or more of the following: peptide nucleic acids (achiral or chiral PNA); deoxyribonucleic acid; ribonucleic acid; glycol nucleic acid; threose nucleic acid; locked nucleic acid; phosphorothioate oligonucleotide; or phosphorodiamidate morpholino oligomer; protein; peptide; peptide or protein having non-natural amino acids; enzyme; antibody; single-domain antibody (nanobody); aptamer; drug molecule; small molecule; compound; cell; or one or more combinations of the above.

[0093] The above detection portion may be, for example, one or more of the following: fluorescent labeling; luminescence labeling; chromophore labeling; chemiluminescence labeling; radioactive labeling, enzyme labeling; or visual labeling (e.g., metallic labeling (e.g., gold)); microspheres (magnetic, fluorescent, silica, etc.); nanoparticles (carbon nanotubes, quantum dots, etc.); biotin labeling; avidin labeling; digoxigenin, cholesterol, or dinitrophenyl labeling; horseradish peroxidase labeling; or one or more combinations of the above.

[0094] In various examples, the linker may be 1 to 120 atoms long; it may contain one or more elements: C, N, O, S, P, and Si; and it may be in a chain containing one or a combination of the following bonds: single / double / triple bonds, amide bonds, ester bonds, disulfide bonds, imino groups, ether bonds, thioether bonds, and thioester bonds.

[0095] The above left-handed PNA sequences are programmable to enable multiplexing, in that their length and / or monomer sequence can be changed to ensure that they interact only with surface-specific complementary PNAs and not with each other or other nucleic acid sequences. The present invention provides, for example, the following items: (Item 1) A method for enriching a target in a sample, wherein the method is A step of introducing a probe containing a left-handed PNA molecule attached to a capture portion into a sample containing the target analyte; The process of capturing the target analyte in the aforementioned capture section; and A step of binding the left-handed PNA molecule with a complementary PNA molecule containing a left-handed chiral structure, wherein the complementary PNA molecule is bound to a surface and thereby enriches the target analyte. A method that includes (Item 2) The surface is the method according to item 1, including beads. (Item 3) The method according to item 2, wherein the beads are magnetic beads. (Item 4) The method according to item 1, wherein the surface includes a fixed substrate. (Item 5) The method according to item 1, wherein the left-handed PNA molecule includes a linker that connects a portion of the left-handed PNA molecule, which contains a nucleotide sequence, to a targeting portion. (Item 6) The method according to item 1, further comprising the step of introducing a second probe into the sample, wherein the second probe includes a left-handed PNA, a targeting portion, and a detection portion. (Item 7) The method according to item 6, wherein the targeting portion is a right-handed targeted PNA. (Item 8) The method according to item 1, wherein the capture portion comprises an antibody or antibody fragment. (Item 9) The method according to item 1, comprising the step of constructing a chain having multiple links provided by a left-handed PNA pair, wherein the chain comprises some or all of a target, an antibody, and a detection portion. (Item 10) The method described above is the method described in item 1, performed at a temperature in the range of 18°C ​​to 80°C. (Item 11) The method described above is the method described in item 10, which is carried out at a temperature of approximately 37°C. (Item 12) The target analyte is RNA, as described in item 1. (Item 13) The RNA is the method described in item 12, including 18s ribosomal RNA. (Item 14) The method according to item 1, wherein the surface includes the surface of a bead, and the bead is thermally melted after the steps of the method to provide a clean capture surface for reuse in further assays. (Item 15) The method according to item 1, wherein the capture portion comprises one or more of the following: achiral or chiral PNA, deoxyribonucleic acid, ribonucleic acid, glycol nucleic acid, threose nucleic acid, locked nucleic acid, phosphorothioate oligonucleotide, or phosphorodiamidate morpholino oligomer, protein, peptide, peptide or protein having unnatural amino acids, enzyme, antibody, single-domain antibody (nanobody), aptamer, drug molecule, small molecule, compound, cell, or one or more combinations of the above. (Item 16) The method according to item 1, wherein the detection portion is linked to the left-handed PNA molecule, and the detection portion comprises one or more of the following: fluorescent labeling, luminescence labeling, chromophore labeling, chemiluminescence labeling, radioactive labeling, enzyme labeling, or visual labeling (e.g., metallic labeling such as gold), microspheres, nanoparticles, biotin labeling, avidin labeling, digoxigenin, cholesterol, or dinitrophenyl labeling, horseradish peroxidase labeling, or one or more combinations of the above. (Item 17) The method according to item 1, wherein the left-handed PNA molecule comprises a linker having a length of 1 to 120 atoms and / or having one or more of the elements: C, N, O, S, P, and Si, and / or being in a chain containing one or a combination of the following bonds: single bond, double bond, triple bond, amide bond, ester bond, disulfide bond, imino group, ether bond, thioether bond, and thioester bond. (Item 18) The method according to item 1, wherein the PNA molecule is programmable to enable multiplexing. (Item 19) The method according to item 1, wherein the PNA molecule is modified by altering the cyclic skeleton to induce a left-handed helical structure.

Claims

1. A method for enriching a target in a sample, wherein the method is A step of introducing a probe containing a left-handed PNA molecule attached to a capture portion into a sample containing the target analyte; The process of capturing the target analyte in the aforementioned capture portion; and A step of binding the left-handed PNA molecule with a complementary PNA molecule containing a left-handed chiral structure, wherein the complementary PNA molecule is bound to a surface and thereby enriches the target analyte. A method that includes.

2. The method according to claim 1, wherein the surface includes beads.

3. The method according to claim 2, wherein the beads are magnetic beads.

4. The method according to claim 1, wherein the surface includes a fixed substrate.

5. The method according to claim 1, wherein the left-handed PNA molecule includes a linker that connects the left-handed PNA molecule, which contains a nucleotide sequence, to a targeting portion.

6. The method according to claim 1, further comprising the step of introducing a second probe into the sample, wherein the second probe includes a left-handed PNA, a targeting portion, and a detection portion.

7. The method according to claim 6, wherein the targeting portion is a right-handed targeted PNA.

8. The method according to claim 1, wherein the capture portion comprises an antibody or an antibody fragment.

9. The method according to claim 1, wherein the method is carried out at a temperature in the range of 18°C ​​to 80°C.

10. The method according to claim 9, wherein the method is carried out at a temperature of approximately 37°C.

11. The method according to claim 1, wherein the target analyte includes RNA.

12. The method according to claim 11, wherein the RNA includes 18s ribosomal RNA.

13. The method according to claim 1, wherein the surface includes the surface of a bead, and the bead is thermally melted after the steps of the method to provide a clean capture surface for reuse in a further assay.

14. The method according to claim 1, wherein the capture portion comprises one or more of the following: achiral or chiral PNA, deoxyribonucleic acid, ribonucleic acid, glycol nucleic acid, threose nucleic acid, locked nucleic acid, phosphorothioate oligonucleotide, phosphorodiamidate morpholino oligomer, protein, peptide, peptide or protein having non-natural amino acids, enzyme, antibody, single-domain antibody, and aptamer.

15. The method according to claim 1, wherein the detection portion is linked to the left-handed PNA molecule, and the detection portion comprises one or more of the following: fluorescent labeling, luminescence labeling, chromophore labeling, chemiluminescence labeling, radioactive labeling, enzyme labeling, visual labeling, metallic labeling, microspheres, nanoparticles, biotin labeling, avidin labeling, digoxigenin, cholesterol, or dinitrophenyl labeling, horseradish peroxidase labeling, or one or more combinations of the above.

16. The method according to claim 1, wherein the left-handed PNA molecule includes a linker, the linker having a length of 1 to 120 atoms and / or having one or more of the elements: C, N, O, S, P, and Si, and / or being in a chain containing one or a combination of the following bonds: single bond, double bond, triple bond, amide bond, ester bond, disulfide bond, imino group, ether bond, thioether bond, and thioester bond.

17. The method according to claim 1, wherein the PNA molecule is modified by altering the cyclic skeleton to induce a left-handed helical structure.

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