Hybrid full LNA oligonucleotides

Abstract

The present invention relates to hybrid full LNA oligonucleotides. The present report relates to hybridizing single stranded (ss-) oligonucleotides consisting entirely of locked nucleic acid (LNA) monomers. This document shows hybridization experiments of pairs of fully complementary ss-oligonucleotides that fail to form double strands within a given time interval. The present report provides a method of identifying such incompatible oligonucleotide pairs. In another aspect, the present report provides pairs of complementary ss-oligonucleotides capable of rapidly forming double strands. The present report also provides methods of identifying and selecting compatible oligonucleotide pairs. In another aspect, the present report provides the use of a compatible oligonucleotide pair as a binding partner in binding assays, such as receptor-based assays.

Description

[0001] This application is a divisional application of the invention patent application entitled “Hybrid All-LNA Oligonucleotide”, which entered China with international application number 202080042051.4 and international application date PCT / EP2020 / 065654 on June 5, 2020. Technical Field

[0002] This report relates to hybridization of single-stranded (ss-) oligonucleotides composed entirely of locked nucleic acid (LNA) monomers. This document presents hybridization experiments with pairs of fully complementary ss-oligonucleotides that fail to form a duplex within a given time interval. This report provides methods for identifying such incompatible oligonucleotide pairs. In another aspect, this report provides pairs of complementary ss-oligonucleotides that can rapidly form a duplex. This report also provides methods for identifying and selecting compatible oligonucleotide pairs. In another aspect, this report provides the use of compatible oligonucleotide pairs as binding partners in binding assays (e.g., in immunoassays). Specific embodiments are discussed in which compatible LNA oligonucleotide pairs are used to immobilize analyte-specific capture molecules for detecting or determining analytes in samples in an assay. Background Art

[0003] Of particular interest are general biochemical applications in which the two partners of a binding pair function functionally through specific interactions of molecular recognition and their eventual linkage to one another. For example, in immunoassays, the biotin:(strept)avidin binding pair is frequently used to immobilize analyte-specific capture receptors on a solid phase. This report conceptualizes, explains, and details applications such as immunoassays utilizing alternative binding pairs. Specifically, alternative binding pairs composed of two single-stranded LNA oligonucleotides capable of forming a duplex via hybridization offer a technical alternative to the biotin:(strept)avidin binding pair.

[0004] The focus of the present disclosure is on the means of anchoring the capture receptor on the solid phase during the immunoassay process. In particular, the present disclosure focuses on binding pairs that promote the immobilization of analyte-specific capture receptors in the presence of a sample containing analyte, and / or can anchor the detection complex after complex formation. The binding pairs in immunoassays need to have specific technical characteristics. First, the interaction of the two binding partners must be specific. In addition, the kinetics of forming the binding partner connection must ensure that the two separated partners of the binding pair interact and ultimately associate (i.e., bind to each other) at a high speed. In addition, the connection of the two binding partners is expected to be stable once formed. In addition, the binding partner must be suitable for chemical conjugation with other molecules (e.g., analyte-specific receptors and stationary phase surfaces) for use in immunoassays.

[0005] It is important to recognize that in immunoassays, receptors, and often also the analyte to be detected, retain their conformation and function only under certain conditions. Such conditions may vary depending on the specific receptor or analyte under consideration; therefore, the receptor molecule or analyte can only tolerate limited deviations from these conditions. Such conditions may include, but are not limited to, a buffered aqueous solution having a pH value in the range of about pH 6 to about pH 8, one or more dissolved salts, one or more auxiliary substances (e.g., selected from stabilizers, scavengers, preservatives, detergents), a total amount of solutes of about 200 to about 500 mosm / kg, the absence of denaturing compounds (e.g., certain non-aqueous solvents), helical destabilizing agents (e.g., formamide and chaotropes), and preferred storage and / or assay temperatures (ranging from 0°C to 40°C), to name a few. However, it is important to note any ingredients and / or conditions that would result in denaturation of the analyte to be assayed or the analyte-specific receptor used in a particular assay.

[0006] The isolated binding partners of the binding pair are required to be suitable for conjugation, specifically to the capture molecule, i.e., the receptor, and to the surface of the stationary phase, without losing their ability to specifically associate and bind to each other. With respect to conjugates in immunoassays, each isolated binding partner of the surrogate binding pair must be functional under the assay conditions. The same reasoning applies to all other required materials to which the binding partner is conjugated, such as, but not limited to, the analyte, support material, stationary phase, and other substances or compounds that may be present during the assay.

[0007] Previously, it has been proposed to use single-stranded oligonucleotides with complementary sequences, i.e., oligonucleotides that can form duplexes by hybridization, as binding tools to connect macromolecules or molecules to a stationary phase. EP 0488152 discloses a heterogeneous immunoassay using a stationary phase in which an analyte-specific capture antibody is fixed to the stationary phase by connecting a nucleic acid duplex of the antibody and the stationary phase. In one embodiment, a hybridized oligonucleotide is shown to be attached to the antibody, while a complementary oligonucleotide is attached to the stationary phase, thereby forming a connection duplex. Similar disclosures are provided in documents EP 0698792, WO 1995 / 024649, WO 1998 / 029736, and EP 0905517. WO 2013 / 188756 discloses methods and compositions for flow cytometry, comprising an antibody conjugated to a first oligonucleotide, an oligosphere conjugated to a second oligonucleotide having the same sequence as the first oligonucleotide, and an oligonucleotide probe having a label and a third sequence complementary to the first and second oligonucleotides. In a specific embodiment, the oligosphere is magnetic. This document reports specific uses of the oligosphere as a reference in standardized procedures.

[0008] Modified oligonucleotides, such as peptide nucleic acids (PNA) and locked nucleic acids (LNA), have been investigated for use in a range of fundamental biochemical applications. LNAs possess a methylene linkage between the 2′-oxygen and 4′-carbon atoms of the ribose moiety, which locks the sugar in a C3-endo conformation, hence the term "locked nucleic acid." In technical applications involving duplex formation by hybridization of oligonucleotides containing LNA monomers with complementary target sequences, this chemical modification confers nuclease resistance, as well as increased affinity and specificity for the oligonucleotide target. LNA monomers are provided as 2′-O, 4′-C—methylene-(D-ribofuranosyl) nucleoside monomers (Singh S.K. et al. Chem. Commun. 4 (1998) 455-456; Koskin AA et al. Tetrahedron 54 (1998) 3607-3630; Wengel J. Acc. Chem. Res. 32 (1999) 301-310). Furthermore, WO 1998 / 39352 discloses locked nucleic acid (LNA) structures. Through chemical synthesis, single chains consisting only of LNA nucleoside analog monomers ("all-LNA") can be synthesized.

[0009] Mixed DNA-LNA oligonucleotides comprising DNA and LNA monomers have enhanced thermal stability when hybridized with complementary DNA and RNA. In fact, compared with other high-affinity nucleic acid mimics that have been synthesized, for example, peptide nucleic acids (PNAs), hexitol nucleic acids (HNAs), and 2′-fluoro N3′-phosphoramidates, LNAs exhibit extraordinary binding affinity. Christensen U. et al. reported the hybridization kinetics of LNA-DNA mixed oligonucleotides (also referred to as “hybrids”) (Biochem J 354 (2001) 481-484). Eichert A. et al. reported the crystal structure of a fully locked nucleic acid duplex consisting of two complementary ss-oligonucleotides, each of which consists of 7 LNA monomers (Nucleic Acids Research 38 (2010) 6729-6736).

[0010] In most cases, single-stranded hybrid LNA / DNA oligonucleotides (LNA / DNA and LNA / RNA, i.e. hybrid single strand) have been analyzed. There are few reports on the characterization of hybrid single-stranded oligonucleotides made only of LNA monomers (i.e., "all-LNA" single-stranded oligonucleotides) published by BP et al. (Analyst 130 (2005) 1634-1638). Eze NA et al. (Biomacromolecules 18 (2017) 1086-1096) reported that the association rate of DNA / LNA hybrids and DNA probes was less than 10 5 M -1 s -1 . According to these authors, replacing one or more DNA monomers with LNA monomers does not seem to affect the hybridization kinetics in solution, given that one-third of the monomers are replaceable. Childs JL (PNAS 99 (2002) 11091-11096) reported an all-LNA octamer (TACCTTTC) that was able to inhibit the self-splicing of the Candida albicans group I intron in vitro in a concentration-dependent manner. To anneal the octamer to the target RNA, the octamer was heated to 68°C and then cooled to 37°C. It was found that the annealed LNA oligomers disrupted the tertiary structure of the intron, thereby affecting its biological function.

[0011] WO 2000 / 066604 and WO 2000 / 056746 disclose certain stereoisomers of LNA nucleoside monomers.

[0012] WO 1999 / 14226 proposes the use of oligonucleotides containing LNA monomers in the construction of affinity pairs for attachment to a target molecule and a solid support. However, the technical problems associated with hybridization of complementary all-LNA single strands are also known in the art. The LNA user manual written by Jesper Wengel and published by Exiqon mentions the tendency of single-stranded LNA-containing oligonucleotides to form intramolecular LNA:LNA duplexes, also known as self-hybridization. Therefore, the document considers secondary structure as an application limitation, i.e., as a technical obstacle ("LNA hybridization" in: "Locked Nucleic Acid Technology"). TM=(1) Thermodynamic analysis of hybridization of oligonucleotide analogs consisting solely of LNAs is therefore largely empirical, and sequence prediction of complementary all-LNA oligomer hybrid pairs without a prior denaturation step (e.g., heating to remove intramolecular secondary structure prior to hybridization) appears impossible so far.

[0013] Predictions of the thermodynamic behavior of LNA-containing oligonucleotides were aided by specialized computer programs cited by Tolstrup N et al. (Nucleic Acids Research 31 (2003) 3758-3762). However, the report explicitly cited higher prediction errors for LNA oligonucleotides, due to the more complex nature of these oligonucleotides rather than a lack of experimental data. Furthermore, the disclosed algorithm does not appear to provide guidance for the design of complementary pairs of all-LNA oligonucleotides. Similar conclusions can be drawn from a recent publication on the molecular thermodynamics of LNA:LNA base pairs in DNA / LNA hybrid oligonucleotides (Fakhfakh K et al. American Institute of Chemical Engineers Journal 61 (2015) 2711-2731).

[0014] Specifically, this report demonstrates that complementary single-stranded oligonucleotides composed solely of LNA monomers are indeed unpredictable in their ability to form duplex molecules via Watson-Crick base pairing. Therefore, in order to provide technically suitable alternatives to biotin:(strept)avidin binding pairs for specific applications using such molecular recognition, technical means are needed to select and provide alternative binding pairs; for the purposes of this report, such binding pairs are desired to consist of complementary single-stranded oligonucleotides comprising solely of LNA monomers, wherein

[0015] - Oligonucleotide pairs must contain complementary sequences, and the complementary sequences must be capable of duplex formation and Watson-Crick base pairing;

[0016] - the oligonucleotide pair must not require any denaturation treatment before the actual use of the binding pair in an application under storage and conventional biochemical application conditions in molecular recognition; this translates into a technical requirement that each oligonucleotide of the binding pair must be free of any secondary structures formed intermolecularly or intramolecularly that would significantly reduce the ability of the respective oligonucleotide to align with its binding partner (i.e. the complementary oligonucleotide or the complementary sequence therein) and to form a duplex;

[0017] -Under the conditions of conventional biochemical applications, single-strand pairs must be able to form Watson-

[0018] Crick pairs the oligonucleotide duplexes while ensuring sufficient specificity for molecular recognition;

[0019] - It is desirable that the duplex formed by the complementary oligonucleotide pair is sufficiently stable and preferably irreversibly formed during a given biochemical application using molecular recognition of the binding pair.

[0020] Therefore, a general objective of this report is to identify and provide binding pairs of single-stranded, all-LNA oligonucleotides that can hybridize without a prior denaturation step, thereby forming duplex molecules via Watson-Crick base pairing under suitable assay conditions for use as binding pairs in analyte detection assays. In other words, the goal is to seek binding pairs that can form duplexes under non-denaturing conditions, more specifically, under conditions compatible with the function of analyte-specific receptors in analyte detection assays (such as, but not limited to, immunoassays). Importantly, the goal is to seek single-stranded, all-LNA oligonucleotides that can be stored under ambient conditions, or even refrigerated, without forming inter- or intramolecular secondary structures that could inhibit hybridization and duplex formation between complementary oligonucleotides. Furthermore, the goal is to seek single-stranded, all-LNA oligonucleotides that can hybridize to each other under assay conditions (e.g., in aqueous solution at ambient temperature, such as room temperature), without prior denaturation. The absence of denaturation specifically refers to the removal of any inter- or intramolecular secondary structures that could inhibit hybridization and duplex formation between complementary oligonucleotides used as binding pairs in analyte detection assays. Summary of the Invention

[0021] The present disclosure unexpectedly provides, in a first aspect related to all other aspects and embodiments disclosed herein, a pair of isolated ss-oligonucleotides, each oligonucleotide consisting of 5 to 15 LNA monomers, the isolated ss-oligonucleotides being capable of forming antiparallel duplexes with each other in aqueous solution in the absence of denaturing conditions before or during duplex formation. This report further discloses another embodiment of the first aspect, which is a pair of isolated ss-oligonucleotides, each oligonucleotide consisting of 5 to 15 LNA monomers, the isolated ss-oligonucleotides being capable of forming antiparallel duplexes comprising 5 to 15 consecutive base pairs with each other in aqueous solution in the absence of denaturing conditions before or during duplex formation. This report further discloses another embodiment of the first aspect, which is a pair of isolated ss-oligonucleotides, each oligonucleotide consisting of 5 to 15 LNA monomers, and the isolated ss-oligonucleotides are capable of forming an antiparallel duplex containing 5 to 7 consecutive base pairs with each other in aqueous solution in the absence of denaturing conditions before or during duplex formation. This report further discloses another embodiment of the first aspect, which is a pair of isolated ss-oligonucleotides, each oligonucleotide consisting of 5 to 7 LNA monomers, and the isolated ss-oligonucleotides are capable of forming an antiparallel duplex containing 5 to 7 consecutive base pairs with each other in aqueous solution in the absence of denaturing conditions before or during duplex formation.

[0022] In a second aspect related to all other aspects and embodiments disclosed herein, the present disclosure provides a method for selecting and providing a binding pair of single-stranded all-LNA oligonucleotides capable of forming an antiparallel duplex having 5 to 15 consecutive base pairs in aqueous solution at a temperature of 0° C. to 40° C., the method comprising the steps of:

[0023] (a) providing a first single-stranded (=ss-) oligonucleotide consisting of 5 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0024] (b) providing a second ss-oligonucleotide composed of 5 to 15 LNA monomers, the second ss-oligonucleotide consisting of at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence of the second ss-oligonucleotide, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation, and the complementarity predicts the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 5 to 15 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other;

[0025] (c) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0026] (d) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex;

[0027] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0028] (f) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide, selecting the binding pair;

[0029] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0030] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0031] In a second aspect related to all other aspects and embodiments disclosed herein, the present disclosure provides a liquid composition comprising an aqueous solvent and a binding pair consisting of a first single-stranded oligonucleotide and a second single-stranded oligonucleotide,

[0032] wherein each oligonucleotide consists of 5 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of said monomers forming a first nucleobase sequence of the first oligonucleotide and a second nucleobase sequence of the second oligonucleotide,

[0033] wherein the first nucleobase sequence and the second nucleobase sequence are selected so that the first oligonucleotide and the second oligonucleotide are capable of forming an antiparallel duplex having 5 to 15 consecutive Watson-Crick base pairs at a temperature of 0°C to 40°C,

[0034] And it is characterized in that the binding pair can be obtained by the method according to the first aspect disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram depicting the method for screening compatible all-LNA oligonucleotide binding pairs (Example 2). The black bars represent the number of individual single-stranded molecules, double-stranded molecules or other complex molecules.

[0036] The first (1) and second (2) single stranded oligonucleotides are in contact with each other.

[0037] A: Neither oligonucleotide features intramolecular or inter-molecular secondary structure, and both are capable of unhindered molecular recognition of their respective partners; therefore, duplexes are the predominantly abundant product, with single strands either undetectable or present in negligible amounts (expected result).

[0038] B: At least one of the two oligonucleotides is characterized by an intermolecular or intramolecular secondary structure; therefore, the number of duplexes is small and most single strands remain; duplexes are formed,

[0039] But the speed is reduced (not the expected result).

[0040] C: Both oligonucleotides feature inter- or intramolecular secondary structures; therefore, even after long incubation periods, no duplex is formed or only a trace amount of duplex is formed, and single strands remain (not an expected result).

[0041] Figure 2 HPLC analysis of single-chain LNA 1 (SEQ ID NO: 1; Example 2); the main peak indicated a retention time of 3.353 min.

[0042] Figure 3 HPLC analysis of single-chain LNA 2 (SEQ ID NO: 2; Example 2); the minor peak indicated a retention time of 6.440 min, and the major peak indicated a retention time of 7.145 min.

[0043] Figure 4 HPLC analysis of the mixed LNA 1 and LNA 2, immediately injected into the HPLC system (Example 2); the minor peak indicated a retention time of 1.671 min, and the major peak indicated a retention time of 6.641 min.

[0044] Figure 5HPLC analysis of mixed LNA1 and LNA2 after heat denaturation before injection (Example 2); positive control: duplex formation; minor peak indicated at retention time 1.710 min, major peak indicated at retention time 6.656 min.

[0045] Figure 6 HPLC analysis of single-chain LNA 3 (SEQ ID NO: 5; Example 2); the main peak indicated a retention time of 3.353 min.

[0046] Figure 7 HPLC analysis of single-chain LNA 4 (SEQ ID NO: 6; Example 2); the minor peak indicated a retention time of 6.440 min, and the major peak indicated a retention time of 7.145 min.

[0047] Figure 8 HPLC analysis of mixed LNA 3 and LNA 4, immediately injected into the HPLC system (Example 2); duplex formation was slow (ratio < 0.05); the first peak indicated a retention time of 3.387 min, the main peak indicated a retention time of 7.157 min.

[0048] Figure 9 HPLC analysis of mixed LNA 3 and LNA 4, injected after 50 min (Example 2); duplex formation was slow (ratio = 0.05); first peak indicated a retention time of 3.365 min, second peak indicated a retention time of 6.871 min, third peak indicated a retention time of 7.148 min.

[0049] Figure 10 HPLC analysis of mixed LNA 3 and LNA 4 after heat denaturation before injection (Example 2); positive control: duplex formation; major peak indicated at a retention time of 6.882 min.

[0050] Figure 11 HPLC analysis of single-chain LNA 5'-Bi-Heg-accaac-3' (5'-modified SEQ ID NO: 20); the main peak indicated a retention time of 6.184 min.

[0051] Figure 12 HPLC analysis of single-chain LNA 5'-gttggt-3' (SEQ ID NO: 16); the minor peak indicated a retention time of 1.496 min, and the major peak indicated a retention time of 1.865 min.

[0052] Figure 13HPLC analysis of a mixture of LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO: 20) and 5′-gttggt-3′ (SEQ ID NO: 16) (mixed at room temperature and injected immediately); the main peak indicated a retention time of 6.568 min. Rapid duplex formation (100% duplex formation: ratio 1.0). 5′-gttggt-3′ (SEQ ID NO: 14) was used for duplex formation, and some residual single-stranded LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO: 20) was detectable.

[0053] Figure 14 HPLC analysis of a mixture of LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO: 20) and 5′-gttggt-3′ (SEQ ID NO: 16) (stored at +4°C to +6°C, mixed, and injected immediately); the main peak indicated a retention time of 6.588 min, and another indicated a retention time of 7.056 min. Rapid duplex formation (100% duplex formation: ratio 1.0). 5′-gttggt-3′ (SEQ ID NO: 16) was used for duplex formation, and a small amount of residual single-stranded LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO: 20) was detectable.

[0054] Figure 15 HPLC analysis of a mixture of LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO: 20) and 5′-gttggt-3′ (SEQ ID NO: 16) (stored at 0°C and mixed (ice bath) immediately before injection); the main peak indicated a retention time of 6.552 min. Rapid duplex formation (100% duplex formation: ratio 1.0). 5′-gttggt-3′ (SEQ ID NO: 16) was used for duplex formation, and a small amount of residual single-stranded LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO: 20) was detectable.

[0055] Figure 16HPLC analysis of mixed LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO: 20) and 5′-gttggt-3′ (SEQ ID NO: 16) (stored at -10°C and mixed (MgCl2 / ice bath) and injected immediately); the main peak indicated a retention time of 6.547 min. Rapid duplex formation (100% duplex formation: ratio 1.0). 5′-gttggt-3′ (SEQ ID NO: 16) was used for duplex formation, and a small amount of residual single-stranded LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO: 20) was detectable.

[0056] Figure 17 HPLC analysis of mixed LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO: 20) and 5′-gttggt-3′ (SEQ ID NO: 16) after heat denaturation and annealing before injection; the main peak indicated a retention time of 6.583 min, and the other indicated a retention time of 6.967 min. Positive control for duplex formation.

[0057] Figure 18 HPLC analysis of single-chain LNA 5'-Bi-Heg-cgtcaggcagttcag-3' (5' modified SEQ ID NO: 47); the main peak indicated a retention time of 6.840 min.

[0058] Figure 19 HPLC analysis of single-chain LNA 5'-ctgaactgcctgacg-3' (SEQ ID NO: 48); the main peak indicated a retention time of 3.488 min.

[0059] Figure 20 HPLC analysis of a mixed LNA 5′-Bi-Heg-cgtcaggcagttcag-3′ (SEQ ID NO: 47) / 5′-ctgaactgcctgacg-3′ (SEQ ID NO: 48) (mixed at room temperature and injected immediately); the first peak indicated a retention time of 3.594 min, and the corresponding second peak indicated a retention time of 6.580 min. Sequence pairs with slow duplex formation were identified. Duplex formation was slow (ratio < 0.5).

[0060] Figure 21HPLC analysis of mixed LNA 5′-Bi-Heg-cgtcaggcagttcag-3′ (SEQ ID NO: 47) / 5′-ctgaactgcctgacg-3′ (SEQ ID NO: 48) (stored at 0°C and mixed (ice bath) and injected immediately); the first peak indicated a retention time of 3.541 min, and the corresponding second peak indicated a retention time of 6.853 min. Duplex formation was slow (ratio <0.5).

[0061] Figure 22 HPLC analysis of mixed LNA 5′-Bi-Heg-cgtcaggcagttcag-3′ (SEQ ID NO: 47) / 5′-ctgaactgcctgacg-3′ (SEQ ID NO: 48) (stored at -10°C and mixed (MgCl2 / ice bath) and injected immediately); the first peak indicated a retention time of 3.516 min, and the corresponding second peak indicated a retention time of 6.848 min. Duplex formation was slow (ratio <0.5).

[0062] Figure 23 HPLC analysis of mixed LNA 5'-Bi-Heg-cgtcaggcagttcag-3' (SEQ ID NO: 47) / 5'-ctgaactgcctgacg-3' (SEQ ID NO: 48) after heat denaturation and annealing before injection; the main peak indicated a retention time of 6.580 min. Positive control: duplex formation.

[0063] Figure 24 A: Schematic diagram of the Biacore sensor used in Example 4.

[0064] B: The components are as shown in A.

[0065] 1: Sensor surface

[0066] 2: Streptavidin attached to the sensor surface

[0067] 3: Biotin

[0068] 4: Linker molecule covalently links the first ss-oligonucleotide to biotin.

[0069] 5: First ss-oligonucleotide

[0070] 6: Second ss-oligonucleotide

[0071] a: Depicts the process of contacting the sensor with the first ss-oligonucleotide when the second ss-oligonucleotide is attached.

[0072] The situation when the trigger

[0073] b: Depicted is the result where two ss-oligonucleotides are compatible and form a duplex under non-denaturing conditions; as a result, the bound second ss-oligonucleotide causes a change that can be detected by the Biacore instrument.

[0074] Figure 25-50 Results of Example 4.

[0075] Figure 51 Schematic diagram of the Biacore experiment in Example 5.

[0076] Figures 52-53 show the results of Example 5.

[0077] Figure 54 Schematic diagram of the Biacore experiment in Example 6.

[0078] Figure 55: Binding of LNA structure to Bi-LNA structure; Binding constant at 25°C

[0079] Figure 56: Binding of LNA structure to Bi-LNA structure; Binding constant at 37°C DETAILED DESCRIPTION

[0080] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0081] The articles "a" and "an" can refer to one or more (i.e., to at least one) of the grammatical object of the article. For example, "an item" refers to one item (a single item) or more than one item (a plurality of items). If "a" refers to a member that is part of a pair of two members, "a" means one member of the pair or both members of the pair, i.e., a single member of the pair or both members together.

[0082] It will be further understood that when the root terms "include" and / or "have" are used in this specification, they specify the presence of the recited features, matters, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of at least one other feature, integer, step, operation, element, component and / or group thereof. In a similar manner, "with" also specifies the presence of the recited features, etc.

[0083] As used herein, the terms "comprises / comprising / contains / containing / includes / including," "has / having," or any other variations thereof, are intended to cover a non-exclusive inclusion, i.e., to represent an open list of features. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited to only those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus. In contrast, "consists of / consisting of," or any other variations thereof, designates a closed list of features. Notably, a closed list of a given feature is understood to represent a specific embodiment of the open list of those features.

[0084] As used herein, unless expressly stated otherwise, "or" refers to an inclusive or rather than an exclusive or. For example, any of the following may satisfy condition A or B: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0085] As used herein, "substantially," "relatively," "generally," "typically," "about," and "approximately" are relative modifiers intended to indicate permissible variations in the characteristic so modified. They are not intended to limit the absolute value or property to which they modify, but rather to approximate or approximate such physical or functional property. If not otherwise specified, the term "about" in combination with a numerical value n ("about n") is understood to mean a value x in the interval given by ±5% of the numerical value, i.e., n-0.05*n≤x≤n+0.05*n. If the term "about" is combined with a numerical value n to describe an embodiment of the present invention, then, if not otherwise specified, that value of n is the most preferred.

[0086] In this detailed description, references to "one embodiment," "an embodiment," or "in an embodiment" mean that the referenced features are included in at least one embodiment of the technology in accordance with all aspects of the present disclosure. Furthermore, separate references to "one embodiment," "an embodiment," or "an embodiment" are not necessarily to the same embodiment; however, these embodiments are not mutually exclusive unless otherwise specified and unless obvious to one skilled in the art. Therefore, the technology in accordance with the present disclosure in all its aspects can include any kind of combination and / or integration of the embodiments described herein.

[0087] As used herein, the term "solid phase" refers to a variety of materials commonly used by those skilled in the art to isolate molecules, including solids, semi-solids, gels, films, membranes, nets, felts, composite materials, particles, paper, etc. The solid phase can be non-porous or porous. Suitable solid phases include those developed and / or used as solid phases in solid phase binding assays. See, for example, Immunoassay, Chapter 9, edited by EP Dianiandis and TK Christopoulos, Academic Press: New York, 1996, which is incorporated herein by reference. Examples of suitable solid phases include membrane filters, cellulose-based paper, beads (including polymers, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels and porous plates. See, for example, Leon et al., Bioorg. Med. Chem. Lett. 8:2997, 1998; Kessler et al., Agnew. Lett. 42:515, 2001; Papanikos et al., J. Am. Chem. Soc. 123: 2176, 2001; Gottschling et al., Bioorg. Med. Chem. Lett. 11: 2997, 2001.

[0088] The surfaces of the solid phases described above may be modified to provide attachment sites, for example by bromoacetylation, silanization, addition of amino groups using nitric acid, and attachment of intermediate proteins, dendrimers, and / or star polymers. This list is not meant to be limiting, and any method known to those skilled in the art may be used.

[0089] Particle-based analyte-specific binding assays are widely used, for example, in certain turbidimetric assays, certain latex agglutination assays, and many sensitive sandwich-type assays using a variety of labeling or detection technologies.

[0090] A particle is an embodiment of a solid phase. As used herein, a "particle" refers to a small, localized object to which a physical property such as volume, mass, or average size can be attributed. Microparticles can therefore be symmetrical, spherical, substantially spherical, or spherical, or of irregular, asymmetric shape or form. The size of the particles contemplated by the present invention can vary. In one embodiment, spherical shapes are used, for example, microparticles having diameters in the nanometer and micrometer range. In one embodiment, the microparticles used in accordance with the methods of the present disclosure have a diameter of 50 nanometers to 20 microns. In another embodiment, the microparticles have a diameter of between 100 nm and 10 μm. In one embodiment, the microparticles used in accordance with the methods of the present disclosure have a diameter of 200 nm to 5 μm or 750 nm to 5 μm.

[0091] Microparticles as defined above may comprise or consist of any suitable material known to those skilled in the art, for example they may comprise or consist of an inorganic or organic material. Typically, they may comprise or consist of a metal or metal alloy, or an organic material, or consist of or consist of a carbohydrate element, or consist of or consist of a carbohydrate element. Examples of materials envisioned for microparticles include agarose, polystyrene, latex, polyvinyl alcohol, silicon dioxide, and ferromagnetic metals, alloys, or composites. In one embodiment, the microparticles are magnetic or ferromagnetic metals, alloys, or compositions. In another embodiment, the material may have specific properties, for example, be hydrophobic or hydrophilic. Such microparticles are typically dispersed in aqueous solution and retain a small negative surface charge, thereby keeping the microparticles separate and avoiding nonspecific aggregation.

[0092] In one embodiment of the present invention, the microparticles are paramagnetic microparticles, and the separation of such microparticles in the measurement methods according to the present disclosure is facilitated by magnetic forces. A magnetic force is applied to pull the paramagnetic or magnetic particles out of the solution / suspension and retain them as desired, while the liquid of the solution / suspension can be removed and the particles can be washed, for example. The microparticles used in the methods according to the present invention are coated with the first member of the specific binding pair.

[0093] In general, the term "receptor" refers to any compound or composition that is capable of recognizing a specific spatial and polar organization of a target molecule (i.e., an epitope site of an analyte). Thus, the term "analyte-specific receptor" as referred to herein includes analyte-specific reactants that are capable of binding or complexing with an analyte. This includes, but is not limited to, antibodies, specifically monoclonal antibodies or antibody fragments. Such receptors can act as traps for analytes, for example to immobilize analytes. The epitope recognized by the binding antibody is followed by a labeled antibody specific for another epitope of the analyte. Other receptors are known to those skilled in the art. With reference to this disclosure, those skilled in the art will understand the specific uses of various receptors in receptor-based assays.

[0094] An "analyte" can be any molecule that can be bound by an analyte-specific receptor. In one embodiment, the analyte in the present disclosure is a nucleic acid (DNA or RNA) molecule, a peptide, a protein, a drug molecule, a hormone, or a vitamin. In one embodiment, the analyte in the present disclosure is a peptide, a protein, a drug molecule, a hormone, or a vitamin. In another embodiment, the analyte includes several variants, in one embodiment, different genotypes, isozymes, isoforms, serotypes, or mutants of the analyte. In one embodiment, the analyte is an antigen of an infectious agent. Examples of infectious agents are viruses, bacteria, and protozoan pathogens that infect humans. In one embodiment, the analyte is a viral antigen, in one embodiment, a hepatitis virus antigen or a human retrovirus antigen. In one embodiment, the analyte is a hepatitis C virus, a hepatitis B virus, or an HIV antigen.

[0095] In the context of the present disclosure, the term "antibody" relates to intact immunoglobulin molecules, in particular IgM, IgD, IgE, IgA or IgG, as well as parts of such immunoglobulin molecules, such as Fab-fragments or V L -、V H - or CDR-region. In addition, the term relates to modified and / or altered antibodies, such as chimeric and humanized antibodies. The term also relates to modified or altered monoclonal or polyclonal antibodies and recombinantly or synthetically produced / synthesized antibodies. The term also relates to whole antibodies and antibody fragments / portions thereof, such as isolated light and heavy chains, Fab, Fab / c, Fv, Fab', F(ab')2. The term "antibody" also includes antibody derivatives, bifunctional antibodies and antibody constructs, such as single-chain Fv (scFv), bispecific scFv or antibody fusion proteins.

[0096] "Detectable labels" include moieties that are detectable or can become detectable. The skilled artisan knows that labels are compounds or compositions that provide a detectable signal in conjunction with a physical activation (or excitation) or chemical agent and that can be modified to reduce or increase a specific signal.

[0097] Specific embodiments of detectable labels include labels that can be detected by a variety of commercially available instruments that utilize electrochemiluminescence (ECL) for analytical measurement. Species that can induce emission of ECL (ECL-active species) have been used as ECL labels. Examples of ECL labels include: i) organometallic compounds, wherein the metal is from, for example, Group VIII noble metals, including organometallic compounds containing Ru and Os, such as ruthenium terpyridine (RuBpy) moieties and ii) luminol and related compounds. Species that participate in the ECL process together with the ECL label are referred to herein as ECL co-reactants. Common co-reactants include tertiary amines (see, for example, US5,846,485), oxalates, and persulfates for ECL from RuBpy and hydrogen peroxide for ECL from luminol (see, for example, US5,240,863). The light generated by the ECL label can be used as a reporter signal in diagnostic procedures (Bard et al. al., US5,238,808). For example, an ECL label can be covalently coupled to a binding agent, such as an antibody, a nucleic acid probe, a receptor, or a ligand; the participation of the binding agent in the binding interaction can be monitored by measuring the ECL emitted from the ECL label. Alternatively, the ECL signal from an ECL-active compound can be indicative of the chemical environment (see, e.g., US5,641,623, which describes an ECL assay that monitors the formation or destruction of ECL co-reactants). For more background on ECL, ECL labels, ECL assays, and instruments for performing ECL assays, see US5,093,268; US5,147,806; US5,324,457; US5,591,581; US5,597,910; US5, 641,623; US5,643,713; US5,679,519; US5,705,402; US5,846,485; US5,866,434; US5,786,141; US5,731,147; US6,066,448; US6,136,268; US5,776,672; US5,308,754; US5,240,863; US6,207,369 and US5,589,136; and WO99 / 63347, WO00 / 03233, WO99 / 58962, WO99 / 32662, WO99 / 14599, WO98 / 12539, WO97 / 36931 and WO98 / 57154.

[0098] According to general knowledge in the field of biochemistry, a "binding pair" is understood to be a set of two different partners, namely, a first and a second partner, or a species of partner, or a first and a second member of a pair, or a first and a second species. Under non-denaturing conditions, the partners are capable of specifically recognizing the other species of partner at the molecular level. Following recognition, the partners of the binding pair form a stable non-covalent intermolecular bond, linking the first partner to the second partner. When selecting the species of partners to create a binding pair, it is important that each partner does not form an association with another partner of the same species. That is, no stable intramolecular bonds occur between the two first partners or between the two second partners.

[0099] In this document, a punctuation mark (“:”) between the first and second members of a binding pair can be used to indicate a specific connection or the ability to form such a specific connection between the first and second members of the binding pair, thus being represented by “member 1:member 2”. Typically, the first and second members belong to different species, i.e., the first and second members are not the same compound. Thus, depending on the context, “member 1:member 2” can mean that member 1 and member 2 can form a binding pair, and member 1 is capable of specifically recognizing and binding to member 2; or, depending on the context, “member 1:member 2” can mean that member 1 and member 2 are a linked pair. It should also be understood that, unless specifically described differently, members include not only members that are isolated compounds, but also members that are linked to another entity, e.g., forming part of another entity. For example, a “(strept)avidin:biotin” (= “biotin:(strept)avidin”) binding pair is well known to those skilled in the art. Biotin or a biotin moiety on the one hand and (strept)avidin or (strept)avidin coupled to another structure on the other hand represent the two members of this exemplary binding pair.

[0100] Single-stranded nucleic acids are polymers composed of monomeric units called nucleotides. Each nucleotide is composed of a phosphate, a sugar, and a nucleobase. Nucleotide chains in nucleic acids are linked by 3′, 5′ phosphodiester bonds. This means that the 5′-phosphate group of one nucleotide is esterified by the 3′-hydroxyl group of an adjacent nucleotide.

[0101] Single-stranded " oligonucleotide " is a kind of short nucleic acid, is generally made up of up to about 15 nucleotide monomers, and these monomers are connected by the phosphodiester bond between the 3 ' carbon atom of a sugar molecule and the 5 ' carbon atom of another sugar molecule.The monomer (general sense) included in the oligonucleotide can be not only naturally occurring monomers, but also non-natural monomers, also referred to as nucleotide analogs. In a non-limiting manner, exemplary analogs include sugar moieties other than ribose or deoxyribose, particularly ribose in which the sugar ring is "locked" by the methylene bridge connecting 2 '-O atom and 4 '-C atom. For the purposes of this disclosure, the term nucleotide includes natural and non-natural nucleotides as monomers in oligonucleotides. Therefore, according to this definition, the oligonucleotide can be composed entirely of natural or non-natural monomers, or it can be composed of their mixtures. In addition, it should be understood that different types of non-natural monomers (such as PNA, D-LNA, L-LNA, homologous DNA (containing hexose), HNA (containing hexitol, hexitol nucleic acid), L-DNA, etc.) can be included in the oligonucleotide, if not otherwise specified.

[0102] Non-natural monomers can include nucleobases, which themselves can be naturally occurring nucleobases or their non-natural analogs. "Nucleobases" are nitrogenous unsaturated hydrocarbon compounds and include planar heterocyclic moieties. Naturally occurring nucleobases can be divided into two main forms: purines and pyrimidines. Although purines and pyrimidines are both heterocyclic aromatic compounds, they can be distinguished based on their chemical structure. Purines appear in the form of two carbon rings, while pyrimidines appear in the form of one carbon ring. Purines have a pyrimidine ring fused to an imidazole ring. Pyrimidines have only one pyrimidine ring, purines have four nitrogen atoms, and pyrimidines have two. Nucleosides are formed when nucleobases are connected to a sugar moiety, which is typically a five-carbon ribose or deoxyribose or its derivatives (e.g., locked ribose). Therefore, nucleosides are glycosylamines and include, for example, cytidine, uridine, adenosine, guanosine, thymidine, and inosine. In these examples, the anomeric carbon of the five-carbon sugar is connected to the N9 of purine or the N1 of pyrimidine by a glycosidic bond.

[0103] Nucleosides are components of nucleotides that further include a phosphate moiety or its derivatives or functional analogs. Nucleotides are the monomeric units of single-stranded nucleic acids. In double-stranded nucleic acids such as DNA, nucleobases are paired. The two complementary nucleobases are connected by hydrogen bonds.

[0104] The term "nucleobase" includes canonical and non-canonical naturally occurring nucleobases and their analogs. A large number of non-naturally occurring nucleobases are known. For the purposes of this disclosure, these nucleobase analogs are specifically considered to be embodiments in which a nucleobase analog as part of a first oligonucleotide chain can form one or more hydrogen bonds with another adjacent nucleobase in a second oligonucleotide chain, wherein the two oligonucleotide chains are paired and form a duplex, specifically an antiparallel duplex. Typically, the nucleobases of the base pairs in the duplex are in a planar orientation. For the purposes of this disclosure, a non-limiting compilation of nucleobases includes compounds selected from the group consisting of: N 4 -acetylcytosine, 5-acetyluracil, 4-amino-6-chloropyrimidine, 4-amino-5-fluoro-2-methoxypyrimidine, 6-amino-1-methyluracil, 5-aminoorotic acid, 5-aminouracil, 6-aminouracil, 6-azauracil, N 4 -benzoylcytosine, 5-bromouracil, 5-chlorouracil, 6-chlorouracil, 6-chloromethyluracil, 6-chloro-3-methyluracil, cytosine, 5,6-dimethyluracil, 5-ethyluracil, 5-ethynyluracil, 5-fluorocytosine, 5-fluoroorotic acid, 5-fluorouracil, 5-iodo-2,4-dimethoxypyrimidine, 5-iodouracil, isocytosine, 5-methylcytosine, 6-methyl-5-nitrouracil, 2-methylthio-4-pyrimidinol, 5-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyluracil Pyrimidine, 5-nitrouracil, orotic acid, 6-phenyl-2-thiouracil, 6-propyl-2-thiouracil, 2-thiouracil, 4-thiouracil, thymine, 5-(trifluoromethyl)uracil, uracil, adenine, 8-azahypoxanthine, 8-azaguanine, allopurinol, 4-aminopyrazolo[3,4-d]pyrimidine, 2-aminopurine, 2-acetylamino-6-hydroxypurine, 2-amino-6-chloropurine, 2-amino-6-iodopurine, azathioprine, 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine, aminophylline, N 6 -Benzyl adenine, N 6-benzoyl adenine, 6-benzyloxypurine, 8-bromotheoline, 8-bromo-3-methylxanthine, 8-bromo-7-(2-butyn-1-yl)-3-methylxanthine, 6-chloropurine, 8-chlorotheoline, 6-chloro-2-fluoropurine, 6-chloro-7-deazapurine, 2-chloroadenine, 6-chloro-7-iodo-7-deazapurine, 2,6-diaminopurine, 2,6-dichloropurine, 6 -(dimethylamino)purine, 2,6-dichloro-7-deazapurine, 5,6-dichlorobenzimidazole hydrochloride, 7-deazahypoxanthine, 2-fluoroadenine, guanine, hypoxanthine, isoguanine, 3-iodo-1H-pyrazolo-[3,4-d]pyrimidin-4-amine, kinetin, 6-mercaptopurine, 6-methoxypurine, 3-methylxanthine, 1-methylxanthine, 3-methyladenine, O 6 -(cyclohexylmethyl)guanine, 6-thioguanine, 2-thioxanthine, xanthine, 5-propynyl-uracil, 5-propynyl-cytidine, 7-deazaadenine, 7-deazaguanine, 5-propynyl-uracil, 5-propynyl-cytidine, 7-deazaadenine, 7-deazaguanine, 7-propynyl-7-deazaadenine, 7-propynyl-7-deazaguanine and derivatives thereof.

[0105] Complementary single-stranded oligonucleotides or polynucleotides can form double-stranded ("duplex") nucleic acids. Duplex formation, also referred to as "hybridization," refers to the formation of partially or fully double-stranded (duplex) nucleic acids (e.g., DNA:DNA, DNA:RNA, RNA:RNA, LNA:DNA, LNA:LNA, etc.) through sequence-specific interaction between two at least partially complementary single-stranded nucleic acids, as examples of binding pairs. The terms "association" or "detachment (denaturation)" of complementary single-stranded nucleic acids and "annealing" of duplexes are often used to describe hybridization between fully complementary strands.

[0106] Hybridization in aqueous solution, also known as "annealing," is an integral part of the present disclosure. With respect to hybridized duplex molecules of oligonucleotides or polynucleotides (including analogs thereof), the skilled artisan understands that melting temperature, hybridization rate, and dissociation rate are interrelated with temperature.

[0107] According to common sense, a "Watson-Crick base pair" is a single non-covalent crosslink in a double-stranded nucleic acid helix (=duplex), where each single strand of the duplex is an oligonucleotide. Thus, in an exemplary embodiment of a duplex, the two oligonucleotide strands are crosslinked via purine and pyrimidine base pairs that protrude inward from the oligonucleotide backbone sugars and are linked by hydrogen bonds, such as adenine pairing with thymine and cytosine pairing with guanine. Consistent with the above, the nucleobases can be naturally occurring nucleobases or analogs thereof, as long as the pair of nucleobases can interact complementary to form a single non-covalent crosslink (duplex) in the double-stranded nucleic acid helix.

[0108] It is well known that secondary structural motifs in single-stranded nucleic acids often impair the intended hybridization reaction (e.g., as discussed by Koehler RT & Peyret N. Comput Biol Chem. 29 (2005) 393-397). This finding also applies to ss-oligonucleotides, including single-stranded LNA oligonucleotides. Secondary structure can arise through internal folding of single-stranded molecules, driven by intramolecular interactions (e.g., hydrogen bonds or hydrophobic interactions). In the specific case of a first single-stranded oligonucleotide, a certain folded structure may be thermodynamically favored, where the folded structure then interferes with the unimpeded presentation of the nucleobase sequence to the complementary second oligonucleotide. Therefore, efforts are necessary to predict and avoid such structures. Conversely, secondary structure in the target binding site may also impair hybridization. Therefore, it is necessary to assess the secondary structure of both partners of a binding pair consisting of oligonucleotides. Several challenges confound this goal, including imperfect empirical rules and prediction parameters, and the poor scalability of folding algorithms across sequence lengths.

[0109] In addition, in the member of identical oligonucleotide kind, i.e. the oligonucleotide that shares identical core base sequence, there may be one or more parts that can be partially complementary, thereby potentially causing intermolecular interaction by the Watson-Crick base pairing of one or more core bases. Otherwise, there may also be one or more parts in the sequence, which can cause intermolecular interaction by the intermolecular interaction of non-Watson-Crick (e.g. Hoogsteen) base pairing or other forms. In the case of intramolecular folding (see above), the intramolecular interaction between the member of the first single-stranded oligonucleotide can result in a thermodynamically favorable structure, wherein such a structure then interferes with the unhindered presentation of the core base sequence to the complementary second oligonucleotide.

[0110] If unwanted inter- or intramolecular structures are present, they can be eliminated by denaturation. As is known from the use of ss-oligonucleotides in the field of polymerase chain reaction (PCR) technology, the annealing step is typically preceded by a heating step, during which the oligonucleotides are denatured, i.e., their intramolecular secondary structures are disrupted. The heating step is typically followed by a gradual and controlled temperature reduction, intended to provide suitable annealing conditions between the oligonucleotide and the target sequence. However, PCR is a process involving sufficiently thermostable reaction partners, such as oligonucleotide primers, nucleoside triphosphates, salts, buffers, and thermostable polymerases. However, other processes in which oligonucleotide annealing can be effective prohibit heating or other types of denaturing treatments, as these processes may involve denaturation-sensitive components that may irreversibly degrade. This is particularly (but not exclusively) true for analyte detection assays, in which protein analyte-specific receptors (e.g., antibodies) play a key functional role. Therefore, the present disclosure and reports of surprising findings, which exclusively involve technical settings that utilize heat, particularly incubation at temperatures above 68° C. (e.g., Childs JLPNAS 99 (2002) 11091-11096), are not possible. For practical reasons, temperatures above 40° C. are undesirable in most assays, such as, but not limited to, immunoassays. That is, for practical applications of binding pairs composed of complementary oligonucleotides, particularly desirable conditions are between 0° C. and 40° C. Under these conditions, any technical application must be unaffected by intra- or intermolecular structure, as may be the case with isolated binding partners of an oligonucleotide binding pair.

[0111] Other options for denaturing nucleic acids (including oligonucleotides) are known to the skilled artisan from reports on DNA. Several methods for denaturing DNA are known in the art, including heating, incubation under alkaline conditions equivalent to an aqueous solution of more than 0.01 mol / L NaOH (pH 12 or higher), incubation in the presence of dimethyl sulfoxide (DMSO), incubation in the presence of formamide, incubation in the presence of chaotropic compounds, and incubation in the presence of sonication. Although it remains to be demonstrated that such treatments provide conditions for the production and / or stabilization of not only ss-DNA but also ss-LNA, it is clear that they are not required in assays for detecting analytes, in which protein analyte-specific receptors, such as antibodies, play a key functional role.

[0112] Therefore, "denaturing conditions" that may, on the one hand, be able to counteract unwanted intramolecular and intermolecular structures in all-LNA ss-oligonucleotide species in aqueous solution, but on the other hand are to be avoided in any aspect and embodiment of the technical method presented in this report, are selected from the group consisting of: applying a temperature above 40°C, applying a temperature above 68°C (heating), applying ultrasonic treatment, incubating under alkaline conditions equivalent to an aqueous solution of more than 0.01 mol / L NaOH, incubating in the presence of dimethyl sulfoxide (DMSO) at a concentration capable of destroying intramolecular and intermolecular structures, incubating in the presence of formamide at a concentration capable of destroying intramolecular and intermolecular structures, incubating in the presence of a chaotropic compound at a concentration capable of destroying intramolecular and intermolecular structures, and mixtures thereof. For the purposes of this report, embodiments in the absence of denaturing conditions (= embodiments under non-denaturing conditions) are the absence and / or lack of any application of temperatures above 40°C, application of temperatures above 68°C (heating), application of ultrasound, incubation under alkaline conditions equivalent to more than 0.01 mol / L NaOH aqueous solution, incubation in the presence of dimethyl sulfoxide (DMSO) at a concentration capable of destroying intra- and intermolecular structures, incubation in the presence of formamide at a concentration capable of destroying intra- and intermolecular structures, incubation in the presence of chaotropic compounds at a concentration capable of destroying intra- and intermolecular structures, and mixtures thereof.

[0113] Importantly, under native conditions, each partner of a binding pair is not expected to form any intramolecular bonds that would prevent it from forming bonds with the other species of partner. As previously described and exemplified, in such undesirable situations, intramolecular folding and stabilization of one fold within a partner species can result in a secondary structure that is sufficiently stable under native conditions to inhibit or prevent the desired intramolecular bonding of the two different species of the binding pair.

[0114] All-LNA ss-oligonucleotides containing five or more monomers have properties that cannot be reliably predicted using the tools available to those skilled in the art, taking into account non-denaturing conditions and specifically excluding any application of the denaturing treatments detailed herein. For practical reasons, this study was limited to ss-oligonucleotides composed of up to 15 LNA monomers to identify those capable of forming a hybrid duplex from two separate single-stranded species in the absence of denaturing conditions. This means that each member of the binding pair must be fully free of any inter- or intramolecular structure. This report demonstrates that this cannot be taken for granted for complementary all-LNA oligonucleotides.

[0115] Accordingly, the present disclosure unexpectedly provides, in a first aspect related to all other aspects and embodiments disclosed herein, pairs of isolated ss-oligonucleotides, each composed of 5 to 15 LNA monomers, capable of forming antiparallel duplexes with each other in aqueous solution in the absence of denaturing conditions prior to duplex formation. Independent of any theoretical and / or computer-implemented models of uncertain reliability, such pairs can be unexpectedly identified and provided by the following method of the second aspect. To the best of the authors' knowledge, such a method has not been previously demonstrated or even suggested.

[0116] Thus, in a second aspect related to all other aspects and embodiments disclosed herein, the present disclosure provides a method for selecting and providing a binding pair of single-stranded all-LNA oligonucleotides capable of forming an antiparallel duplex having 5 to 15 consecutive base pairs in aqueous solution at a temperature of 0° C. to 40° C., the method comprising the steps of:

[0117] (a) Providing a first single strand (=ss-) consisting of 5 to 15 locked nucleic acid (=LNA) monomers

[0118] an oligonucleotide, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0119] (b) providing a second ss-oligonucleotide composed of 5 to 15 LNA monomers, the second ss-oligonucleotide consisting of at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence of the second ss-oligonucleotide, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation, and the complementarity predicts the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 5 to 15 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other;

[0120] (c) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0121] (d) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex;

[0122] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0123] (f) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide, selecting the binding pair;

[0124] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0125] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0126] In one embodiment, specifically, steps (c) and (d) are performed in the absence of conditions designated as "denaturing conditions" as described above.

[0127] A specific embodiment of the second aspect, which is relevant to all other aspects and embodiments disclosed herein, provides a method for selecting and providing a binding pair of single-stranded all-LNA oligonucleotides capable of forming an antiparallel duplex having 5 to 7 consecutive base pairs in aqueous solution at a temperature of 0° C. to 40° C., the method comprising the steps of:

[0128] (a) providing a first single-stranded (=ss-) oligonucleotide consisting of 5 to 7 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0129] (b) providing a second ss-oligonucleotide consisting of 5 to 15 LNA monomers, the second ss-oligonucleotide consisting of at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence of the second ss-oligonucleotide, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation, and the complementarity predicts the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 5 to 7 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other;

[0130] (c) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0131] (d) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex;

[0132] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0133] (f) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide, selecting the binding pair;

[0134] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0135] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0136] In one embodiment, specifically, steps (c) and (d) are performed in the absence of conditions designated as "denaturing conditions" as described above.

[0137] Each single-stranded oligonucleotide is composed of monomers, wherein each monomer is a ribonucleoside analog, wherein in the ribose portion of the ribonucleoside analog, a methylene group links the 2'-oxygen and 4'-carbon atoms, thereby locking the ribose in a C3-endo conformation. The all-LNA ss-oligonucleotides according to the present disclosure can be synthesized using standard techniques using building blocks such as protected phosphoramidite chemistry of individual LNA nucleosides.

[0138] Each LNA monomer comprises a nucleobase, wherein the nucleobase is selected from canonical or non-canonical naturally occurring nucleobases and non-naturally occurring nucleobases. In one embodiment, the LNA monomer comprises a nucleobase selected from the group consisting of: N 4 -acetylcytosine, 5-acetyluracil, 4-amino-6-chloropyrimidine, 4-amino-5-fluoro-2-methoxypyrimidine, 6-amino-1-methyluracil, 5-aminoorotic acid, 5-aminouracil, 6-aminouracil, 6-azauracil, N 4-benzoylcytosine, 5-bromouracil, 5-chlorouracil, 6-chlorouracil, 6-chloromethyluracil, 6-chloro-3-methyluracil, cytosine, 5,6-dimethyluracil, 5-ethyluracil, 5-ethynyluracil, 5-fluorocytosine, 5-fluoroorotic acid, 5-fluorouracil, 5-iodo-2,4-dimethoxypyrimidine, 5-iodouracil, isocytosine, 5-methylcytosine, 6-methyl-5-nitrouracil, 2-methylthio-4-pyrimidinol, 5-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyluracil Pyrimidine, 5-nitrouracil, orotic acid, 6-phenyl-2-thiouracil, 6-propyl-2-thiouracil, 2-thiouracil, 4-thiouracil, thymine, 5-(trifluoromethyl)uracil, uracil, adenine, 8-azahypoxanthine, 8-azaguanine, allopurinol, 4-aminopyrazolo[3,4-d]pyrimidine, 2-aminopurine, 2-acetylamino-6-hydroxypurine, 2-amino-6-chloropurine, 2-amino-6-iodopurine, azathioprine, 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine, aminophylline, N 6 -Benzyl adenine, N 6 -Benzoyladenine, 6-benzyloxypurine, 8-bromotheoline, 8-bromo-3-methylxanthine, 8-bromo-7-(2-butyn-1-yl)-3-methylxanthine, 6-chloropurine, 8-chlorotheoline, 6-chloro-2-fluoropurine, 6-chloro-7-deazapurine, 2-chloroadenine, 6-chloro-7-iodo-7-deazapurine, 2,6-diaminopurine, 2,6-dichloropurine, 6-(dimethylamino) Purine, 2,6-dichloro-7-deazapurine, 5,6-dichlorobenzimidazole hydrochloride, 7-deazahypoxanthine, 2-fluoroadenine, guanine, hypoxanthine, 9-(2-hydroxyethyl)adenine, isoguanine, 3-iodo-1H-pyrazolo-[3,4-d]pyrimidin-4-amine, kinetin, 6-mercaptopurine, 6-methoxypurine, 3-methylxanthine, 1-methylxanthine, 3-methyladenine, O 6 -(cyclohexylmethyl)guanine, 6-thioguanine, 2-thioxanthine, xanthine, 5-propynyl-uracil, 5-propynyl-cytidine, 7-deazaadenine, 7-deazaguanine, 7-propynyl-7-deazaadenine, 7-propynyl-7-deazaguanine and derivatives thereof.

[0139] The all-LNA ss-oligonucleotide according to all aspects and embodiments disclosed herein may comprise a number of monomers, the number of which is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. In one embodiment of all aspects and embodiments disclosed herein, the first ss-oligonucleotide consists of 8 to 15 monomers (i.e., a number selected from 8, 9, 10, 11, 12, 13, 14, and 15 monomers). In another embodiment of all aspects and embodiments disclosed herein, the first ss-oligonucleotide consists of 9 to 11 monomers (i.e., a number selected from 9, 10, and 11 monomers), and in a more specific embodiment of all aspects and embodiments disclosed herein, the first ss-oligonucleotide consists of 9 monomers.

[0140] Initial experiments used these oligonucleotide sizes to provide the following combination: first, high binding specificity for the binding partner; second, favorable speed at which the two partners of the binding pair hybridize and form a duplex; and third, stable duplex formation with essentially no detectable tendency to re-dissociate into single strands. Surprisingly, it was found that even binding pairs of all-LNA ss-oligonucleotides with complementary nucleobase sequences consisting of 7, 6, or even 5 consecutive LNA monomers met the criteria of sufficient specificity, pairing speed, and duplex stability. Even more surprisingly, these criteria were met under specific environmental conditions, a prerequisite for using binding pairs of all-LNA ss-oligonucleotides as a molecular recognition tool in assays for detecting target analytes, such as, but not limited to, immunoassays. Therefore, a specific embodiment related to all other aspects disclosed herein is a method for selecting and providing binding pairs of single-stranded all-LNA oligonucleotides capable of forming antiparallel duplexes with 5, 6, or 7 consecutive base pairs in aqueous solution at temperatures between 0°C and 40°C, which method is described as a specific embodiment.

[0141] Another specific embodiment related to all other aspects and embodiments disclosed herein is a pair of isolated ss-oligonucleotides, each comprised of 5 to 7 LNA monomers, the isolated ss-oligonucleotides being capable of forming antiparallel duplexes with each other in aqueous solution in the absence of denaturing conditions prior to duplex formation. Another embodiment related to all other aspects is a pair of ss-oligonucleotides sharing complementary nucleobase sequences of 5, 6, or 7 LNA monomers contained in each ss-oligonucleotide, the bound pair being capable of forming all-LNA duplexes in aqueous solution in the absence of denaturing conditions. As described above, the absence of denaturing conditions means that none of the ss-oligonucleotides undergo denaturation prior to duplex formation, nor is a denaturing treatment part of the incubation after the members of the bound pair are brought into contact with each other in aqueous solution.

[0142] Shorter LNA oligomers can be less complex and more economical to synthesize, and they provide an ideal source of complementary binding partners for molecular recognition in aqueous solution.

[0143] In certain embodiments relevant to all aspects of this report, a member of a binding pair is an all-LNA ss-oligonucleotide, wherein the all-LNA ss-oligonucleotide is a fragment of a larger all-LNA ss-oligonucleotide, the larger ss-oligonucleotide being a member, partner, or species of the binding pair selected and provided by the method for selecting and providing a single-stranded all-LNA binding pair according to the second aspect provided herein. In a specific embodiment thereof, the larger oligonucleotide comprises 8 to 15 LNA monomers, and the fragment comprises a contiguous nucleobase subsequence of the larger fragment, wherein the fragment comprises 5 to 7 LNA monomers. Thus, in one embodiment, the binding pair consists of a first and a second all-LNA ss-oligonucleotide, each comprising 5, 6, or 7 LNA monomers, the nucleobase sequences of the first and second all-LNA ss-oligonucleotides being complementary, thereby being capable of forming an antiparallel duplex having 5, 6, or 7 base pairs, wherein each member of the binding pair is a fragment of a larger all-LNA single-stranded oligonucleotide selected and provided by the method according to the second aspect provided herein. In one embodiment, the first and second all-LNA ss-oligonucleotides each consist of 5 monomers. In another embodiment, the first and second all-LNA ss-oligonucleotides each consist of 6 monomers. In yet another embodiment, the first and second all-LNA ss-oligonucleotides each consist of 7 monomers.

[0144] Thus, the present disclosure, in a third aspect, particularly relating to the second aspect but also to all other aspects and embodiments disclosed herein, provides a method for selecting and providing a binding pair of single-stranded all-LNA oligonucleotides capable of forming an antiparallel duplex having 5 to 7 consecutive base pairs in aqueous solution at a temperature of 0° C. to 40° C., the method comprising the steps of:

[0145] (a) Providing a first single strand (=ss-) consisting of 8 to 15 locked nucleic acid (=LNA) monomers

[0146] an oligonucleotide, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0147] (b) providing a second ss-oligonucleotide consisting of 8 to 15 LNA monomers, the second ss-oligonucleotide consisting of at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence of the second ss-oligonucleotide, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation, and the complementarity predicts the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 8 to 15 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other;

[0148] (c) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0149] (d) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex;

[0150] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0151] (f) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide, selecting the binding pair;

[0152] (g) selecting a first contiguous nucleobase subsequence from the oligonucleotides of the binding pair selected in step (f), thereby generating a fragment of the oligonucleotide consisting of 5 to 7 LNA monomers;

[0153] (h) optionally selecting a second contiguous nucleobase subsequence from the other oligonucleotide of the binding pair selected in step (f), wherein the second subsequence is complementary to the first subsequence of step (g), thereby generating a further oligonucleotide consisting of a fragment of 5 to 7 LNA monomers;

[0154] (i) synthesizing the ss-oligonucleotide fragment of step (g) and the ss-oligonucleotide fragment of step (h), respectively;

[0155] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0156] In one embodiment, specifically, steps (c) and (d) are performed in the absence of conditions designated as "denaturing conditions" as described above.

[0157] The property of the selected fragments, i.e. their ability to form antiparallel duplexes in aqueous solution at temperatures between 0° C. and 40° C., can be easily verified. Therefore, in a particular embodiment, the method comprises the following additional steps:

[0158] (k) mixing approximately equimolar amounts of said first and second ss-oligonucleotide fragments in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0159] (1) incubating the mixture of (k) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotide fragments as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotide fragments as duplexes;

[0160] (m) If a duplex is detected in step (1) but no ss-oligonucleotide fragment is detectable, then the binding pair is selected.

[0161] This report provides single-stranded all-LNA oligonucleotides as binding pairs, capable of replacing other binding pairs such as biotin and (strept)avidin. That is, with respect to all aspects and embodiments herein, these oligonucleotides are composed of LNA monomers and are capable of duplex formation under native conditions, particularly when provided and optionally stored under native conditions. Thus, each such ss-oligonucleotide, provided and optionally stored under native conditions, is capable of hybridizing with its binding partner under native conditions and retains this quality under native conditions.

[0162] More specifically, in the context of the present disclosure, "denaturing conditions" include any presence or addition of a denaturing agent, as an example, that is capable of reducing the melting temperature of a DNA duplex of 20 base pairs in length and 50% G+C content by 15°C or more.

[0163] Denaturing conditions do not include conditions that must be performed in assays using protein analyte-specific receptors to detect target analytes in order to maintain the desired capacity and functionality of these compounds. That is, under denaturing conditions, the desired capacity and functionality of these compounds may be lost. Furthermore, these non-denaturing conditions do not include any of the following: temperatures exceeding 68°C, application of sonication, incubation under alkaline conditions equivalent to an aqueous solution exceeding 0.01 mol / L NaOH, incubation in the presence of dimethyl sulfoxide (DMSO), incubation in the presence of formamide, incubation in the presence of a chaotropic compound, and mixtures thereof, wherein the application of any of these conditions, if present, disrupts the intra- and intermolecular structure of the individual all-LNA ss-oligonucleotides to such an extent that the intra- and intermolecular structure prevents hybridization and duplex formation of the oligonucleotides with complementary single all-LNA ss-oligonucleotides. Importantly, the all-LNA ss-oligonucleotides taught in this report do not require any such denaturing conditions, neither for the oligonucleotides in isolated form nor for the oligonucleotides as a bound pair, i.e., when one member is in contact with the other.

[0164] The first and second ss-oligonucleotides do not have to be of equal size, ie, do not have to be composed of an equal number of monomers. However, an equal number of monomers constituting the first and second ss-oligonucleotides is a specific embodiment of all aspects and embodiments of the present disclosure.

[0165] As will be appreciated by those skilled in the art, two oligonucleotides are antiparallel if they are parallel to each other but arranged in opposite directions. A specific example is a duplex with two complementary strands extending in opposite directions. Thus, each end of the duplex includes the 5′ end of the first strand adjacent to / aligned with the 3′ end of the opposing second strand. Similar to DNA and RNA, LNAs exhibit Watson-Crick base pairing (Koshkin, AA et al. J Am Chem Soc 120 (1998) 13252-13260).

[0166] The specific Watson-Crick base pairing involving hydrogen bridges forming bases on complementary opposing strands is a well-known feature of those skilled in the art and is widely disclosed in the art. Examples include the classic base pairs adenine:thymine, adenine:uracil, and cytosine:guanine. Other Watson-Crick base pairs include 5-methylcytosine:guanine, 5-hydroxymethylcytosine:guanine, 7-deazaguanine:cytosine, and 5-chlorouracil:7-deazaadenine. Many more are known in the art.

[0167] In order for the two members of a binding pair to join, one must be brought into contact with the other. Following the contacting step, the two members can interact and form a duplex. As described above, denaturing conditions are not required for this purpose. Importantly, after contacting the two different (i.e., first and second) ss-oligonucleotides (see, for example, step (c) of the second aspect disclosed above), step (d) of the method as reported herein provides for an incubation interval of 20 minutes or less. In other words, duplex formation is rapid, and to the extent that duplex formation is possible, the process is substantially complete in 20 minutes or less. In this regard, it should be noted that in all aspects and embodiments disclosed herein, the single-stranded all-LNA binding partners (oligonucleotides) are capable of binding to each other under conditions comparable to those of biotin and (strept)avidin, with specific reference to molecular recognition and binding. In certain embodiments of all aspects and embodiments disclosed herein, the time interval for duplex formation (i.e., after the contacting step) is selected from 1 s to 20 min, 1 s to 15 min, 1 s to 10 min, 1 s to 5 min, 1 s to 1 min, 1 s to 30 s, 1 s to 20 s, 1 s to 10 s, and 1 s to 5 s. A highly desirable and advantageous time interval is selected from 1 s to 10 s and 1 s to 5 s.

[0168] Importantly, prior to step (c), the first ss-oligonucleotide and the second ss-oligonucleotide do not require denaturation treatment, but can be stored or maintained under non-denaturing conditions, particularly at a non-denaturing temperature, while avoiding undesirable consequences. In particular, the ss-oligonucleotides reported herein are characterized by a very low (if not absent) tendency to stably fold into secondary structures that could interfere with the ability of the complementary all-LNA oligonucleotide to form a duplex. In one embodiment of all aspects and embodiments disclosed herein, the first ss-oligonucleotide and the second ss-oligonucleotide are stored and / or maintained at a temperature between -80°C and 40°C, particularly between 0°C and 40°C, and more particularly between 25°C and 37°C. The non-denaturing temperature of a single-stranded all-LNA oligonucleotide comprising 5 to 15 monomers is a temperature below 68°C, more particularly below 40°C.

[0169] In a specific embodiment of all (particularly the second and third) aspects and embodiments disclosed herein in step (c) and / or step (d), the temperature is below 68°C, more particularly below 40°C, and even more particularly between 0°C and 37°C. In the method of the second or third aspect, in step (c), the temperature is selected independently of the temperature in step (d), or vice versa. In a specific embodiment of all aspects and embodiments disclosed herein, the temperature in steps (c) and (d) differs by no more than 5°C, or both steps are performed at the same temperature. In an even more specific embodiment of all aspects and embodiments disclosed herein in step (c) and / or step (d), the temperature is between 25°C and 40°C, more particularly between 25°C and 37°C. In another specific embodiment of all aspects and embodiments disclosed herein, prior to step (c), the first ss-oligonucleotide and the second ss-oligonucleotide are stored and / or maintained at a temperature between -80°C and 40°C, particularly between 0°C and 40°C, more particularly between 25°C and 37°C.

[0170] In another embodiment of all (particularly the second and third) aspects and embodiments disclosed herein, prior to step (c), the first ss-oligonucleotide and the second ss-oligonucleotide are stored and / or maintained in an aqueous solution comprising a buffer that maintains the pH of the solution at pH 6 to pH 8, more particularly pH 6.5 to pH 7.5.

[0171] In a further embodiment of all (particularly the second and third) aspects and embodiments disclosed herein, in step (c), the aqueous solution comprises a buffer that maintains the pH of the solution at pH 6 to pH 8, more particularly at pH 6.5 to pH 7.5. In another embodiment of all (particularly the second and third) aspects and embodiments disclosed herein, in step (c), the aqueous solution comprises a total amount of dissolved substances from 10 mmol / L to 500 mmol / L, more particularly from 200 mmol / L to 300 mmol / L, more particularly from 10 mmol / L to 150 mmol / L, more particularly from 50 mmol / L to 200 mmol / L.

[0172] The conditions described herein for use during the mixing (step (c)) and incubation (step (d)) steps also apply to the conditions for maintaining the separated ss-oligonucleotides. Thus, in one embodiment, prior to step (c), each ss-oligonucleotide of any of steps (a) and (b) is maintained in the absence of denaturing conditions. This includes any embodiment in which each ss-oligonucleotide of any of steps (a) and (b) is pre-incubated in the absence of denaturing conditions prior to step (c). In one embodiment, prior to step (c), each ss-oligonucleotide of any of steps (a) and (b) is maintained in aqueous solution at a temperature of -80°C to 40°C, specifically 0°C to 40°C, more specifically 25°C to 37°C. In another embodiment, prior to step (c), each ss-oligonucleotide of any of steps (a) and (b) is maintained in aqueous solution in the absence of a denaturant compound, specifically in the absence of any of formamide and DMSO.

[0173] The incubation of step (d) provides conditions for duplex formation, provided that the two complementary full LNAss-oligonucleotides are actually able to recognize each other molecularly. The intermolecular and intramolecular structures within one or two species of the suspected binding pair are further discussed. If there is a secondary structure in a species before step (c), for example, the duplex formation in step (d) can be suppressed. The following step (e) is required to determine whether duplex formation has occurred in the absence of denaturation. Step (e) detects and quantifies ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d). In one embodiment of all aspects and embodiments disclosed herein, step (e) includes subjecting the incubated mixture of step (d) to column chromatography with an aqueous solvent as the mobile phase. Therefore, column chromatography can advantageously separate duplex molecules from ss-oligonucleotides. In this regard, suitable column chromatography methods such as HPLC are well known to technicians. However, any alternative method that can distinguish and quantify ss-oligonucleotides and duplexes is also suitable. Such alternative methods include SPR (surface plasmon resonance; for example, Biacore) and electrophoresis.

[0174] If intermolecular and intramolecular structures are present in one or both ss-oligonucleotides before step (c) (which mixes approximately equimolar amounts of the first and second ss-oligonucleotides in aqueous solution), duplex formation is inhibited. If the inhibition is complete, only the presence of the ss-oligonucleotide will be detectable after step (d) (incubation of the mixture). However, inhibition may be incomplete. Therefore, depending on the strength of the intermolecular and intramolecular structures, they may temporarily undergo "unfolding", i.e., certain changes cause the nucleobases of the inhibited first ss-oligonucleotide to become fully exposed. In the unfolded form, the first ss-oligonucleotide is then able to form a duplex with the complementary second ss-oligonucleotide. The same requirements for unfolding also apply to the complementary second ss-oligonucleotide. In any such case, the number of duplexes formed reflects the number of unfolding events during the incubation period (i.e., the incubation time applied in step (d)). Ideally, after step (d), there are essentially no longer any detectable ss-oligonucleotides, and only duplexes are detectable.

[0175] K d The value is the equilibrium dissociation constant between the first and second members of the binding pair. This value provides a quantitative measure of the affinity of the binding partner that characterizes the binding pair. The equilibrium dissociation constant, Kd, ​​is the kd between the first and second members of the binding pair. 解离 / k 缔合 The ratio of K d Inversely proportional to affinity. d The value is related to the concentration of a member that is still sufficient to undergo molecular interaction and bind to another member; K d The lower the value (the lower the concentration), the higher the affinity of the first member for the other member. In one embodiment, the affinity of the binding partners selected by the method of the present invention for each other is K d <1x10 -15 M to >1x10 -5 M, the binding partner consists of a first and a second single-stranded oligonucleotide, each single-stranded oligonucleotide consisting of 5 to 15 locked nucleic acid monomers.

[0176] In a further embodiment, the affinity K of the binding partners selected by the methods herein for each other is d <1x10 -12 M to>1x10 -5 M, the binding partner consists of a first and a second single-stranded oligonucleotide, each single-stranded oligonucleotide consisting of 5 to 15 locked nucleic acid monomers. In a further embodiment, the mutual affinity K of the binding partners selected by the method herein is d 2x10 -12 M to 1x10 -15M, the binding partner consists of a first and a second single-stranded oligonucleotide, each of which consists of 8 to 15 locked nucleic acid monomers.

[0177] In yet another embodiment, the affinity K of the binding partners selected by the methods herein for each other is d 1x10 -12 M to 2x10 -5 M, the binding partner consists of a first and a second single-stranded oligonucleotide, each of which consists of 5 to 7 locked nucleic acid monomers.

[0178] It is worth noting that the K d is substantially influenced by the G+C content of the binding pair. That is, for a binding pair with a given number of complementary base pairs, the affinity of the binding partner generally increases with increasing G+C content.

[0179] In order to exclude any interference of naturally occurring oligonucleotides or polynucleotides with the molecular recognition (i.e., duplex formation) of binding pairs composed of LNA oligonucleotides, stereoisomers of LNA monomers are advantageously used as building blocks for the synthesis of all-LNA ss-oligonucleotides. The basic principle of this approach is to select stereoisomers that prevent the single-stranded oligonucleotide from forming duplexes with naturally occurring oligonucleotides or polynucleotides, in particular, from forming Watson-Crick duplexes with naturally occurring oligonucleotides or polynucleotides.

[0180] In one embodiment of all aspects and embodiments disclosed herein, the first and second ss-oligonucleotides (and any fragments thereof) are composed of β-D-LNA monomers. That is, the first ss-oligonucleotide is composed entirely of β-D-LNA monomers, and the second ss-oligonucleotide is composed entirely of β-D-LNA monomers. In another embodiment of all aspects and embodiments disclosed herein, the first and second ss-oligonucleotides (and any fragments thereof) are composed entirely of β-L-LNA monomers. That is, the first ss-oligonucleotide is composed entirely of β-L-LNA monomers, and the second ss-oligonucleotide is composed entirely of β-L-LNA monomers. The use of β-L-LNA has been shown to be advantageous because it does not interfere with duplex formation in the presence of naturally occurring nucleic acids. In this regard, it should be noted that extensive technical experience regarding hybridization conditions for all-D-LNA and all-L-LNA oligonucleotide pairs is currently quite limited.

[0181] This report discloses pairs of isolated ss-oligonucleotides, each composed of 5 to 15 LNA monomers. The isolated ss-oligonucleotides are capable of forming antiparallel duplexes with each other in aqueous solution in the absence of denaturing conditions before or during duplex formation. The teachings of this report conveniently and advantageously allow for the selection and provision of such single-stranded oligonucleotides whose monomers are LNA monomers. To the best of the authors' knowledge, the method described herein represents the first successful approach to overcome the limitations of known algorithms in the art for predicting hybridization characteristics of all-LNA ss-oligonucleotides. The nucleobase sequence of the first ss-oligonucleotide is complementary to the nucleobase sequence of the second ss-oligonucleotide, allowing the two ss-oligonucleotides to pair in an antiparallel orientation to form a duplex.

[0182] In an embodiment related to all other aspects and embodiments, there is provided a pair of isolated ss-oligonucleotides, each oligonucleotide consisting of 5 to 15 LNA monomers, the isolated ss-oligonucleotides being capable of forming an antiparallel duplex with each other in aqueous solution in the absence of denaturing conditions prior to or during duplex formation, wherein the pair of ss-oligonucleotides is obtainable and / or obtained by performing a method of selecting and providing a binding pair of single-stranded all-LNA oligonucleotides capable of forming an antiparallel duplex having 5 to 15 consecutive base pairs in aqueous solution at a temperature of 0° C. to 40° C., the method comprising the steps of:

[0183] (a) Providing a first single strand (=ss-) consisting of 5 to 15 locked nucleic acid (=LNA) monomers

[0184] an oligonucleotide, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0185] (b) providing a second ss-oligonucleotide composed of 5 to 15 LNA monomers, the second ss-oligonucleotide consisting of at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence of the second ss-oligonucleotide, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation, and the complementarity predicts the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 5 to 15 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other;

[0186] (c) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0187] (d) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex;

[0188] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0189] (f) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide, selecting the binding pair;

[0190] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0191] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0192] In an embodiment related to all other aspects and embodiments, there is provided a pair of isolated ss-oligonucleotides, each oligonucleotide consisting of 5 to 7 LNA monomers, the isolated ss-oligonucleotides being capable of forming an antiparallel duplex with each other in aqueous solution in the absence of denaturing conditions prior to or during duplex formation, wherein the pair of ss-oligonucleotides is obtainable and / or obtained by performing a method of selecting and providing a binding pair of single-stranded all-LNA oligonucleotides capable of forming an antiparallel duplex having 5 to 7 consecutive base pairs in aqueous solution at a temperature of 0° C. to 40° C., the method comprising the steps of:

[0193] (a) Providing a first single strand (=ss-) consisting of 8 to 15 locked nucleic acid (=LNA) monomers

[0194] an oligonucleotide, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0195] (b) providing a second ss-oligonucleotide consisting of 8 to 15 LNA monomers, the second ss-oligonucleotide consisting of at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence of the second ss-oligonucleotide, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation, and the complementarity predicts the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 8 to 15 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other;

[0196] (c) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0197] (d) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex;

[0198] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0199] (f) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide, selecting the binding pair;

[0200] (g) selecting a first contiguous nucleobase subsequence from the oligonucleotides of the binding pair selected in step (f), thereby generating a fragment of the oligonucleotide consisting of 5 to 7 LNA monomers;

[0201] (h) optionally selecting a second contiguous nucleobase subsequence from the other oligonucleotide of the binding pair selected in step (f), wherein the second subsequence is complementary to the first subsequence of step (g), thereby generating a further oligonucleotide consisting of a fragment of 5 to 7 LNA monomers;

[0202] (i) synthesizing the ss-oligonucleotide fragment of step (g) and the ss-oligonucleotide fragment of step (h), respectively;

[0203] Thus, binding pairs of 5 to 7 single-stranded all-LNA oligonucleotides are selected and provided. In one embodiment, specifically, steps (c) and (d) are performed in the absence of conditions designated as "denaturing conditions" as described above. The properties of the selected fragments, i.e., their ability to form antiparallel duplexes in aqueous solution at temperatures between 0°C and 40°C, can be easily verified. Therefore, in a specific embodiment, the method includes the following additional steps:

[0204] (k) mixing approximately equimolar amounts of said first and second ss-oligonucleotide fragments in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0205] (1) incubating the mixture of (k) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotide fragments as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotide fragments as duplexes;

[0206] (m) If a duplex is detected in step (1) but no ss-oligonucleotide fragment is detectable, then the binding pair is selected.

[0207] Each single-stranded all-LNA oligonucleotide can comprise four different nucleobases. In one embodiment of all aspects and embodiments disclosed herein, each ss-oligonucleotide comprises three different nucleobases. In another embodiment of all aspects and embodiments disclosed herein, each ss-oligonucleotide comprises two different nucleobases. In yet another embodiment of all aspects and embodiments disclosed herein, each ss-oligonucleotide comprises only one nucleobase. In this latter embodiment, all nucleobases in the ss-oligonucleotide are identical.

[0208] In one embodiment of all aspects and embodiments disclosed herein, the G+C content of the nucleobases in each ss-oligonucleotide is less than 75%. In a specific embodiment, the G+C content is less than a value selected from 74%, 73%, 72%, 71%, and 70%. In another embodiment of all aspects and embodiments disclosed herein, each LNA monomer in the binding pair comprises a nucleobase selected from the group consisting of adenine, thymine, uracil, guanine, cytosine, and 5-methylcytosine. In a more specific embodiment, in the nucleobases of each ss-oligonucleotide, each cytosine is substituted with 5-methylcytosine.

[0209] In one embodiment of all aspects and embodiments disclosed herein, the binding pair of two separate compatible binding partners is a pair of all-LNA ss-oligonucleotides selected from the group consisting of:

[0210] 5'tgctcctg 3' (SEQ ID NO: 1) and 5'caggagca 3' (SEQ ID NO: 2), 5'tgctcctgt 3' (SEQ ID NO: 9) and 5'acaggagca 3' (SEQ ID NO: 10), 5'gtgcgtct 3' (SEQ ID NO: 11) and 5'agacgcac 3' (SEQ ID NO: 12), and 5'gttggtgt 3' (SEQ ID NO: 13) and 5'acaccaac 3' (SEQ ID NO: 14).

[0211] In one embodiment, the monomers of the ss-oligonucleotides in any selected pair of the aforementioned group are β-D-LNA monomers. In another embodiment, the monomers of the ss-oligonucleotides in any selected pair of the aforementioned group are β-L-LNA monomers.

[0212] Several pairs of such single-stranded all-LNA oligonucleotides have been discovered and are reported herein as exemplary embodiments, i.e., as non-limiting examples of pairs of isolated ss-oligonucleotides capable of binding to each other through hybridization and duplex formation under non-denaturing conditions. Table 1 provides a non-limiting compilation of these. It should be understood that the sequences listed represent all-LNA nucleosides, i.e., oligonucleotides containing only LNA monomers. The sequences are given in the conventional orientation, i.e., from the 5' end to the 3' end.

[0213] Table 1

[0214]

[0215]

[0216] The binding pairs given in Table 1 reflect specific embodiments. It should be understood that any reference to a "first" and "second" member of a binding pair is arbitrary, as the "second" member can be equivalently considered the first member, as long as the second member is replaced by the "first" member in this case. That is, the references to "first" and "second" in the table are arbitrary and their representation can vary. Thus, and by way of example, the binding pair (SEQ ID NO: 16): (SEQ ID NO: 20) is the same as (SEQ ID NO: 20): (SEQ ID NO: 16). In one embodiment of all aspects and embodiments disclosed herein, the binding pair of two separate compatible binding partners is a pair of all-LNA ss-oligonucleotides selected from the group consisting of:

[0217] (SEQ ID NO: 1): (SEQ ID NO: 2),

[0218] (SEQ ID NO: 9): (SEQ ID NO: 10),

[0219] (SEQ ID NO: 11): (SEQ ID NO: 12),

[0220] (SEQ ID NO: 13): (SEQ ID NO: 14),

[0221] (SEQ ID NO: 9): (SEQ ID NO: 15),

[0222] (SEQ ID NO: 16): (SEQ ID NO: 20),

[0223] (SEQ ID NO: 21): (SEQ ID NO: 18),

[0224] (SEQ ID NO: 21): (SEQ ID NO: 20),

[0225] (SEQ ID NO: 21): (SEQ ID NO: 19),

[0226] (SEQ ID NO: 23): (SEQ ID NO: 17),

[0227] (SEQ ID NO:25): (SEQ ID NO:28).

[0228] In one embodiment of all aspects and embodiments disclosed herein, the binding pair of two separate compatible binding partners is the pair of the all-LNA ss-oligonucleotides of SEQ ID NO: 16 and SEQ ID NO: 20. Thus, the binding pair is gttggt:accaac.

[0229] In a specific embodiment, the monomers of the ss-oligonucleotides in any selected pair of the aforementioned group are β-D-LNA monomers. In another specific embodiment, the monomers of the ss-oligonucleotides in any selected pair of the aforementioned group are β-L-LNA monomers.

[0230] In contrast, pairs of single-stranded all-LNA oligonucleotides have been found that are unable or insufficient to form duplexes under non-denaturing conditions. Table 2 provides a non-limiting compilation of these.

[0231] Table 2

[0232]

[0233] The pairing given by SEQ ID NO: 29 and SEQ ID NO: 39 exemplifies a case of minimal sequence complexity, in which one member of the binding pair comprises only four monomers. The binding properties characterizing this particular binding pair were found to be insufficient. One possible explanation could be that the tetramer is too short, thus providing only insufficient intramolecular interactions with the corresponding single chain. This finding contrasts sharply with the case in which the tetramer is replaced by a hexamer (SEQ ID NO: 27 combined with SEQ ID NO: 39).

[0234] With respect to the other pairs of all-LNA oligonucleotides presented in Table 2, it was found that one binding partner always comprised or consisted of the sequence "gcctgacg" (SEQ ID NO: 3). Thus, this particular sequence and its complement appear to negatively impact the ability of such all-LNA oligonucleotides to form duplexes under non-denaturing conditions. This particularly surprising finding may indeed guide one in selecting advantageous single-stranded oligonucleotide pairs having 8 or more monomers. Thus, one embodiment of all other aspects and embodiments provided herein is a pair of isolated ss-oligonucleotides, each consisting of 8 to 15 LNA monomers, the isolated ss-oligonucleotides being capable of forming antiparallel duplexes with each other in aqueous solution in the absence of denaturing conditions prior to or during duplex formation, wherein the pair of ss-oligonucleotides is obtainable and / or obtained by performing a method for selecting and providing binding pairs of single-stranded all-LNA oligonucleotides capable of forming antiparallel duplexes having 8 to 15 consecutive base pairs in aqueous solution at a temperature of 0°C to 40°C, the method comprising the following steps:

[0235] (a) providing a first single strand (=ss-) consisting of 8 to 15 (i.e., a number selected from any one of 8, 9, 10, 11, 12, 13, 14, and 15) locked nucleic acid (=LNA) monomers

[0236] ) oligonucleotide, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence, wherein the first nucleobase sequence does not comprise or consist of a sequence selected from the group consisting of 5'gcctgacg 3' (SEQ ID No: 3) and 5'cgtcaggc 3' (SEQ ID NO: 4);

[0237] (b) providing a second ss-oligonucleotide composed of 8 to 15 LNA monomers, the second ss-oligonucleotide composed of at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence of the second ss-oligonucleotide, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation, and the complementarity predicts the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 8 to 15 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other, wherein the second nucleobase sequence does not comprise or consist of a sequence selected from the group consisting of 5′gcctgacg 3′ (SEQ ID NO: 3) and 5′cgtcaggc 3′ (SEQ ID NO: 4);

[0238] (c) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0239] (d) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex;

[0240] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0241] (f) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide, selecting the binding pair;

[0242] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0243] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0244] By the method of the second aspect or the method of the third aspect disclosed herein, and further by utilizing any embodiment thereof, including but not limited to the binding pairs shown in Table 1, and alternatively or additionally by selecting the binding pair to exclude sequences selected from 5'gcctgacg 3' (SEQ ID NO: 3) and 5'cgtcaggc 3' (SEQ ID NO: 4) when the number of monomers in one or both oligonucleotides is between 8 and 15, the present disclosure provides an antiparallel all-LNA duplex formed, obtainable, and / or obtained from a pair of non-denaturing complementary single-stranded all-LNA oligonucleotides under non-denaturing conditions at a preselected temperature of 25°C to 40°C. This duplex in aqueous solution can be considered as the fourth aspect of this report. Each oligonucleotide strand in the duplex comprises LNA monomers, the number of LNA monomers being selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15, more specifically the number being selected from the group consisting of 5, 6, and 7. The duplex is formed by complementary Watson-Crick base pairing, and the number of base pairs in the duplex is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15, more specifically any one from 8 to 15 or any one from 5 to 7.

[0245] In a fifth aspect, related to all other aspects and embodiments disclosed herein, the present disclosure provides a liquid composition comprising an aqueous solvent and a binding pair consisting of a first single-stranded oligonucleotide and a second single-stranded oligonucleotide, characterized in that each oligonucleotide consists of 5 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of the monomers forming a first nucleobase sequence of the first oligonucleotide and a second nucleobase sequence of the second oligonucleotide, wherein the first nucleobase sequence and the second nucleobase sequence are selected such that the first oligonucleotide and the second oligonucleotide are capable of forming an antiparallel duplex of 5 to 15 consecutive Watson-Crick base pairs at a temperature of 0°C to 40°C, and characterized in that the binding pair can be obtained by a method according to the second or third aspect of the present disclosure.

[0246] In a specific embodiment of all aspects and embodiments disclosed herein, a liquid composition is provided comprising an aqueous solvent and a binding pair consisting of a first single-stranded oligonucleotide and a second single-stranded oligonucleotide, characterized in that each oligonucleotide consists of 5 to 15, specifically 5 to 7 or 8 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of the monomer forming a first nucleobase sequence of the first oligonucleotide and a second nucleobase sequence of the second oligonucleotide, characterized in that the first nucleobase sequence and the second nucleobase sequence are selected such that the first oligonucleotide and the second oligonucleotide are capable of forming an antiparallel duplex of 5 to 15, specifically 5 to 7 or 8 to 15 consecutive Watson-Crick base pairs at a temperature of 0°C to 40°C, and characterized in that the binding pair is obtainable by a method according to the second or third aspect of the present disclosure.

[0247] In another embodiment of all aspects and embodiments disclosed herein, each oligonucleotide consists of 5 to 15 LNA monomers, characterized in that the first nucleobase sequence and the second nucleobase sequence are selected such that the first oligonucleotide and the second oligonucleotide are capable of forming an antiparallel duplex of 5 or more consecutive Watson-Crick base pairs at a temperature of 0°C to 40°C, and characterized in that the binding pair is obtainable by or according to the method of the second or third aspect and embodiments thereof.

[0248] As a different embodiment, it was further surprisingly discovered that there are even longer complementary all-LNA ss-oligonucleotides that are capable of forming antiparallel duplexes under non-denaturing conditions. Therefore, the present disclosure provides a method for selecting and providing binding pairs of single-stranded all-LNA oligonucleotides capable of forming antiparallel duplexes having 16 to 20 consecutive base pairs in aqueous solution at temperatures between 0°C and 40°C, the method comprising the following steps:

[0249] (a) Providing a first single strand (=ss-) consisting of 16 to 20 locked nucleic acid (=LNA) monomers

[0250] an oligonucleotide, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0251] (b) providing a second ss-oligonucleotide consisting of 16 to 20 LNA monomers, the second ss-oligonucleotide consisting of at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence of the second ss-oligonucleotide, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation, and the complementarity predicts the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 16 to 20 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other;

[0252] (c) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0253] (d) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex;

[0254] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0255] (f) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide, selecting the binding pair;

[0256] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0257] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0258] In one embodiment, specifically, steps (c) and (d) are performed in the absence of conditions designated as "denaturing conditions" as described above.

[0259] A specific embodiment of the second aspect, which is relevant to all other aspects and embodiments disclosed herein, provides a method for selecting and providing a binding pair of single-stranded all-LNA oligonucleotides capable of forming an antiparallel duplex having 5 to 15 consecutive base pairs in aqueous solution at a temperature of 0° C. to 40° C., the method comprising the following steps:

[0260] (a) Providing a first single strand (=ss-) consisting of 5 to 15 locked nucleic acid (=LNA) monomers

[0261] an oligonucleotide, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0262] (b) providing a second ss-oligonucleotide consisting of 16 to 20 LNA monomers, the second ss-oligonucleotide consisting of at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence of the second ss-oligonucleotide, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation, and the complementarity predicts the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 5 to 15 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other;

[0263] (c) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0264] (d) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex;

[0265] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0266] (f) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide, selecting the binding pair;

[0267] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0268] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0269] In one embodiment, specifically, steps (c) and (d) are performed in the absence of conditions designated as "denaturing conditions" as described above.

[0270] Exemplary binding pairs of first and second all-LNA ss-oligonucleotides were identified as shown below in Table 3. These binding pairs are specific examples.

[0271] Table 3

[0272]

[0273] The results showed that none of the sequences shown in Table 3 contained the subsequence gcctgacg (SEQ ID NO: 3) or its complement, discussed further above.

[0274] In one embodiment of all aspects and embodiments disclosed herein, one ss-oligonucleotide of the binding pair is linked (covalently or non-covalently) to a solid phase selected from the group consisting of: magnetic beads, paramagnetic beads, synthetic organic polymer (latex) beads, polysaccharide beads, test tubes, microplate cavities, cuvettes, membranes, scaffold molecules, quartz crystals, thin films, filter paper, discs, and chips. In another embodiment of all aspects and embodiments disclosed herein, one ss-oligonucleotide of the binding pair is linked (covalently or non-covalently) to a molecule selected from the group consisting of peptides, polypeptides, oligonucleotides, polynucleotides, sugars, glycans, haptens, and dyes. In yet another embodiment of all aspects and embodiments disclosed herein, the ss-oligonucleotide is covalently attached to a linker. In yet another embodiment of all aspects and embodiments disclosed herein, the ss-oligonucleotide is covalently attached to an analyte-specific receptor useful in receptor-based assays (such as, but not limited to, immunoassays).

[0275] In general terms, an immunoassay provides one or more receptors capable of specifically binding to a target analyte. Such receptors can be exemplified by analyte-specific immunoglobulins, hence the term immunoassay. However, for the purposes of this disclosure, any other type of analyte-specific receptor is also contemplated. Therefore, the more general term receptor-based assay is appropriate and is used synonymously with the term immunoassay.

[0276] Therefore, a sixth aspect related to all other aspects and embodiments disclosed herein is the use of a pair of single-stranded all-LNA oligonucleotides of the first aspect for determining an analyte in a receptor-based assay, the receptor-based assay comprising an analyte-specific receptor and a solid phase for immobilizing the analyte on the solid phase, wherein the first ss-oligonucleotide of the pair is coupled to the analyte-specific receptor and the second ss-oligonucleotide of the pair is coupled to the solid phase. An embodiment of this aspect is any such use of a pair of single-stranded all-LNA oligonucleotides, wherein the pair of single-stranded all-LNA oligonucleotides can be obtained according to an embodiment of either the second or third aspect of this report.

[0277] In receptor-based assays, the analyte binds to an analyte-specific receptor. The receptor with the bound analyte can be immobilized on a solid phase via a duplex formed by pairs of single-stranded all-LNA oligonucleotides. Consequently, a complex is formed comprising the solid phase, the duplex, the receptor, and the analyte. In a further step, the immobilized analyte can be detected, for example using a separate analyte-specific binding agent that is itself labeled or whose presence can be detected by other means. In competitive assay formats, no further binding agent is required. Instead, a predetermined amount of labeled analyte is added and competes for binding with an unlabeled analyte, the presence and / or concentration of which is to be detected.

[0278] Typically, the analyte is contained in a sample, wherein the sample is a complex mixture of different molecules. For the purposes of this disclosure, liquid samples are considered. The liquid sample comprises a liquid phase, i.e., it comprises a liquid solvent, which is typically an aqueous solvent. In the aqueous solvent, multiple molecules exist in a dissolved state. Therefore, in a specific embodiment, the sample is in a liquid aggregate state and is a single-phase homogeneous mixture. In another specific embodiment, the sample comprises an insoluble portion and is therefore a multiphase mixture, and the analyte is uniformly distributed in the sample. Typically, the analyte is contained in the mixture in a dissolved form, and one or more other molecules are present in the mixture in a dissolved form.

[0279] About detection being present in liquid sample or thinking to be present in the target analyte of described liquid sample, in basic step, specifically bind to described analyte.Specific binding means that analyte-specific receptor exists or adds, wherein said receptor has binding affinity and binding specificity to analyte, and described binding affinity and binding specificity are high for described target analyte, and are low or non-existent for other molecules also present in described sample.In a specific embodiment (and illustrating many existing determinations), the compound comprising the receptor that can be specifically bound to analyte is added to described sample.Importantly, described sample and described mixture of described compound comprising receptor must provide the condition that allows described receptor to interact specifically with described target analyte in described sample.This is included in described mixture, and condition must allow described receptor to actually combine with described analyte, and it is expected that described receptor is stabilized with described combined target analyte.At the same time, it is expected that described sample and described mixture of described compound do not promote or stabilize other molecule and described receptor or with the non-specific binding of compound overall comprising described receptor.

[0280] Subsequently, the analyte is immobilized. Immobilization is an important step in the detection process because it can separate the analyte from the surrounding complex mixture, specifically from other molecules in the sample. Immobilization requires a stationary phase to which the target analyte adheres. Once immobilized, the analyte can be isolated from the mixture by phase separation. The analyte is then isolated (i.e., purified) from the mixture and then detected.

[0281] In view of receptor-based assays and the immobilization step, it is necessary to provide a solid phase and establish a connection between the solid phase and the target analyte. It is desirable to establish the connection during a self-assembly process.

[0282] Immunoassays are well-established bioanalytical procedures, in particular embodiments, in which the detection or quantification of an analyte depends on the reaction of the analyte with at least one analyte-specific receptor, thereby forming an analyte:receptor complex. A non-limiting example is the reaction between an antigen and an antibody, respectively.

[0283] Thus, a seventh aspect, related to all other aspects and embodiments disclosed herein, is a method of performing a receptor-based assay to determine an analyte, the method comprising the steps of contacting the analyte with an analyte-specific receptor to which is attached a first member of a pair of isolated ss-oligonucleotides of the first aspect described herein, and contacting a solid phase to which is attached a second member of the pair; incubating to form a complex comprising the solid phase, the analyte-specific receptor bound to the solid phase, and the analyte bound to the analyte-specific receptor, wherein an antiparallel duplex is formed, the duplex consisting of the first and second members of the pair, wherein the duplex connects the analyte-specific receptor in the complex to the solid phase; and then detecting the analyte bound to the complex to determine the analyte. In one embodiment, the latter detection step can be performed, for example, using a labeled analyte-specific antibody capable of binding to the analyte in the complex, also known in the art as a "sandwich" assay.

[0284] Specific embodiments of "sandwich" immunoassays can be used for analytes with multiple recognition epitopes (i.e., more than one recognition epitope). Therefore, a sandwich assay requires at least two receptors that are bound to non-overlapping epitopes on the analyte. In a "heterogeneous sandwich immunoassay," one of the receptors has the function of an analyte-specific capture receptor; the receptor is immobilized on a stationary phase (during the assay). The second analyte-specific receptor is provided in a dissolved form in a liquid phase. Once the analyte binds to the first and second receptors (receptor-1: analyte: receptor-2), a sandwich complex is formed. The sandwich complex is also referred to as a "detection complex." In the detection complex, the analyte is sandwiched between the receptors, i.e., in this complex, the analyte represents the connecting element between the first receptor and the second receptor.

[0285] The term "heterogeneous" (as opposed to "homogeneous") refers to two basic and independent steps in the assay process. In the first step, a detection complex containing the label is formed and immobilized, but unbound label still surrounds the complex. Before determining the label-dependent signal, the unbound label is washed away from the immobilized detection complex, thus representing the second step. In contrast, homogeneous assays generate an analyte-dependent detectable signal through a one-step incubation and do not require a wash step.

[0286] In heterogeneous assays, the solid phase is functionalized so that before contact with the analyte, the solid phase may already have a functional capture receptor (first receptor) bound to its surface; or the surface of the solid phase is functionalized so that the first receptor can be anchored after the first receptor reacts (i.e., binds) with the analyte. In the latter case, the anchoring process must not interfere with the ability of the receptor to specifically capture and bind to the analyte. The second analyte-specific receptor present in the liquid phase is used to detect the bound analyte, i.e., the analyte that has been fixed or immobilized on the solid phase. Therefore, in an immunoassay, the analyte is allowed to bind to the first (capture) and second (detection) receptors. A "detection complex" is thereby formed, in which the analyte is sandwiched between the capture receptor and the detection receptor. In a typical embodiment, the detection receptor is labeled before contact with the analyte; alternatively, after the analyte binds, the label is specifically attached to the detection receptor. When the detection complex is fixed on the stationary phase, the amount of detectable label on the stationary phase corresponds to the amount of analyte sandwiched in the middle. After removal of unbound label by a washing step, the immobilized label can be detected, indicating the presence and amount of analyte.

[0287] Any necessary wash steps in heterogeneous immunoassays require that the non-covalent linkage of the first and second binding partners be sufficiently stable. However, the desired degree of stability of the linkage depends on the intensity of the wash steps to be applied. Importantly and surprisingly, the binding pairs demonstrated herein are well suited to facilitate the immobilization step in immunoassays. That is, in immunoassays, the first binding partner of the binding pair of all-LNA oligonucleotides attached to a solid phase and the second binding partner of the binding pair bound to an analyte-specific capture receptor are well suited to facilitate the immobilization of the receptor on the solid phase. Similarly, this immobilization also works advantageously for the capture receptor bound to the target analyte and the detection complex.

[0288] Another well-known embodiment is the competitive immunoassay, which in its simplest form differs from the sandwich format by the lack of the second detection receptor. Instead, a sample with the analyte is mixed with an artificially produced labeled analog that can cross-react with the analyte-specific receptor. In the assay, the analyte and the analog compete for binding to a fixed or immobilized capture receptor. After the binding step, the higher the amount of fixed label, the lower the amount of unlabeled analyte that can compete with the capture receptor. The fixed label is determined after the washing step. Therefore, the amount of label detectable on the stationary phase is inversely proportional to the amount of analyte initially present in the sample.

[0289] In all aspects and embodiments disclosed herein, the binding force in the duplex formed by the all-LNA oligonucleotide pair will exceed any binding force binding the analyte to any analyte-specific (capture and / or detection) receptor in an assay for analyte detection (e.g., but not limited to, an immunoassay). In cases where fine-tuning of the binding force in the duplex is desired, the binding pair can be selected so as to provide Watson-Crick pairing regions of varying lengths and / or base pair compositions. More generally, with respect to the first and second components connected to each other by the all-LNA binding pair, complementary single-stranded all-LNA oligonucleotide pairs can be provided and selected according to the specific technical requirements.

[0290] The binding force in a duplex of Watson-Crick paired complementary all-LNA oligonucleotides with a given number of paired nucleobases can be fine-tuned by changing a single base pair in the duplex (e.g., replacing an A:T base pair with a C:G base pair), or by lengthening or shortening the duplex. In any case, the fine-tuned single-stranded all-LNA pairs need to be subjected to a method for selecting and providing single-stranded all-LNA oligonucleotide binding pairs capable of forming antiparallel duplexes with 5 to 15 consecutive base pairs in aqueous solution at 0°C to 40°C, as disclosed elsewhere in this document.

[0291] However, an eighth aspect that is relevant to all other aspects and embodiments disclosed herein is a kit for performing a receptor-based assay to determine an analyte, the kit comprising in a first container an analyte-specific receptor to which is linked a first member of a pair of isolated ss-oligonucleotides according to the first aspect disclosed herein, or to which is linked a first member of a pair of isolated ss-oligonucleotides obtained by a method according to the second or third aspect disclosed herein, the kit further comprising in a second container a solid phase to which is linked a second member of the pair.

[0292] Even more generally, a kit for non-covalently linking a first and a second component is provided, wherein the kit comprises in a first separate compartment a first component to which is linked a first member of a pair of an isolated ss-oligonucleotide according to the first aspect as disclosed herein, or to which is linked a first member obtained by a method according to the second aspect or the third aspect as disclosed herein, the kit further comprising in a second container a second component to which is linked the second member of the pair.

[0293] It will be appreciated that the binding pairs disclosed in this report can serve as general alternatives to established binding pairs such as biotin:(strept)avidin. Thus, the all-LNA binding pairs can be used to link first and second components, which are respectively linked to the first and second members of a complementary ss-oligonucleotide pair capable of forming a duplex under non-denaturing conditions. Those skilled in the art will be well aware of numerous applications beyond the analyte detection assays described in greater detail herein, but also extending, for example, to in situ analysis of target antigens in tissue samples.

[0294] In principle, the binding pairs disclosed herein can also provide separate different binding pairs in the same aqueous solution, thus opening the way for multiplexed assays. Due to the identification of different binding pairs of oligonucleotides that do not share complementary base sequences, many different additional applications become feasible.

[0295] Specific aspects and embodiments include the following more formal bulleted list. This numbered bulleted list forms part of the original disclosure of this report.

[0296] 1. A method for selecting and providing a binding pair of single-stranded all-LNA oligonucleotides capable of forming an antiparallel duplex having 5 to 15 consecutive base pairs in aqueous solution at a temperature of 0°C to 40°C, the method comprising the following steps:

[0297] (a) providing a first single-stranded (=ss-) oligonucleotide consisting of 5 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0298] (b) providing a second ss-oligonucleotide consisting of 5 to 15 LNA monomers, the second ss-oligonucleotide comprising at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation and predicting the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 5 to 15 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other;

[0299] (c) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0300] (d) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex;

[0301] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0302] (f) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide, selecting the binding pair;

[0303] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0304] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0305] 2. The method according to item 1, wherein in step (a), when the number of monomers of the first oligonucleotide is 8 to 15, the first nucleobase sequence is not a sequence selected from 5′gcctgacg 3′ (SEQ ID NO: 3) and 5′cgtcaggc 3′ (SEQ ID NO: 4) or is not composed thereof.

[0306] 3. The method according to any one of items 1 and 2, wherein when the number of monomers of the first oligonucleotide is 8 to 15, the first nucleobase sequence is not a sequence comprising or consisting of a sequence selected from 5′gcctgacg 3′ (SEQ ID NO: 3) and 5′cgtcaggc 3′ (SEQ ID NO: 4).

[0307] 4. The method according to any one of items 1 to 3, wherein before step (e), the mixture obtained in step (d) is subjected to an additional step of separating ss-oligonucleotides and duplex oligonucleotides.

[0308] 5. A method according to any one of items 1 to 4, wherein steps (c) and (d) are carried out at a non-denaturing temperature, in particular at a temperature selected from the group consisting of 0°C to 5°C, 5°C to 10°C, 10°C to 15°C, 15°C to 20°C, 20°C to 25°C, 25°C to 30°C, 30°C to 35°C and 35°C to 40°C.

[0309] 6. The process according to any one of items 1 to 5, wherein steps (c) and (d) are carried out at a temperature of 25°C to 37°C.

[0310] 7. The method according to any one of items 1 to 6, wherein prior to step (c), each ss-oligonucleotide of any of steps (a) and (b) is maintained in the absence of denaturing conditions.

[0311] 8. The method of claim 7, wherein prior to step (c), each ss-oligonucleotide of any of steps (a) and (b) is maintained at a temperature of -80°C to 40°C, specifically 0°C to 40°C, more specifically 25°C to 37°C in aqueous solution.

[0312] 9. A method according to claim 7, wherein prior to step (c), each ss-oligonucleotide of any of steps (a) and (b) is maintained in aqueous solution in the absence of a denaturant compound capable of reducing the melting temperature of a DNA duplex of 20 base pairs in length and a G+C content of 50% by at least 15°C, specifically in the absence of either formamide and dimethyl sulfoxide.

[0313] 10. The method according to any one of items 1 to 9, wherein in step (d), the time interval is selected from the group consisting of 1 s to 20 min, 1 s to 5 min, 1 s to 60 s and 1 s to 30 s.

[0314] 11. The process according to any one of items 1 to 10, wherein the mixture of step (c) comprises a buffer which maintains the pH of the mixture at pH 6 to pH 8, more particularly at pH 6.5 to pH 7.5.

[0315] 12. A method according to any one of items 1 to 11, wherein the mixture of step (c) contains an amount of dissolved substance of about 10 mmol / L to about 1000 mmol / L, specifically about 10 mmol / L to about 500 mmol / L, more specifically about 200 mmol / L to about 300 mmol / L.

[0316] 13. A method according to any one of items 1 to 12, wherein steps (c) and (d) are carried out in the absence of a denaturant compound capable of reducing the melting temperature of a DNA duplex with a length of 20 base pairs and a G+C content of 50% by at least 15°C, more specifically in the absence of either formamide and dimethyl sulfoxide.

[0317] 14. The method according to any one of items 1 to 13, wherein each LNA monomer comprises a nucleobase selected from the group consisting of: N 4 -acetylcytosine, 5-acetyluracil, 4-amino-6-chloropyrimidine, 4-amino-5-fluoro-2-methoxypyrimidine, 6-amino-1-methyluracil, 5-aminoorotic acid, 5-aminouracil, 6-aminouracil, 6-azauracil, N 4 -benzoylcytosine, 5-bromouracil, 5-chlorouracil, 6-chlorouracil, 6-chloromethyluracil, 6-chloro-3-methyluracil, cytosine, 5,6-dimethyluracil, 5-ethyluracil, 5-ethynyluracil, 5-fluorocytosine, 5-fluoroorotic acid, 5-fluorouracil, 5-iodo-2,4-dimethoxypyrimidine, 5-iodouracil, isocytosine, 5-methylcytosine, 6-methyl-5-nitrouracil, 2-methylthio-4-pyrimidinol, 5-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl Uracil, 5-nitrouracil, orotic acid, 6-phenyl-2-thiouracil, 6-propyl-2-thiouracil, 2-thiouracil, 4-thiouracil, thymine, 5-(trifluoromethyl)uracil, uracil, adenine, 8-azahypoxanthine, 8-azaguanine, allopurinol, 4-aminopyrazolo[3,4-ρ]pyrimidine, 2-aminopurine, 2-acetylamino-6-hydroxypurine, 2-amino-6-chloropurine, 2-amino-6-iodopurine, azathioprine, 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine, aminophylline, N 6 -Benzyl adenine, N 6-benzoyl adenine, 6-benzyloxypurine, 8-bromotheoline, 8-bromo-3-methylxanthine, 8-bromo-7-(2-butyn-1-yl)-3-methylxanthine, 6-chloropurine, 8-chlorotheoline, 6-chloro-2-fluoropurine, 6-chloro-7-deazapurine, 2-chloroadenine, 6-chloro-7-iodo-7-deazapurine, 2,6-diaminopurine, 2,6-dichloropurine, 6 -(dimethylamino)purine, 2,6-dichloro-7-deazapurine, 5,6-dichlorobenzimidazole hydrochloride, 7-deazahypoxanthine, 2-fluoroadenine, guanine, hypoxanthine, isoguanine, 3-iodo-1H-pyrazolo-[3,4-d]pyrimidin-4-amine, kinetin, 6-mercaptopurine, 6-methoxypurine, 3-methylxanthine, 1-methylxanthine, 3-methyladenine, O 6 -(cyclohexylmethyl)guanine, 6-thioguanine, 2-thioxanthine, xanthine, 5-propynyl-uracil, 5-propynyl-cytidine, 7-deazaadenine, 7-deazaguanine, 7-propynyl-7-deazaadenine, 7-propynyl-7-deazaguanine and derivatives thereof.

[0318] 15. The method according to item 14, wherein each LNA monomer comprises a nucleobase selected from the group consisting of adenine, thymine, uracil, guanine, cytosine and 5-methylcytosine.

[0319] 16. The method according to any one of items 1 to 15, wherein prior to step (e), the incubation mixture of step (d) is subjected to a step of separating ss-oligonucleotides and double-stranded oligonucleotides.

[0320] 17. The method according to item 16, wherein the incubation mixture of step (d) is subjected to column chromatography and / or electrophoresis.

[0321] 18. The method according to any one of items 1 to 17, wherein the monomers of the ss-oligonucleotide of any of steps (a) and (b) are β-D-LNA monomers.

[0322] 19. The method according to any one of items 1 to 17, wherein the monomer of the ss-oligonucleotide of any of steps (a) and (b) is a β-L-LNA monomer.

[0323] 20. A pair of isolated complementary ss-oligonucleotides, each ss-oligonucleotide consisting of 5 to 15 LNA monomers, the isolated ss-oligonucleotides being capable of forming antiparallel duplexes with each other in aqueous solution in the absence of denaturing conditions prior to or during duplex formation.

[0324] 21. A pair of isolated complementary ss-oligonucleotides, each ss-oligonucleotide consisting of 8 to 15 LNA monomers, the isolated ss-oligonucleotides being capable of forming antiparallel duplexes with each other in aqueous solution in the absence of denaturing conditions prior to or during duplex formation, wherein each ss-oligonucleotide does not comprise a sequence selected from the group consisting of 5′gcctgacg 3′ (SEQ ID NO: 3) and 5′cgtcaggc 3′ (SEQ ID NO: 4).

[0325] 22. A pair of isolated complementary ss-oligonucleotides, each ss-oligonucleotide consisting of 5 to 7 LNA monomers, the isolated ss-oligonucleotides being capable of forming antiparallel duplexes with each other in aqueous solution in the absence of denaturing conditions prior to or during duplex formation.

[0326] 23. The isolated pair of complementary ss-oligonucleotides according to any one of items 20 to 22, wherein said pair of ss-oligonucleotides is obtained by performing the method according to any one of items 1 to 19.

[0327] 24. A pair of isolated complementary ss-oligonucleotides, wherein said pair is selected from the group consisting of

[0328] (SEQ ID NO: 1): (SEQ ID NO: 2),

[0329] (SEQ ID NO: 9): (SEQ ID NO: 10),

[0330] (SEQ ID NO: 11): (SEQ ID NO: 12),

[0331] (SEQ ID NO: 13): (SEQ ID NO: 14),

[0332] (SEQ ID NO: 9): (SEQ ID NO: 15),

[0333] (SEQ ID NO: 16): (SEQ ID NO: 20),

[0334] (SEQ ID NO: 21): (SEQ ID NO: 18),

[0335] (SEQ ID NO: 21): (SEQ ID NO: 20),

[0336] (SEQ ID NO: 21): (SEQ ID NO: 19),

[0337] (SEQ ID NO: 23): (SEQ ID NO: 17),

[0338] (SEQ ID NO:25): (SEQ ID NO:28).

[0339] 25. A pair of isolated complementary ss-oligonucleotides according to item 24, wherein said pair is selected from the group consisting of

[0340] (SEQ ID NO: 1): (SEQ ID NO: 2),

[0341] (SEQ ID NO: 28): (SEQ ID NO: 24),

[0342] (SEQ ID NO: 16): (SEQ ID NO: 20).

[0343] 26. The pair of isolated complementary ss-oligonucleotides according to any one of items 20 to 25, wherein the first ss-oligonucleotide of the pair is ligated to a first target and the second ss-oligonucleotide is ligated to a second target.

[0344] 27. A pair of isolated complementary ss-oligonucleotides according to item 26, wherein the ss-oligonucleotides are covalently or non-covalently linked to their respective targets.

[0345] 28. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 and 27, wherein

[0346] The targets are independently selected from the group consisting of solid phase, biomolecules, and chemically synthesized compounds.

[0347] 29. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 to 28, wherein

[0348] The targets are independently selected from the group consisting of amino acids or analogs thereof, peptides, polypeptides, proteins, nucleobases, nucleosides, oligonucleotides, nucleic acids, lipids, and analyte-specific receptors including antibodies, antibody derivatives, and antibody fragments.

[0349] 30. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 to 28, wherein

[0350] The targets are independently selected from the group consisting of peptides, polypeptides, proteins, steroid or non-steroid hormones, haptens, and conjugates thereof.

[0351] 31. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 to 28, wherein

[0352] The target comprises a conjugate composed of a plurality of different molecules selected from the group consisting of peptides, polypeptides, proteins, steroid or nonsteroid hormones, haptens, nucleobases, nucleosides, oligonucleotides, nucleic acids, lipids, analyte-specific receptors, analytes, cross-linking agents, and mixtures thereof.

[0353] 32. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 to 28, wherein

[0354] The target comprises a solid phase.

[0355] 33. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 to 28, wherein

[0356] The target comprises a detectable label.

[0357] 34. A method for forming antiparallel full LNA duplexes in the absence of denaturing conditions, the method comprising the steps of:

[0358] (a) providing a first and a second member of a pair of single-stranded all-LNA oligonucleotides according to any one of items 20 to 33, respectively, wherein each single-stranded all-LNA oligonucleotide is kept separately dissolved in an aqueous solution in the absence of a denaturing agent and at a temperature of 0° C. to 40° C.;

[0359] (b) contacting the pair of single-stranded all-LNA oligonucleotides with each other at a temperature of 0° C. to 40° C. in the absence of a denaturing agent;

[0360] An antiparallel all-LNA duplex is thereby formed.

[0361] 35. An antiparallel duplex formed by the first and second members of a pair of single-stranded all-LNA oligonucleotides, wherein the antiparallel duplex is obtained by the method of item 34.

[0362] 36. Use of a pair of single-stranded all-LNA oligonucleotides according to any one of items 20 to 33 for linking a first and a second component in a complex in a liquid aqueous medium, wherein the first component is linked to the first member of the pair and the second component is linked to the second member of the pair.

[0363] 37. Use of a pair of single-stranded all-LNA oligonucleotides according to any one of items 20 to 33 in a receptor-based assay for determining an analyte, the receptor-based assay comprising an analyte-specific receptor and a solid phase for immobilizing the analyte on the solid phase, wherein the first ss-oligonucleotide of the pair is coupled to the analyte-specific receptor and the second ss-oligonucleotide of the pair is coupled to the solid phase.

[0364] 38. A kit for performing a receptor-based assay to determine an analyte, the kit comprising in a first container an analyte-specific receptor to which is linked a first member of a pair of isolated ss-oligonucleotides according to any one of items 20 to 33, the kit further comprising in a second container a solid phase to which is linked a second member of the pair.

[0365] 39. A method for performing a receptor-based assay for determining an analyte, the method comprising the steps of: contacting the analyte with an analyte-specific receptor to which is linked a first member of a pair of isolated ss-oligonucleotides of any one of items 20 to 33, and with a solid phase to which is linked a second member of the pair; incubating to form a complex comprising the solid phase, the analyte-specific receptor bound to the solid phase, and the analyte bound to the analyte-specific receptor, wherein an antiparallel duplex is formed, the duplex consisting of the first and second members of the pair, wherein the duplex connects the analyte-specific receptor in the complex and the solid phase; and then detecting the analyte bound to the complex to determine the analyte.

[0366] With respect to all aspects and embodiments disclosed herein, the skilled artisan understands that the analyte-specific receptor specifically comprises an antibody or an antibody fragment.

[0367] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications can be made to the procedures set forth without departing from the spirit of the present invention.

[0368] Example 1

[0369] Synthesis of LNA oligonucleotides

[0370] LNA oligonucleotides were synthesized on an ABI 394 DNA synthesizer at a 1 μmol scale using a standard automated stationary phase DNA synthesis procedure and applying phosphoramidite chemistry. Glen UnySupport PS (Glen Research catalog number 26-5040) and LNA phosphoramidites (Qiagen / Exiqon catalog numbers 33970 (LNA-A(Bz), 339702 (LNA-T), 339705 (LNA-mC(Bz), and 339706 (LNA-G(dmf); β-L-LNA analogs were synthesized similarly to D-β-LNA phosphoramidites (Carbosynth, cat. number MG05247) starting from L-glucose according to AA Koshkin et al., J. Org. Chem 2001, 66, 8504-8512), and spacer phosphoramidite 18 (Glen Research catalog number 10-1918) and 5′-biotin phosphoramidite (Glen Research Cat. No. 10-5950) was used as a building block. All phosphoramidites were at a concentration of 0, 1 M in DNA grade acetonitrile. Standard DNA cycles with extended coupling time (180 seconds), extended oxidation time (45 seconds) and detritylation time (85 seconds) as well as standard synthesis reagents and solvents were used to assemble LNA oligonucleotides. 5'-biotinylated LNA oligonucleotides were synthesized as DMToff, while unmodified LNA oligonucleotides were synthesized as DMTon. The LNA oligonucleotides were then cleaved from the support by concentrated ammonia using a standard cleavage procedure. Residual protecting groups were cleaved by treatment with concentrated ammonia (8 h at 56°C). The crude LNA oligonucleotides were evaporated and purified by RP HPLC (column: PRP-1, 7 μm, 250x21.5 mm (Hamilton, product number 79352) or XBridge BEH C18 OBD, 5 μm, 10 x 250 mm (Waters product number 186008167)) was eluted using a 0.1 M triethylammonium acetate / acetonitrile gradient at pH 7. Product fractions were pooled and desalted by dialysis against water (MWCO 1000, SpectraPor 6, product number 132638) for 3 days, thereby also cleaving the DMT group of the DMTon-purified oligonucleotide. Finally, the LNA oligonucleotide was lyophilized.

[0371] The yield ranged from 85 nmol to 360 nmol.

[0372] The LNA oligonucleotides were analyzed by RP18 HPLC (Chromolith RP18e, Merck product number 1.02129.0001) using a 0.1 M triethylammonium acetate / acetonitrile gradient at pH 7. Typical purity was ≥90%. The identity of the LNA oligonucleotides was confirmed by LC-MS analysis.

[0373] Each species of oligonucleotide was synthesized and stored separately.

[0374] Example 2

[0375] RP-HPLC analysis identifies LNA oligonucleotide sequences capable of forming duplexes without prior denaturation

[0376] a) General method:

[0377] The LNA oligonucleotides from Example 1 were dissolved in buffer (0.01 M Hepes pH 7.4, 0.15 M NaCl) and analyzed on an RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate pH 7 / acetonitrile gradient (8% to 24% acetonitrile in 10 minutes; detection at 260 nm).

[0378] The strand and the corresponding reverse strand LNA oligonucleotides were mixed in equimolar concentrations at room temperature (rt) and immediately analyzed on a RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate / acetonitrile gradient at pH 7 (8% to 24% B in 10 min; detection at 260 nm).

[0379] In a first control experiment, the strand and the corresponding reverse strand LNA oligonucleotides were mixed in equimolar concentrations at room temperature, incubated for 1 hour at room temperature, and then analyzed on a RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate / acetonitrile gradient at pH 7 (8% to 25% acetonitrile in 10 minutes; detection at 260 nm).

[0380] In a second control experiment showing duplex formation (positive control), the strand and the corresponding reverse strand LNA oligonucleotide were mixed in equimolar concentrations at room temperature, heat denatured at 95°C (10 minutes), and after reaching room temperature, analyzed again on an RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate / acetonitrile gradient at pH 7 (8% to 24% acetonitrile in 10 minutes; detection at 260 nm).

[0381] Duplex formation can be detected by the formation of a new peak at a different retention time compared to the individual single-stranded LNA oligonucleotides. In a positive control, the mixed strands are heat-denatured before injection to generate duplexes. By mixing the strands and counterstrands at room temperature without prior denaturation and then injecting them over time, the kinetics of duplex formation can be monitored.

[0382] An LNA sequence is determined to be capable of rapid duplex formation if, without prior denaturation, after annealing at room temperature for 5 to 60 minutes, the HPLC% ratio of the formed duplex to one of the two single-stranded LNAs (corrected by the extinction coefficient; if the two strands are not completely equimolar, a higher ratio value is considered) is ≥ 0.9 (HPLC% corrected by the extinction coefficient; the hyperchromicity of the duplex is not taken into account).

[0383] b) Identification of rapidly duplex-forming sequences

[0384] LNA 1: 5'-tgctcctg-3' (SEQ ID NO 1)

[0385] LNA 2: 5'-Bi-Heg-caggagca-3' (5'-modified SEQ ID NO 2)

[0386] Heg is hexaethylene glycol

[0387] Bi is a biotin label attached via the carboxyl functional group of the biotin valeric acid moiety

[0388] The results are as follows Figure 2-10 shown.

[0389] c) Identification of slowly duplex-forming sequences

[0390] For a 10-bp hybridization experiment, the following ratio calculations were performed

[0391] LNA 3: 5′-CTGCCTGACG-3′

[0392] LNA 4 (conjugate): 5'-Bi-Heg-cgtcaggcag-3'

[0393]

[0394] HPLC%*ε -1 *1000(LNA 3 / LNA 4 duplex) / HPLC%*ε -1 *1000(LNA 3 single chain)=0.023 / 0.456=0.05

[0395] HPLC%*ε -1 *1000(LNA 3 / LNA 4 duplex) / HPLC%*ε -1 *1000(LNA 4 single chain)=0.023 / 0.457=0.05

[0396] Example 3

[0397] RP-HPLC analysis identifies LNA oligonucleotide sequences capable of forming duplexes without prior denaturation

[0398] a) General method:

[0399] The LNA oligonucleotides from Example 1 were dissolved in buffer (0.01 M Hepes pH 7.4, 0.15 M NaCl) and analyzed on an RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate pH 7 / acetonitrile gradient (8% to 24% acetonitrile in 10 minutes; detection at 260 nm).

[0400] The strand and the corresponding anti-strand LNA oligonucleotides (i.e., the first and second oligonucleotides) were mixed in equimolar concentrations at rt (rt) or at a temperature selected from 0°C to 40°C and immediately analyzed on a RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate / acetonitrile gradient at pH 7 (8-24% B in 10 min; detection at 260 nm).

[0401] In one type of experiment, the strand and the corresponding reverse strand LNA oligonucleotide were mixed at equimolar concentrations at room temperature and incubated at room temperature for 1 hour. The analysis was then immediately performed on an RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate / acetonitrile gradient (8-25% acetonitrile over 10 minutes; detection at 260 nm). Other experiments were performed at different temperatures. The temperature range was 0°C to 70°C, more specifically 0°C to 5°C, 0°C to 5°C, 0°C to 10°C, 0°C to 20°C, 0°C to 30°C, 5°C to 10°C, 10°C to 15°C, 15°C to 20°C, 20°C to 25°C, 25°C to 30°C, 30°C to 35°C, and 35°C to 40°C. Chromatographic analysis was performed at room temperature.

[0402] In a control experiment showing duplex formation (positive control), the strand and the corresponding reverse strand LNA oligonucleotide were mixed in equimolar concentrations at rt, heat denatured at 95°C (10 min), and cooled back to room temperature before analysis on an RP18 HPLC (Chromolith RP18e, Merck product number 1.02129.0001) using a 0.1 M triethylammonium acetate / acetonitrile gradient at pH 7 (8-24% acetonitrile in 10 min; detection at 260 nm).

[0403] Duplex formation is detected if a new peak appears at a different retention time than the peak corresponding to a single single-stranded LNA oligonucleotide. In a positive control (control experiment, see above), the mixed strand and anti-strand were heat-denatured before injection to destroy any structures that might have pores and could prevent duplex formation. Therefore, after heat denaturation, duplexes are obtained. Without prior denaturation, the strand and anti-strand LNAs were mixed at room temperature and injected time-dependently to monitor the kinetics of duplex formation.

[0404] The ability of LNA sequences to rapidly form duplexes was analyzed. In an exemplary, but non-limiting, case, a positive result was obtained if the HPLC % ratio of the duplex formed to one of the two single-stranded LNAs after 5-60 min of warming at room temperature (corrected by the extinction coefficient; higher ratio values ​​are considered if the two strands are not completely equimolar) was >0.9. No prior denaturation was performed (HPLC % corrected by the extinction coefficient; the hyperchromicity of the duplex was not considered).

[0405] b) Identification of exemplary sequence pairs capable of rapid duplex formation under non-denaturing conditions

[0406] Initial experiments produced a first LNA oligonucleotide 5'-tgctcctg-3' (SEQ ID NO: 1) and a second LNA oligonucleotide Bi-Heg-5'-caggagca-3' (5' modified SEQ ID NO: 2).

[0407] Heg is hexaethylene glycol

[0408] Bi = biotin label attached via the carboxyl functionality of the biotin valeric acid moiety These and other results are reflected in the figures.

[0409] The presence of the HEG moiety was found to have no effect on the hybridization properties of the corresponding oligonucleotides. No differences were found between the β-D-LNA oligonucleotide pairs and the β-L-LNA oligonucleotide pairs with regard to nucleobase sequence and hybridization properties.

[0410] The following table provides further exemplary results for oligonucleotide pairs that can be kept separate under non-denaturing conditions and that are capable of forming duplexes under non-denaturing conditions when contacted with each other.

[0411] The 15-mer 5'caccaacacaccaac 3' (SEQ ID NO 32, tested as a 5'-modified Bi-Heg molecule) and the 15-mer 5'gttggtgtgttggtg 3' (SEQ ID NO 31) showed rapid duplex formation upon contact with each other.

[0412] Number of Watson-Crick complementary base pairs: 5

[0413] It was found that 5′ggaag 3′ / 5′cttcc 3′ has rapid duplex formation;

[0414] (SEQ ID NOs: 34 and 33, respectively).

[0415] It was found that 5′ggagc 3′ / 5′gctcc 3′ has rapid duplex formation;

[0416] (SEQ ID NOs: 43 and 42, respectively).

[0417] Number of Watson-Crick complementary base pairs: 6

[0418] It was found that 5′accaac 3′ / 5′gttggt 3′ has rapid duplex formation;

[0419] (SEQ ID NOs: 20 and 16, respectively).

[0420] It was found that 5′tttttt 3′ / 5′aaaaaa 3′ had rapid duplex formation;

[0421] (SEQ ID NOs: 27 and 39, respectively).

[0422] It was found that 5′ggagca 3′ / 5′tgctcc 3′ has rapid duplex formation;

[0423] (SEQ ID NOs: 45 and 44, respectively).

[0424] It was found that 5′ctgtca 3′ / 5′tgacag 3′ has rapid duplex formation;

[0425] (SEQ ID NOs: 40 and 41, respectively).

[0426] It was found that 5′ggaaga 3′ / 5′tcttcc 3′ has rapid duplex formation;

[0427] (SEQ ID NOs: 36 and 35, respectively).

[0428] Number of Watson-Crick complementary base pairs: 8

[0429] It was found that 5′caggagca 3′ / 5′tgctcctg 3′ had rapid duplex formation;

[0430] (SEQ ID NO: 2 and 1, respectively).

[0431] Number of Watson-Crick complementary base pairs: 9

[0432] It was found that 5′ggaagagaa 3′ / 5′ttctcttcc 3′ had rapid duplex formation;

[0433] (SEQ ID NOs: 38 and 37, respectively).

[0434] Number of Watson-Crick complementary base pairs: 15

[0435] It was found that 5′caccaacacaccaac 3′ / 5′gttggtgtgttggtg 3′ had rapid duplex formation;

[0436] (SEQ ID NO: 32 and 31, respectively).

[0437] Typically, the first oligonucleotide of the binding pair described above was used as a Bi-Heg conjugate as described in the initial experiments.

[0438] For instructions, see Figure 11-17 .

[0439] c) Identification of slowly duplex-forming sequences

[0440] Number of possible Watson-Crick complementary base pairs: 10

[0441] It was found that 5′cgtcaggcag 3′ / 5′ctgcctgacg 3′ had slow duplex formation;

[0442] (SEQ ID NOs: 6 and 5, respectively).

[0443] Number of possible Watson-Crick complementary base pairs: 15

[0444] 5'cgtcaggcagttcag 3' / 5'ctgaactgcctgacg 3' was found to have slow duplex formation; (SEQ ID NOs: 47 and 48, respectively).

[0445] For instructions, see Figure 18-20 and Figure 21-23 .

[0446] Figure 20 Identification of binding pairs showing slow hybridization is illustrated. The ratios of the mixed LNA 5'-Bi-Heg-cgtcaggcagttcag-3' (5'-modified SEQ ID NO: 47) and 5'-ctgaactgcctgacg-3' (SEQ ID NO: 48) combinations were calculated.

[0447]

[0448] HPLC%*ε -1 *1000(LNA duplex) / HPLC%*ε -1 *1000(LNA single chain)=0.104 / 0.237=0.44

[0449] Example 4

[0450] Biospecific interaction analysis, contact of an immobilized first LNA oligonucleotide with a second LNA oligonucleotide, three different motifs; kinetic characterization at 25°C and 37°C

[0451] a) Project Overview

[0452] · 12 different oligonucleotide LNA sequences were designed and terminally biotinylated (Bi-LNA sequences)

[0453] The binding of 7 LNA oligonucleotide sequences to 12 immobilized Bi-LNA sequences was analyzed at 25°C / 37°C

[0454] 3 min association time and 30 min dissociation time, respectively, with a flow rate of 60 μl / min

[0455] Pre-incubate the LNA samples overnight at RT (room temperature) in slightly alkaline buffer (chemical inactivation) as recommended by the manufacturer

[0456] Test setup such as Figure 24 shown

[0457] b) Technical procedures

[0458] Kinetic studies were performed on a GE Healthcare Biacore 8k instrument.

[0459] A Biacore Biotin Capture Kit S Series sensor (Catalog No. 28-9202-34) was installed in the instrument and hydrodynamic processing and pretreatment were performed according to the manufacturer's instructions. The system buffer was HBS-T (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% TWEEN 20). The sample buffer was the system buffer. The biotin capture reagent provided by the manufacturer, GE Healthcare, was diluted 1:50 in the system buffer and injected into all measurement flow cells at a rate of 10 μl / min for a period of 60 seconds. Reference cells were not immobilized and maintained as blank controls. 10 nM of the corresponding biotinylated ligand was injected at a rate of 30 μl / min to obtain ligand capture levels between 4 RU and 30 RU. A concentration series of analytes in solution was injected at a rate of 60 μl / min for an association time of 3 min. Dissociation was monitored for 5 minutes. High affinity interactions were monitored for a dissociation time of 30 minutes. The analyte concentration series was 0 nM (buffer), 0.11 nM, 0.33 nM, 3 nM, 9 nM, 27 nM. In another embodiment, 0 nM, 0.56 nM, 1.67 nM, 5 nM, 15 nM, and 45 nM. The CAP sensor was fully regenerated by injecting 100 mM NaOH for 1 minute. Kinetic data were determined using Biacore evaluation software.

[0460] c) Results

[0461] Seven different LNA oligonucleotides representing three different sequence motifs were analyzed for binding to 12 different complementary biotinylated LNA oligonucleotides with different sequence lengths (Bi-LNA) at two different temperatures: 25°C and 37°C.

[0462] 0.05% Tween 20 was used as a detergent in the SPR measurements.

[0463] Initial molar ratios MR = 1.0-0.7 (data not shown) indicated 1:1 binding, and MR decreased during the assay cycle (MR = 0.5-0.3), most likely due to inactivation of the streptavidin surface by the harsh alkaline pH used in the regeneration step.

[0464] Motif "Sequence 1"

[0465] 9-mer LNA shows binding to complementary Bi-LNA 7-9-mer with motif 1

[0466] At 37°C, 'Bi-(HEG)4-5'caggagca 3' (5'-modified SEQ ID NO: 2)' and 'Bi-(HEG)4-5'caggagc 3' (5'-modified SEQ ID NO: 15) showed high complex stability with the complementary oligonucleotide 5'tgctcctgt 3' (SEQ ID NO: 9)' with and without the (HEG)4-MH-5'-tag.

[0467] t / 2diss (Bi-LNA 7&8-mer / 9-mer)=>247 / 228 minutes,

[0468] t / 2diss (Bi-LNA 7 & 8-mer / 9-mer with Heg4-MH5') => 734 / 800 min, resulting in high affinity (K D =6-9pM):

[0469] Hybridization with "sequence 2"-LNA showed weaker binding to sequence 1 Bi-LNA 7-9mers

[0470] No binding was detected for the 9-mer A sequence '5'aaaaaaaaa'3' (SEQ ID NO: 28) including the (HEG)4-MH-5'-tag.

[0471] Motif "Sequence 2"

[0472] Bi-LNAs representing motif 2 of different lengths (12-mer, 10-mer, 8-mer, 7-mer and 6-mer) did not show any binding to LNA motifs 1 or 3.

[0473] Motif 2-LNA binding of different lengths showed comparable complex formation for 6-8mers, whereas complex formation for 12mers was only slightly slower.

[0474] Complex stability changes: Bi-LNA 7-mer showed the highest complex stability in these experiments, followed by Bi-LNA 8-mer

[0475] t / 2diss (Bi-LNA 7-mer / 6-mer) = 238 minutes,

[0476] t / 2diss (Bi-LNA 7-mer / 7-mer) = 720 minutes,

[0477] t / 2diss (Bi-LNA 7-mer / 8-mer) = 644 minutes,

[0478] t / 2diss (Bi-LNA 8-mer / 7-mer) = 545 minutes,

[0479] t / 2diSs (Bi-LNA 8mer / 8mer) = 433 minutes, resulting in high affinity (K D =1-5pM)

[0480] Motif 2 is estimated to be better than motif 1

[0481] Motif "Sequence 3"

[0482] A 5' modified poly A sequence (HEG) 4-MH-5' sequence 5'aaaaaaaaa 3' (SEQ ID NO: 28) was used as a negative control. This control did not show any binding to any of the biotinylated sequences 1 and 2. However, specific binding was detected using the complementary poly T sequence from "Group 3".

[0483] At 37°C, as the LNA length increased 1000-fold from 6-9mers (t / 2diss = 1 to >1160 minutes), the complex stability increased significantly and complex formation decreased 55-fold, resulting in affinities in the range of KD = 300pM-10pM - ssL-DNA hybridization slowed as the length increased from 6mers to 9mers, and the complex stability remained high; overhangs with >2 unpaired nucleotides significantly reduced the stability of the poly A / poly T paired complex.

[0484] d) Conclusion

[0485] Given the goal of providing an alternative to the streptavidin:biotin binding pair, it was important to choose a low affinity (hopefully in the pM range) binding pair that already has a very fast association rate constant at 25°C and a persistently high complex stability at 37°C.

[0486] All-LNA oligonucleotide duplexes with four to five complementary LNA nucleobase pairings representing the motif "sequence 2" meet the requirements for the desired binding pair as they show rapid association to saturation and high complex stability.

[0487] To ensure rapid association, it is desirable that the binding pair be as short as possible to avoid time-consuming "false priming" intermediates. 'Bi-(HEG)4-5'caccaac 3' (7-mer oligonucleotide, SEQ ID NO: 19) in combination with 5'gttggt 3" and Bi-(HEG)4-MH-5'gttggtgt 3' (5'-modified SEQ ID NO: 16)' showed complex formation and complex stability, t / 2diss = 720 and 644 min, respectively, at 37°C, resulting in high affinity (K D =2 pM). 'Bi-(HEG)4-5'acaccaac 3' (8-mer, 5'-modified SEQ ID NO: 14) bound to (HEG)4-MH-5'gttggtg 3' (5'-modified SEQ ID NO: 21) and (HEG)4-MH-5'gttggtgt 3' (5'-modified SEQ ID NO: 13)', showing sufficient complex formation and complex stability, with t / 2diss = 545 and 433 minutes, respectively, at 37°C, with high affinity (KD = 2 pM).

[0488] LNAs with different lengths and the motif "sequence 1" did not bind. As a negative control, the sequence 5'aaaaaaaaaa 3' (SEQ ID NO: 28) including a (HEG)4-MH-5'-tag was tested and no measurable intermolecular interaction was observed.

[0489] Example 5

[0490] Biospecific interaction analysis

[0491] a) Overview of the protocol and assay setup

[0492] Reversible capture of streptavidin conjugates by CAP-kit

[0493] Streptavidin conjugated to complementary ss-LNA

[0494] Reversible binding of oligonucleotides to pre-immobilized ss-LNA oligonucleotides on SCM

[0495] Determination:

[0496] Capture level (CL), association rate constant k a ,

[0497] Dissociation rate constant k d ,

[0498] Dissociation equilibrium constant K o

[0499] Molar ratio (MR)

[0500] Test setup such as Figure 51 As shown in A

[0501] Four free LNA constructs "Motif 2" and LNA-Fab with different lengths (6-8mer and 12mer) were analyzed <tsh>-conjugate (LNA-Fab <tsh>= binding of an antibody Fab fragment specific for the TSH antigen) to a complementary Bi-LNA sequence at 25° / 37°C.

[0502] Bi-LNA sequences are captured as ligands on the CAP chip via a reversible biotin capture kit;

[0503] Free LNA or LNA-Fab <tsh>-Conjugates used as analytes in solution

[0504] Hybridization was analyzed using an association time of 3 minutes and a dissociation time of 30 minutes,

[0505] Flow rate 60 μl / min

[0506] C(free LNA)=9-0.1nM, c(LNA-Fab <tsh>-conjugate) = 45-0.6 nM, c (12聚体LNA / Fab < TsH>-缀合物) =45-0.6nM

[0507] As suggested by the customer, LNA samples were pre-incubated in a weak alkaline buffer at RT overnight (chemical inactivation)

[0508] B) Reagent: sequence "Motif 2"

[0509] Biotinylated ligand

[0510] 'Bi-(HEG)4-5'accaac 3' (SEQ ID NO: 20)

[0511] BMO28.542740, GO4094, ID 6681, 6-mer, MW 3.8 kDa

[0512] 'Bi-(HEG)4-5'caccaac 3' (SEQ ID NO: 19)

[0513] BMO 28.542739, GO4093, ID 6681, 7-mer, MW 4.1 kDa

[0514] 'Bi-(HEG)4-5'acaccaac 3' (SEQ ID NO: 14)

[0515] BMO28.542738, GO4092, ID 6680, octamer, MW 4.4 kDa

[0516] 'Bi-(HEG)4-5'caacacaccaac 3' (SEQ ID NO: 52)

[0517] BMO 28.542742, GO4096, ID 6684, 12-mer, MW 5.8 kDa

[0518] Analytes

[0519] 2300 / 103(HEG)4-MH-5'gttggt 3'(SEQ ID NO: 16)'

[0520] BMO28.170333, AO581, ID 6719, 6-mer, MW 3.8 kDa

[0521] 2300 / 104(HEG)4-MH-5'gttggtg 3'(SEQ ID NO: 21)'

[0522] BMO28.170334, AO582, ID 6720, 7-mer, MW 4.1 kDa

[0523] 2300 / 105(HEG)4-MH-5'gttggtgt 3'(SEQ ID NO: 13)'

[0524] BMO28.170335, AO583, ID 6721, octamer, MW 4.4 kDa

[0525] 2300 / 102(HEG)4-MH-5'gttggtgtgttg 3'(SEQ ID NO: 53)'

[0526] BMO28.542727, GO4073, ID 6653, 12-mer, MW 5.8 kDa

[0527] mAb <tsh>M-Tu1.20-F(ab′)2-SATP-D-LNA-conjugate

[0528] 2331 / 111mAb <tsh>M-Tu1.20-F(ab′)2-SATP-D-LNA-5′gttggt 3′ (SEQ ID NO: 16), 6-mer, MW 104 kDa

[0529] 2331 / 112mAb <tsh>M-Tu 1.20-F(ab')2-SATP-D-LNA-5'gttggtg 3' (SEQ ID NO: 21), 7-mer, MW 104 kDa

[0530] 2331 / 113mAb <tsh>M-Tu1.20-F(ab′)2-SATP-D-LNA-5′gttggtgt3′ (SEQ ID NO: 13), 8-mer, MW 104 kDa

[0531] 2331 / 114mAb <tsh>M-Tu1.20-F(ab′)2-SATP-D-LNA-5′gttggtgtgttg 3′(SEQ IDNO:53),12-member,MW 106kDa

[0532] mAb <tsh>M-Tu1.20-F(ab')2 represents the F(ab')2 fragment of a monoclonal antibody specific for TSH, which is human thyroid stimulating hormone. D-LNA represents that the oligonucleotide with the subsequent nucleobase sequence is composed of D-LNA monomers.

[0533] c) Results

[0534] The four different 5′-modified LNA oligonucleotides given above, as well as those having the same corresponding sequence but containing a F(ab′)2 <tsh>The oligonucleotides in the conjugates represented the sequence "Motif 2" with lengths of 6-mer, 7-mer, 8-mer and 12-mer. All assays were performed at 25° / 37°C in conjunction with their complementary Bi-LNA sequences.

[0535] We also analyzed LNA-Fab hybridized with <tsh>TSH binding of the conjugate (TSH is the analyte).

[0536] Sequence "motif 2"

[0537] Bi-LNAs of different lengths showed comparable complex formation, with complex stabilities ranging from t / 2diss 154 to >232 min, pM affinity range (K D At 37°C, complex formation is in the pM affinity range (K D Hybridization kinetics of free oligonucleotides at 25°C and 37°C are mass-transfer limited (data shown in red), corrected for MTL using an SW scrubber. Molar ratios (MR) of 0.8-1.1 indicate 1:1 hybridization stoichiometry at both temperatures. Supersaturation of the 12-mer associating phase at 25°C.

[0538] Compared with unconjugated LNA oligonucleotides, LNA-Fab <tsh>The conjugates showed a 2-4 fold slowing of complex formation. <tsh>-No MTL during hybridization of LNA-conjugate. Complex stability t / 2diss >232 min, resulting in pM affinity range (K D =22-11 pM). At 37°C, FAb <tsh>Compared with free LNA of the same length, there was no significant difference in complex formation between the LNA conjugates (7-mer, 8-mer and 12-mer) and the free LNA. / 2diss >232 minutes, with affinity in the pM range (K D <12-14 pM).

[0539] LNA-Fab <tsh>The molar ratio MR of the conjugates was 0.1-0.6, indicating substoichiometric 1:1 binding. <tsh>Conjugates of TSH and LNA-Fab <tsh>The binding of the conjugates (6-8mer and 12mer) showed comparable kinetics, rapid complex formation and sufficient complex formation, t / 2diss 31-33 minutes, resulting in affinity K D =0.7nM

[0540] The binding constant is within the range of known affinities for this interaction.

[0541] A molar ratio (MR) of 1.8 / 1.9 showed a fully functional stoichiometric 2:1 binding, indicating binding of a functional conjugate.

[0542] It was found that the hybridized Fab conjugate showed the full antigen binding activity of the antibody portion of the conjugate. The results are shown in Figure 52.

[0543] For the following data, see also Figure 51 C

[0544]

[0545] Table: Molar epitope accessibility matrix showing hTK sandwich formation by four anti-hTK antibodies. EA =1, completely independent epitope, MR EA <1 overlapping epitope.

[0546] Antibody A can form immune complexes with 23C11, 6C6, and 4H4, which share the same epitope.

[0547] d) Alternatives (see Figure 51 B, result Figure 53)

[0548] TSH was analyzed at 37°C with pre-hybridized LNA-FAbs of different lengths (6-mer, 7-mer, 8-mer, and 12-mer). <tsh>Conjugation of conjugates

[0549] Assay format is shown on slide 10 and binding curves are shown on slide 11

[0550] ·3Bi-LNA sequence irreversibly binds to the SA chip on Fc2-4

[0551] LNA-Fab <tsh>The conjugates (6-mer, 7-mer and 8-mer) were pre-hybridized with their complementary Bi-LNA at 37°C.

[0552] TSH was used as the analyte solution with an association time of 3 minutes and a dissociation time of 5 minutes.

[0553] Flow rate 60 μl / min, C TSH =270nM

[0554] The results are shown in Figure 53.

[0555] Example 6

[0556] Biospecific interaction analysis

[0557] a) Overview of the protocol and assay setup

[0558] A schematic overview of the experiment is as follows Figure 54 shown

[0559] Analysis of binding of free LNA constructs of different lengths (5-mer, 6-mer, 9-mer or 15-mer) and sequences to complementary Bi-LNA sequences (4-6-mer, 9-mer or 15-mer) at 25° / 37°C

[0560] Bi-LNA sequences are captured as ligands on the CAP chip via a reversible biotin capture kit;

[0561] Free LNA was used as the analyte in solution

[0562] Hybridization was analyzed using an association time of 3 minutes and a dissociation time of 30 minutes, with a flow rate of 60 μl / min.

[0563] C(free LNA) = optimized for each interaction

[0564] Measurements: Capture level (CL), association rate constant k a , dissociation rate constant k d , dissociation equilibrium constant K D , molar ratio (MR).

[0565] SA-conjugates are reversibly captured by the CAP-kit, where streptavidin (=SA) is conjugated to a complementary ss-LNA oligonucleotide which reversibly binds to the pre-immobilized ss-LNA oligonucleotide.

[0566] b) Reagents

[0567] Biotinylated ligand

[0568] 2387 / L01 Bi-(HEG)-5'accaac 3' (SEQ ID NO: 20)

[0569] BMO28.170341, AO591, ID 6730, 6-mer, MW 2.71 kDa

[0570] 2387 / L02 Bi-(HEG)-5'cacaccaac 3' (SEQ ID NO: 30)

[0571] BMO28.170342, AO592, ID 6731, 9-mer, MW 3.71 kDa

[0572] 2387 / L03 Bi-(HEG)-5'caccaacaccaac 3' (SEQ ID NO: 54)

[0573] BMO28.170343, AO593, ID6732, 15-mer, MW 5.73 kDa

[0574] 2387 / L04 Bi-(HEG)-5'ggaag 3' (SEQ ID NO: 34)

[0575] BMO28.170347, AO597, ID6736, 5-mer, MW 2.44 kDa

[0576] 2387 / L05 Bi-(HEG)-5'ggaaga 3' (SEQ ID NO: 36)

[0577] BMO28.170348, AO598, ID 6737, 6-mer, MW 2.78 kDa

[0578] 2387 / L06 Bi-(HEG)-5'ggaagagaa 3' (SEQ ID NO: 38)

[0579] BMO28.170349, AO599, ID 6738, 9-mer, MW 3.82 kDa

[0580] 2300 / 12 Bi-(HEG)4-5'tttttt 3' (SEQ ID NO: 27)

[0581] BMO28.170336, AO584, ID 6722, 6-mer, MW 3.71 kDa

[0582] 2387 / L08 Bi-(HEG)-5'ctgtca 3' (SEQ ID NO: 40)

[0583] BMO 28.170354, AO604, ID 6743, 6-mer, MW 2.71 kDa

[0584] 2387 / L09 Bi-(HEG)-5'cgtcaggcagttcag 3' (SEQ ID NO: 55)

[0585] BMO28.170356, AO606, ID 6745, 15-mer, MW 5.12 kDa

[0586] 2387 / L10 Bi-(HEG)-5'ggagc 3' (SEQ ID NO: 43)

[0587] BMO28.170358, AO608, ID 6747, 5-mer, MW 2.43 kDa

[0588] 2387 / L11 Bi-(HEG)-5'ggagca 3' (SEQ ID NO: 45)

[0589] BMO 28.170360, AO610, ID 6749, 6-mer, MW 2.77 kDa

[0590] 2387 / L12 Bi-(HEG)4-5'-ccaac 3' (SEQ ID NO: 46)

[0591] BMO 28.542748, GO4105, ID 6764, 5-mer, MW 3.40 kDa

[0592] 2387 / L13 Bi-(HEG)4-5'caac 3' (SEQ ID NO: 56)

[0593] BMO 28.542749, GO4106, ID 6765, tetramer, MW 3.07 kDa

[0594] 2387 / L14 Bi-(HEG)4-5'ttttt 3' (SEQ ID NO: 57)

[0595] BMO28.542750, GO4107, ID 67665-mer, MW 3.38 kDa

[0596] 2387 / L15 Bi-(HEG)4-5'tttt 3' (SEQ ID NO: 58)

[0597] BMO28.542751, GO4108, ID 6768 4-mer, MW 3.05 kDa

[0598] Analytes

[0599] 2387 / A01 3'-TGG TTG-5'

[0600] BMO28.170344, AO594, ID, 6733, 6-mer, MW 2.01 kDa

[0601] 2387 / A023'-GTG TGG TTG-5'

[0602] BMO28.170345, AO595 / ID 6734, 9-mer, MW 3.05 kDa

[0603] 2387 / A033'-GTG GTT GTG TGG GTT-5'

[0604] BMO28.170346, AO596, ID 6735, 15-mer, MW 5.12 kDa

[0605] 2387 / A043'-CCT TC-5'

[0606] BMO28.170350, AO600, ID 6739, 5-mer, MW 1.60 kDa

[0607] 2387 / A053-'CCT-TCT-5'

[0608] BMO28.170351, AO601, ID 6740, 6-mer, MW 1.93 kDa

[0609] 2387 / A063'-CCT TCT CTT-5'

[0610] BMO28.170352, AO602, ID 6741, 9-mer, MW 2.92 kDa

[0611] 2387 / A073'-AAA AAA-5'

[0612] BMO 28.170353, AO603, ID 6742, 6-mer, MW 1.99 kDa

[0613] 2387 / A083'-GAC AGT-5'

[0614] BMO28.170355, AO605, ID 6744, 6-mer, MW 2.00 kDa

[0615] 2387 / A093'-GCA GTC CGT CAA GTC-5'

[0616] BMO28.170357, AO607, ID 6746, 15-mer, MW 5.04 kDa

[0617] 2387 / A103'-CCT CG-5'

[0618] BMO 28.170359, AO609, ID 6748, 5-mer, MW 1.62 kDa

[0619] 2387 / A113'-CCT CGT-5'

[0620] BMO 28.170361, AO611, ID 6750, 6-mer, MW 1.95 kDa

[0621] c) Results

[0622] Eleven LNAs with different sequences were analyzed for binding to their complementary Bi-LNA sequences. In addition, two Bi-LNAs of different lengths (5-mer and 4-mer) were analyzed in combination with free LNA 6-mers at 25° / 37°C.

[0623] Sequence "motif 2" (2387 / L01-L03) & "Short Motif 2" (2387 / L12 & L13) 6-mer & 9-mer Bi-LNA 5'-Bi-Heg-ACC AAC-3' and 5'-Bi-Heg-CAC ACC AAC-3' showed high affinity binding to their complementary LNA 6-mer 3'-TGGTTG-5' and 9-mer 3'-GTG TGG TTG-5'. The kinetics were characterized by rapid hybridization and sustained high complex stability at 25°C and 37°C. / 2diss =160 to >232 minutes, pM affinity range (K D = 1-9 pM); hybridization kinetics at 25°C and 37°C were mass transfer limited and also corrected for MTL.

[0624] A molar ratio (MR) of 1.1 / 1.2 indicates a 1:1 hybridization stoichiometry for the 6-mer pair at both temperatures; an MR of 1.3 / 1.5 indicates an overstoichiometry for the 9-mer pair. Compared to the 6-mer and 9-mer, the Bi-LNA 15-mer exhibits slower hybridization kinetics, resulting in slightly lower affinity. D = 24 / 53 pM at both temperatures; MR 1.3 indicates slightly superstoichiometric binding.

[0625] When bound to the 5-mer (2387 / L12)Bi-LNA 5′Bi-4x(HEG)-CCA AC-3′, the free 6-mer-LNA3′-TGG TTG-5′ showed a decrease in complex stability. / 2diss = 50 min, a 2-digit pM affinity range, and upon binding to the tetramer (2387 / L13)5'Bi-4x(HEG)-CAA C-3', the complex stability decreased to t / 2diss <1 minute, resulting in double-digit nM affinities. MRs of 1.1-1.3 indicate slightly superstoichiometric binding. Thus, overhangs with one or two mismatched nucleotides can be explained in this case, reducing the stability of the complex to some extent.

[0626] "Motif 3" Poly-T control "short" (2300 / 12 and 2387 / L14&L15); 3 Poly-T-controlled Bi-LNA of different lengths (5- & -6-mers) showed typical fast association / dissociation curves for binding to poly-A-6-mers, with complex half-lives t / 2diss In <2 minutes, Bi-LNA 4-mer showed only weak / no binding to poly A-6-mer.

[0627] "Motif 4" (2387 / L04-L06) 5-mer, 6-mer or 9-mer 5'-Bi-Heg-GGAAG-3', 5'-Bi-Heg-GGA AGA-3' or 5'-Bi-Heg-GGA AGA GAA-3' were not as good as "motif 2" in binding to their complementary LNA. At 25°C, the half-life of the complex was t / 2diss Between 20 and 65 minutes, 2-3 digit pM affinities were generated.

[0628] "Motif 5" Binding of (2387 / L08) 5'-Bi-Heg-CTG TCA-3' to the complementary LNA 3'-GAC AGT-5' shows slightly slower hybridization than the 6-mer of "Motif 2," with complex stability in the double-digit pM affinities at both 25°C and 37°C. A molar ratio of 0.3 / 0.4 indicates substoichiometric binding.

[0629] "Motif 6" (2387 / L09) 15-mer 5′-Bi-Heg-CGT CAG GCA GTT CAG-3′ in combination with 3′-GCA GTCCGT CAA GTC-5′ shows a single-digit nM affinity interaction resulting from slowed hybridization and reduced complex stability; a molar ratio of 0.1 / 0.4 indicates substoichiometric binding.

[0630] "Motif 7" (2387 / L10 and L11)

[0631] The 5-mer 5′-Bi-Heg-GGA GC-3′ binds to its complementary LNA better than the 6-mer 5′-Bi-Heg-GGA GCA-3′; at 25°C, the half-life of the complex is t / 2diss Between 174 and 62 minutes, the affinity ranged from 32 / 186 pM, with the 5-mer showing a 29 pM interaction at 37°C, and a molar ratio MR 0.5 / 0.3 indicating substoichiometric binding at 25°C, which increased at 37°C (MR 1.2 / 0.6).

[0632] d) Conclusion

[0633] The Bi-LNAs 5′-Bi-Heg-ACC AAC-3′ and 5′-Bi-Heg-CAC ACC AAC-3′ ("Motif 2") both displayed high affinity for their complementary LNA 6-mers 3′-TGG TTG-5′ and 9-mers 3′-GTG TGG TTG-5′, respectively. The molar ratios demonstrated fully functional 1:1 LNA hybridization. 5′-Bi-Heg-ACC AAC-3′ / 3′-TGGTTG-5′ exhibited slightly improved hybridization kinetics compared to 5′-Bi-(HEG)4-ACCAAC-3′ / 3'-TGG-TTG-5′-Heg4-MH-5′, due to a 2-fold increase in complex stability.

[0634] According to the supplier's information, 1000 RU = 1 ng / mm 2 The absolute density of Bi-LNA5′-Bi-Heg-ACC AAC-3′ captured on the sensor surface used in the experiment was 11 fmol / mm 2 (30pg / mm 2 ). In the hydrogel, this is assumed to correspond to a concentration of 0.3 mg / mL.

[0635] The present invention relates to the following projects:

[0636] 1. A method for selecting and providing a binding pair of single-stranded all-LNA oligonucleotides capable of forming an antiparallel duplex having 5 to 15 consecutive base pairs in aqueous solution at a temperature of 0°C to 40°C, the method comprising the following steps:

[0637] (h) providing a first single strand (=ss-) consisting of 5 to 15 locked nucleic acid (=LNA) monomers

[0638] an oligonucleotide, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0639] (i) providing a second ss-oligonucleotide consisting of 5 to 15 LNA monomers, the second ss-oligonucleotide comprising at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation and predicting the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 5 to 15 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other;

[0640] (i) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.;

[0641] (k) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex;

[0642] (1) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0643] (m) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is greater than the molar amount of the ss-oligonucleotide, selecting the binding pair;

[0644] (n) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0645] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0646] 2. The method according to item 1, wherein before step (e), the mixture obtained in step (d) is subjected to an additional step of separating ss-oligonucleotides and duplex oligonucleotides.

[0647] 3. The method according to any one of items 1 and 2, wherein steps (c) and (d) are carried out at a non-denaturing temperature, specifically at a temperature selected from the group consisting of 0°C to 5°C, 5°C to 10°C, 10°C to 15°C, 15°C to 20°C, 20°C to 25°C, 25°C to 30°C, 30°C to 35°C and 35°C to 40°C.

[0648] 4. The method according to any one of items 1 to 3, wherein prior to step (c), each ss-oligonucleotide of any of steps (a) and (b) is maintained in the absence of denaturing conditions.

[0649] 5. The method according to item 4, wherein prior to step (c), each ss-oligonucleotide of any of steps (a) and (b) is maintained at a temperature of -80°C to 40°C, specifically 0°C to 40°C, more specifically 25°C to 37°C in aqueous solution.

[0650] 6. The method according to any one of items 1 to 5, wherein in step (d), the time interval is selected from the group consisting of 1 second to 20 minutes, 1 second to 5 minutes, 1 second to 60 seconds and 1 second to 30 seconds.

[0651] 7. The method according to any one of items 1 to 6, wherein steps (c) and (d) are carried out in the absence of a denaturant compound capable of lowering the melting temperature of a DNA duplex of 20 base pairs in length and a G+C content of 50% by at least 15°C, more specifically in the absence of either formamide and dimethyl sulfoxide.

[0652] 8. The method according to any one of items 1 to 7, wherein each LNA monomer comprises a nucleobase selected from the group consisting of: N 4 -acetylcytosine, 5-acetyluracil, 4-amino-6-chloropyrimidine, 4-amino-5-fluoro-2-methoxypyrimidine, 6-amino-1-methyluracil, 5-aminoorotic acid, 5-aminouracil, 6-aminouracil, 6-azauracil, N 4 -Benzoylcytosine, 5-bromouracil, 5-chlorouracil, 6-chlorouracil,

[0653] 6-chloromethyluracil, 6-chloro-3-methyluracil, cytosine, 5,6-dimethyluracil,

[0654] 5-Ethyluracil, 5-Ethynyluracil, 5-Fluorocytosine, 5-Fluororotic acid, 5-Fluorouracil, 5-Iodo-2,4-dimethoxypyrimidine, 5-Iodouracil, Isocytosine, 5-Methylcytosine, 6-Methyl-5-nitrouracil, 2-Methylthio-4-pyrimidinol, 5-Methyl-2-thiouracil, 6-Methyl-2-thiouracil, 6-Methyluracil, 5-Nitrouracil, Orotic acid,

[0655] 6-phenyl-2-thiouracil, 6-propyl-2-thiouracil, 2-thiouracil, 4-thiouracil, thymine, 5-(trifluoromethyl)uracil, uracil, adenine, 8-azahypoxanthine, 8-azaguanine, allopurinol, 4-aminopyrazolo[3,4-clopyrimidine, 2-aminopurine, 2-acetylamino-6-hydroxypurine, 2-amino-6-chloropurine, 2-amino-6-iodopurine, azathioprine, 4-amino-6-hydroxypyrazolo[3,4-clopyrimidine, aminophylline, N 6 -Benzyl adenine, N 6 -Benzoyladenine, 6-benzyloxypurine, 8-bromotheoline, 8-bromo-3-methylxanthine, 8-bromo-7-(2-butyn-1-yl)-3-methylxanthine, 6-chloropurine, 8-chlorotheoline, 6-chloro-2-fluoropurine, 6-chloro-7-deazapurine, 2-chloroadenine, 6-chloro-7-iodo-7-deazapurine, 2,6-diaminopurine, 2,6-dichloropurine, 6-(dimethylamino) Purine, 2,6-dichloro-7-deazapurine, 5,6-dichlorobenzimidazole hydrochloride, 7-deazahypoxanthine, 2-fluoroadenine, guanine, hypoxanthine, 9-(2-hydroxyethyl)adenine, isoguanine, 3-iodo-1H-pyrazolo-[3,4-d]pyrimidin-4-amine, kinetin, 6-mercaptopurine, 6-methoxypurine, 3-methylxanthine, 1-methylxanthine, 3-methyladenine, O 6 -(cyclohexylmethyl)guanine, 6-thioguanine, 2-thioxanthine, xanthine, 5-propynyl-uracil, 5-propynyl-cytidine, 7-deazaadenine, 7-deazaguanine, 7-propynyl-7-deazaadenine, 7-propynyl-7-deazaguanine and derivatives thereof.

[0656] 9. The method according to item 8, wherein each LNA monomer comprises a nucleobase selected from the group consisting of adenine, thymine, uracil, guanine, cytosine and 5-methylcytosine.

[0657] 10. The method according to any one of items 8 and 9, wherein one or more cytosines, where present, are replaced by 5-methylcytosine.

[0658] 11. The method according to item 10, wherein each cytosine is replaced by 5-methylcytosine.

[0659] 12. The method according to any one of items 1 to 11, wherein the monomer of the ss-oligonucleotide in any one of steps (a) and (b) is a β-L-LNA monomer.

[0660] 13. A pair of isolated complementary ss-oligonucleotides, each ss-oligonucleotide consisting of 5 to 15 LNA monomers, the isolated ss-oligonucleotides being capable of forming antiparallel duplexes with each other in aqueous solution in the absence of denaturing conditions prior to or during duplex formation.

[0661] 14. The pair of isolated complementary ss-oligonucleotides according to item 11, wherein the pair of ss-oligonucleotides is obtained by performing the method according to any one of items 1 to 12.

[0662] 15. The pair of isolated complementary ss-oligonucleotides according to any one of items 13 and 14, wherein the pair is selected from the group consisting of

[0663] (SEQ ID NO: 1): (SEQ ID NO: 2),

[0664] (SEQ ID NO: 9): (SEQ ID NO: 10),

[0665] (SEQ ID NO: 11): (SEQ ID NO: 12),

[0666] (SEQ ID NO: 13): (SEQ ID NO: 14),

[0667] (SEQ ID NO: 15): (SEQ ID NO: 16),

[0668] (SEQ ID NO: 16): (SEQ ID NO: 20),

[0669] (SEQ ID NO: 17): (SEQ ID NO: 18),

[0670] (SEQ ID NO: 19): (SEQ ID NO: 20),

[0671] (SEQ ID NO: 21): (SEQ ID NO: 22),

[0672] (SEQ ID NO: 23): (SEQ ID NO: 24),

[0673] (SEQ ID NO:25): (SEQ ID NO:26).

[0674] 16. The pair of isolated complementary ss-oligonucleotides according to any one of items 13 to 15, wherein the first ss-oligonucleotide of the pair is ligated to a first target and the second ss-oligonucleotide is ligated to a second target.

[0675] 17. The pair of isolated complementary ss-oligonucleotides according to item 16, wherein the targets are independently selected from the group consisting of a solid phase, a biomolecule and a chemically synthesized compound.

[0676] 18. A method for forming antiparallel full LNA duplexes in the absence of denaturing conditions, the method comprising the steps of:

[0677] (c) providing a first and a second member of a pair of single-stranded all-LNA oligonucleotides according to item 13 or item 14, respectively, wherein each single-stranded all-LNA oligonucleotide is dissolved in an aqueous solution in the absence of a denaturing agent and maintained at a temperature of 0° C. to 40° C.;

[0678] (d) contacting the pair of single-stranded all-LNA oligonucleotides with each other at a temperature of 0° C. to 40° C. in the absence of a denaturing agent;

[0679] The antiparallel full LNA duplex is thereby formed.

[0680] 19. An antiparallel duplex formed by the first and second members of a pair of single-stranded all-LNA oligonucleotides, wherein the antiparallel duplex is obtained by the method according to item 18.

[0681] 20. Use of a pair of isolated ss-oligonucleotides according to any one of items 13 to 17 in a receptor-based assay for determining an analyte, said receptor-based assay comprising an analyte-specific receptor and a solid phase for immobilizing the analyte on the solid phase, wherein

[0682] The first ss-oligonucleotide of the pair is coupled to the analyte-specific receptor and the second ss-oligonucleotide of the pair is coupled to the solid phase.

[0683] 21. A kit for performing a receptor-based assay for determining an analyte, the kit comprising in a first container an analyte-specific receptor to which is linked a first member of a pair of isolated ss-oligonucleotides according to any one of items 13 to 17, the kit further comprising in a second container a solid phase to which is linked a second member of the pair.

[0684] 22. A method for performing a receptor-based assay to determine an analyte, the method comprising the steps of: contacting the analyte with an analyte-specific receptor to which is linked a first member of a pair of isolated ss-oligonucleotides according to any one of items 13 to 15, and with a solid phase to which is linked a second member of the pair; incubating to form a complex comprising the solid phase, the analyte-specific receptor bound to the solid phase, and the analyte bound to the analyte-specific receptor, wherein an antiparallel duplex is formed, the duplex consisting of the first and second members of the pair, wherein the duplex connects the analyte-specific receptor and the solid phase in the complex; and then detecting the analyte bound to the complex to determine the analyte.

[0685] The sequence listing information of the present invention is as follows:

[0686] Sequence Listing

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

[0706]

[0707]

[0708]

[0709]

[0710]

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721]

[0722] < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh> < / tsh>

Claims

1. A method for selecting and providing a binding pair of single-stranded all-LNA oligonucleotides capable of forming an antiparallel duplex having 5 to 15 consecutive base pairs in aqueous solution at a temperature of 0°C to 40°C, the method comprising the following steps: (a) providing a first single-stranded (=ss-) oligonucleotide consisting of 5 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence; (b) providing a second ss-oligonucleotide consisting of 5 to 15 LNA monomers, the second ss-oligonucleotide comprising at least the same number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence, the second nucleobase sequence comprising or consisting of a nucleobase sequence that is complementary to the first nucleobase sequence in an antiparallel orientation and predicting the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other, the predicted duplex comprising or consisting of 5 to 15 consecutive base pairs, wherein the two bases of each base pair are hydrogen bonded to each other; (c) mixing approximately equimolar amounts of said first and second ss-oligonucleotides in aqueous solution, wherein this step is performed at a non-denaturing temperature, more particularly at a temperature between 0° C. and 40° C.; (d) incubating the mixture of (c) for a time interval of 20 minutes or less, thereby obtaining a mixture still comprising the first and second oligonucleotides as ss-oligonucleotides, or obtaining a mixture comprising or consisting of the first and second oligonucleotides as a duplex; (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by (f) if the presence of a duplex is detectable in step (e) and the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide, selecting the binding pair; (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively; Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

2. The method according to claim 1, wherein prior to step (e), the mixture obtained in step (d) is subjected to an additional step of separating ss-oligonucleotides from duplex oligonucleotides.

3. The method according to any one of claims 1 and 2, wherein steps (c) and (d) are performed at a non-denaturing temperature, specifically at a temperature selected from the group consisting of 0°C to 5°C, 5°C to 10°C, 10°C to 15°C, 15°C to 20°C, 20°C to 25°C, 25°C to 30°C, 30°C to 35°C and 35°C to 40°C.

4. The method according to any one of claims 1 to 3, wherein each ss-oligonucleotide of any of steps (a) and (b) is maintained in the absence of denaturing conditions prior to step (c).

5. The method according to claim 4, wherein before step (c), each ss-oligonucleotide of any of steps (a) and (b) is maintained at a temperature of -80°C to 40°C, specifically 0°C to 40°C, more specifically 25°C to 37°C in aqueous solution.

6. The method according to any one of claims 1 to 5, wherein in step (d), the time interval is selected from the group consisting of 1 second to 20 minutes, 1 second to 5 minutes, 1 second to 60 seconds, and 1 second to 30 seconds.

7. The method according to any one of claims 1 to 6, wherein steps (c) and (d) are carried out in the absence of a denaturant compound capable of lowering the melting temperature of a DNA duplex of 20 base pairs in length and a G+C content of 50% by at least 15°C, more particularly in the absence of either formamide and dimethyl sulfoxide.

8. The method according to any one of claims 1 to 7, wherein each LNA monomer comprises a nucleobase selected from the group consisting of: N 4 -acetylcytosine, 5-acetyluracil, 4-amino-6-chloropyrimidine, 4-amino-5-fluoro-2-methoxypyrimidine, 6-amino-1-methyluracil, 5-aminoorotic acid, 5-aminouracil, 6-aminouracil, 6-azauracil, N 4 Benzoylcytosine, 5-bromouracil, 5-chlorouracil, 6-chlorouracil, 6-chloromethyluracil, 6-chloro-3-methyluracil, cytosine, 5,6-dimethyluracil, 5-ethyluracil, 5-ethynyluracil, 5-fluorocytosine, 5-fluoroorotic acid, 5-fluorouracil, 5-iodo-2,4-dimethoxypyrimidine, 5-iodouracil, isocytosine, 5-methylcytosine, 6-methyl-5-nitrouracil, 2-methylthio-4-pyrimidinol, 5-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl Uracil, 5-nitrouracil, orotic acid, 6-phenyl-2-thiouracil, 6-propyl-2-thiouracil, 2-thiouracil, 4-thiouracil, thymine, 5-(trifluoromethyl)uracil, uracil, adenine, 8-azahypoxanthine, 8-azaguanine, allopurinol, 4-aminopyrazolo[3,4-closed]pyrimidine, 2-aminopurine, 2-acetylamino-6-hydroxypurine, 2-amino-6-chloropurine, 2-amino-6-iodopurine, azathioprine, 4-amino-6-hydroxypyrazolo[3,4-closed]pyrimidine, aminophylline, N 6 -Benzyl adenine, N 6 -Benzoyladenine, 6-benzyloxypurine, 8-bromotheoline, 8-bromo-3-methylxanthine, 8-bromo-7-(2-butyn-1-yl)-3-methylxanthine, 6-chloropurine, 8-chlorotheoline, 6-chloro-2-fluoropurine, 6-chloro-7-deazapurine, 2-chloroadenine, 6-chloro-7-iodo-7-deazapurine, 2,6-diaminopurine, 2,6-dichloropurine, 6-(dimethylamino) Purine, 2,6-dichloro-7-deazapurine, 5,6-dichlorobenzimidazole hydrochloride, 7-deazahypoxanthine, 2-fluoroadenine, guanine, hypoxanthine, 9-(2-hydroxyethyl)adenine, isoguanine, 3-iodo-1H-pyrazolo-[3,4-d]pyrimidin-4-amine, kinetin, 6-mercaptopurine, 6-methoxypurine, 3-methylxanthine, 1-methylxanthine, 3-methyladenine, O 6 -(cyclohexylmethyl)guanine, 6-thioguanine, 2-thioxanthine, xanthine, 5-propynyl-uracil, 5-propynyl-cytidine, 7-deazaadenine, 7-deazaguanine, 7-propynyl-7-deazaadenine, 7-propynyl-7-deazaguanine and derivatives thereof.

9. The method of claim 8, wherein each LNA monomer comprises a nucleobase selected from the group consisting of adenine, thymine, uracil, guanine, cytosine and 5-methylcytosine.

10. The method according to any one of claims 8 and 9, wherein one or more cytosines, where present, are replaced by 5-methylcytosine.

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