Hybridization of all-LNA oligonucleotides

Abstract

This report relates to hybridization of single-stranded (ss-) oligonucleotides consisting entirely of locked nucleic acid (LNA) monomers. This document presents hybridization experiments of pairs of fully complementary ss-oligonucleotides that fail to form duplexes within a given time interval. This report provides methods for identifying such incompatible oligonucleotide pairs. On the other hand, this report provides pairs of complementary ss-oligonucleotides that can quickly form duplexes. This report also provides methods for identifying and selecting compatible oligonucleotide pairs. On the other hand, this report provides the use of compatible oligonucleotide pairs as binding partners in binding assays, such as receptor-based assays.

Description

Technical Field

[0001] This report relates to hybridizing single-stranded (ss-) oligonucleotides consisting entirely of locked nucleic acid (LNA) monomers. This document presents hybridization experiments of 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. On the other hand, this report provides pairs of complementary ss-oligonucleotides that can quickly form a duplex. This report also provides methods for identifying and selecting compatible oligonucleotide pairs. On the other hand, 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 to detect or determine analytes in samples in assays. Background Art

[0002] Of particular interest are general biochemical applications where the two partners of a binding pair function functionally through specific interaction of molecular recognition and their eventual linkage to each other. For example in immunoassays, biotin:(strept)avidin binding pairs are very 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. In particular, alternative binding pairs made of two single-stranded LNA oligonucleotides capable of forming a duplex by hybridization offer a technical alternative to the biotin:(strept)avidin binding pair.

[0003] The focus of the present disclosure is the means of anchoring the capture receptor on the solid phase during the immunoassay. In particular, the present disclosure focuses on binding pairs, which 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 eventually 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.

[0004] 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), spiral destabilizers (e.g., formamide and chaotropes), and preferred storage and / or assay temperatures (range of 0°C to 40°C), to name a few. However, it is important to note any component and / or condition that would result in denaturation of the analyte to be assayed or the analyte-specific receptor used in a particular assay.

[0005] It is required that the separated partners of the binding pair are suitable for conjugation, in particular conjugation to the capture molecule, i.e., the receptor, and conjugation to the stationary phase surface, without losing their ability to specifically associate and bind to each other. With respect to the conjugates in the immunoassay, each separated binding partner of the substituted 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, the carrier material, the stationary phase, and other substances or compounds that may be present during the assay.

[0006] It has been previously proposed to use single-stranded oligonucleotides with complementary sequences, i.e. oligonucleotides capable of forming duplexes by hybridization, as binding pair tools to connect macromolecules or molecules to a stationary phase. EP 0488152 discloses a heterogeneous immunoassay using a stationary phase, which fixes analyte-specific capture antibodies to the stationary phase by connecting the nucleic acid duplexes of the antibody and the stationary phase. In one embodiment, a hybridized oligonucleotide is shown to be attached to the antibody, while the complementary oligonucleotide is attached to the stationary phase, thereby forming a connection duplex. Similar disclosures are provided in documents EP 0698792, WO 1995 / 024649, WO1998 / 029736 and EP 0905517. WO 2013 / 188756 discloses methods and compositions for flow cytometry, the compositions comprising antibodies, oligospheres and oligonucleotide probes, the antibodies being conjugated to a first oligonucleotide, the oligospheres being conjugated to a second oligonucleotide having the same sequence as the first oligonucleotide, the oligonucleotide probes having a label and a third sequence complementary to the first and second oligonucleotides. In a specific embodiment, the oligospheres are magnetic. This document reports specific uses of oligospheres as references in standardized procedures.

[0007] Modified oligonucleotides, such as peptide nucleic acids (PNA) and locked nucleic acids (LNA) have been investigated for a range of basic biochemical applications. LNA has a methylene linker between the 2′-oxygen and 4′-carbon atoms of the ribose moiety, which thereby locks the sugar into a C3-endo conformation, hence the term “locked nucleic acid”. In technical applications involving the formation of duplexes with complementary target sequences by hybridization of oligonucleotides containing LNA monomers, this chemical modification confers nuclease resistance as well as higher affinity and higher specificity for the oligonucleotide target. LNA monomers are provided in the form of 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. By chemical synthesis, single chains consisting only of LNA nucleoside analog monomers ("all-LNA") can be synthesized.

[0008] 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, such as peptide nucleic acids (PNA), hexitol nucleic acids (HNA) and 2'-fluoro N3'-phosphoramidates, LNA exhibits 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 is composed of 7 LNA monomers (Nucleic Acids Research 38 (2010) 6729-6736).

[0009] In most cases, single-stranded hybrid LNA / DNA oligonucleotides (LNA / DNA and LNA / RNA, i.e. hybrid single strand) have been analyzed. There are fewer 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, thus affecting its biological function.

[0010] WO 2000 / 066604 and WO 2000 / 056746 disclose certain stereoisomers of LNA nucleoside monomers.

[0011] WO 1999 / 14226 suggests the use of oligonucleotides with LNA monomers in the construction of affinity pairs to link to a molecule of interest and a solid support. However, the technical problems of hybridization of complementary all-LNA single strands are also known in the art. The LNA-related 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=Thermodynamic analysis of hybridization of oligonucleotide analogs consisting only of LNAs is therefore largely empirical, and sequence prediction of complementary all-LNA oligomer hybridization pairs without a prior denaturation step (e.g. heating to remove intramolecular secondary structures prior to hybridization) seems impossible so far.

[0012] Predictions of the thermodynamic behavior of LNA-containing oligonucleotides were aided by a dedicated computer program cited by Tolstrup N et al. (Nucleic Acids Research 31 (2003) 3758-3762). However, the report explicitly mentions 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 seem to provide guidance in the design of complementary pairs of all-LNA oligonucleotides. The same conclusion 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).

[0013] In particular, this report demonstrates that complementary single-stranded oligonucleotides consisting only of LNA monomers are indeed unpredictable in their ability to form duplex molecules by Watson-Crick base pairing. Therefore, in order to provide technically suitable alternatives to biotin:(strept)avidin binding pairs in specific applications using such molecular recognition, technical means are needed to select and provide alternative binding pairs; for the purposes of this report, it is desired that such binding pairs consist of complementary single-stranded oligonucleotides comprising only LNA monomers, wherein

[0014] - The oligonucleotide pair must contain complementary sequences, and the complementary sequences must be able to form duplexes and Watson-Crick base pairing;

[0015] - 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 needs to be free of any secondary structures formed intermolecularly or intramolecularly, which would significantly reduce the ability of the respective oligonucleotide to align and form a duplex with its binding partner (i.e. the complementary oligonucleotide or the complementary sequence therein);

[0016] - Under the conditions of routine biochemical applications, the single-stranded pairs must be able to form duplexes of Watson-Crick paired oligonucleotides quickly enough while ensuring sufficient specificity for molecular recognition;

[0017] - 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.

[0018] Therefore, one overall goal of this report is to identify and provide binding pairs of single-stranded all-LNA oligonucleotides that are capable of hybridization without a prior denaturation step, thereby being able to form duplex molecules by Watson-Crick base pairing under suitable assay conditions as binding pairs in analyte detection assays. In other words, binding pairs that are capable of forming duplexes under non-denaturing conditions, more specifically, binding pairs that form duplexes under conditions compatible with the function of analyte-specific receptors in analyte detection assays (such as but not limited to immunoassays) are sought. Importantly, single-stranded all-LNA oligonucleotides that can be stored under ambient conditions or even refrigerated without forming intermolecular or intramolecular secondary structures that can inhibit hybridization and duplex formation of complementary oligonucleotides are sought. In addition, single-stranded all-LNA oligonucleotides that can hybridize to each other under assay conditions (e.g., in aqueous solutions at ambient temperatures such as room temperature) without prior denaturation are sought. The absence of denaturation specifically refers to the removal of any intermittent steps of intermolecular or intramolecular secondary structures that can inhibit hybridization and duplex formation of complementary oligonucleotides used as binding pairs in analyte detection assays. Summary of the invention

[0019] The present disclosure unexpectedly provides, in a first aspect related to all other aspects and embodiments disclosed herein, pairs 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 with each other in aqueous solution comprising 5 to 15 consecutive base pairs 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 antiparallel duplexes 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 antiparallel duplexes containing 5 to 7 consecutive base pairs with each other in aqueous solution in the absence of denaturing conditions before or during duplex formation.

[0020] 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 following steps:

[0021] (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;

[0022] (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 complementary to the first nucleobase sequence in an antiparallel orientation, and the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other is predicted by complementarity, the predicted duplex comprising or consisting of 5 to 15 consecutive base pairs, wherein the two bases of each base pair are bound to each other by hydrogen bonds;

[0023] (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 specifically at a temperature of 0° C. to 40° C.;

[0024] (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 duplexes;

[0025] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0026] (f) 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;

[0027] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0028] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0029] 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,

[0030] 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,

[0031] 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.,

[0032] 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

[0033] Figure 1 Schematic diagram describing the screening method for 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.

[0034] The first (1) and second (2) single stranded oligonucleotides are in contact with each other.

[0035] A: Neither oligonucleotide features intramolecular or inter-molecular secondary structures and both are capable of unhindered molecular recognition of their respective partners; therefore, duplexes are the major abundant product and single strands are either undetectable or present in negligible amounts (expected result).

[0036] B: At least one of the two oligonucleotides is characterized by intermolecular or intramolecular secondary structure; therefore, the number of duplexes is low and most single strands remain; duplexes are formed, but at a reduced rate (not the expected result).

[0037] C: Both oligonucleotides are characterized by intermolecular or intramolecular secondary structures; therefore, even after long incubation, duplexes are not formed, or a trace amount of duplexes is formed, and single strands still exist (not an expected result).

[0038] 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.

[0039] 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.

[0040] Figure 4 HPLC analysis of 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.

[0041] Figure 5HPLC analysis of mixed LNA 1 and LNA 2 after heat denaturation before injection (Example 2); positive control: duplex formation; minor peak indicated a retention time of 1.710 min, major peak indicated a retention time of 6.656 min.

[0042] 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.

[0043] 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.

[0044] 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); first peak indicated a retention time of 3.387 min, main peak indicated a retention time of 7.157 min.

[0045] Fig. 9 HPLC analysis of mixed LNA 3 and LNA 4, injected after 50 min (Example 2); duplex formation was slow (ratio = 0.05); the first peak indicated a retention time of 3.365 min, the second peak indicated a retention time of 6.871 min, and the third peak indicated a retention time of 7.148 min.

[0046] Fig.10 HPLC analysis of mixed LNA 3 and LNA 4 after heat denaturation before injection (Example 2); positive control: duplex formation; major peak indicated retention time 6.882 min.

[0047] Fig.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.

[0048] Fig.12 HPLC analysis of single-chain LNA 5'-gttggt-3' (SEQ ID NO: 16); the minor peak indicated a retention time of 1.496, and the major peak indicated a retention time of 1.865 min.

[0049] Fig.13HPLC analysis of mixed LNA 5'-Bi-Heg-accaac-3' (SEQ ID NO: 20) and 5'-gttggt-3' (SEQ ID NO: 16) (mixed at rt 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, some residual single-stranded LNA 5'-Bi-Heg-accaac-3' (SEQ ID NO: 20) could be detected.

[0050] Fig.14 HPLC analysis of mixed LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO: 20) and 5′-gttggt-3′ (SEQ ID NO: 16) (stored and mixed at +4°C to +6°C and injected immediately); the main peak indicated a retention time of 6.588 min, the other 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) could be detected.

[0051] Fig.15 HPLC analysis of mixed LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO: 20) and 5′-gttggt-3′ (SEQ ID NO: 16) (storage and mixing (ice bath) at 0°C and immediate 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.

[0052] Fig.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) could be detected.

[0053] Fig.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.

[0054] Fig.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.

[0055] Fig.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.

[0056] Fig. 20 HPLC analysis of 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); first peak indicated retention time 3.594 min, corresponding peak indicated second retention time 6.580 min. Identification of sequence pairs with slow duplex formation. Duplex formation is slow (ratio < 0.5).

[0057] Fig.21HPLC analysis of mixed LNA 5′-Bi-Heg-cgtcaggcagttcag-3′ (SEQ ID NO: 47) / 5′-ctgaactgcctgacg-3′ (SEQ ID NO: 48) (stored and mixed (ice bath) at 0°C and injected immediately); the first peak indicated a retention time of 3.541 min, the corresponding peak indicated a second retention time of 6.853 min. Duplex formation was slow (ratio < 0.5).

[0058] Fig. 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); first peak indicated retention time 3.516 min, corresponding peak indicated second retention time 6.848 min. Duplex formation was slow (ratio < 0.5).

[0059] Fig.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.

[0060] Fig.24 A: Schematic diagram of the Biacore sensor used in Example 4.

[0061] B: The components are as shown in A.

[0062] 1: Sensor surface

[0063] 2: Streptavidin attached to the sensor surface

[0064] 3. Biotin

[0065] 4: Linker molecule covalently links the first ss-oligonucleotide to biotin.

[0066] 5: First ss-oligonucleotide

[0067] 6: Second ss-oligonucleotide

[0068] a: Depicted is the situation when the second ss-oligonucleotide contacts the sensor to which the first ss-oligonucleotide is attached

[0069] 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.

[0070] Figure 25-50 Results of Example 4.

[0071] Fig.51 Schematic diagram of the Biacore experiment in Example 5.

[0072] Figures 52-53 Results of Example 5.

[0073] Fig.54 Schematic diagram of the Biacore experiment in Example 6.

[0074] Figure 55: Binding of LNA structure to Bi-LNA structure; Binding constant at 25°C

[0075] Figure 56 Binding of LNA structure to Bi-LNA structure; Binding constant at 37°C DETAILED DESCRIPTION

[0076] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0077] The articles "a" and "an" may 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 plural, i.e., a single member of the pair or both members together.

[0078] 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 features, matters, integers, steps, operations, elements and / or components stated, but do not exclude 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 stated features, etc.

[0079] As used herein, the terms "comprises / comprising / contains / containing / includes / including", "has / having" or any other variation 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 includes 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 variation thereof specifies 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.

[0080] 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).

[0081] As used herein, "substantially," "relatively," "generally," "typically," "approximately," and "approximately" are relative modifiers intended to indicate allowable variations with respect to the features so modified. They are not intended to limit the absolute values ​​or properties to which they are modified, but rather to approach or approximate such physical or functional properties. If not otherwise specified, it is understood that the term "about" in combination with a numerical value n ("about n") represents 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, the value of n is most preferred.

[0082] 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" do not necessarily refer to the same embodiment; however, these embodiments are not mutually exclusive unless otherwise stated and unless otherwise apparent to one skilled in the art. Thus, the technology in accordance with the present disclosure in all its aspects may include any kind of combination and / or integration of the embodiments described herein.

[0083] 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.

[0084] The surface of the solid phases as 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.

[0085] 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.

[0086] A particle is an embodiment of a solid phase. As used herein, a "particle" refers to a small, localized object to which physical properties such as volume, mass, or average size can be attributed. Microparticles can therefore be symmetrical, spherical, substantially spherical or spherical, or irregular, asymmetric shapes or forms. The size of the particles contemplated by the present invention can vary. In one embodiment, spherical shapes are used, such as microparticles with diameters in the nanometer and micrometer range. In one embodiment, the microparticles used in the method according to the present disclosure have a diameter of 50 nanometers to 20 microns. In another embodiment, the diameter of the microparticles is between 100nm and 10μm. In one embodiment, the microparticles used in the method according to the present disclosure have a diameter of 200nm to 5μm or 750nm to 5μm.

[0087] 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 inorganic or organic materials or consist of or consist essentially of. Typically, they may comprise or consist of metals or metal alloys, or organic materials or consist essentially of, or comprise carbohydrate elements or consist essentially of. Examples of materials envisioned for microparticles include agarose, polystyrene, latex, polyvinyl alcohol, silicon dioxide and ferromagnetic metals, alloys or composites. In one embodiment, the microparticle is a magnetic or ferromagnetic metal, alloy or composition. In another embodiment, the material may have specific properties, for example, be hydrophobic or hydrophilic. Such microparticles are usually dispersed in aqueous solution and retain a small negative surface charge, thereby keeping the microparticles separated and avoiding nonspecific aggregation.

[0088] In one embodiment of the invention, the microparticles are paramagnetic microparticles and the separation of such microparticles in the measurement method according to the present disclosure is facilitated by magnetic forces. Magnetic forces are 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, for example, washed. The microparticles used in the method according to the invention are coated with the first member of the specific binding pair.

[0089] 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). Therefore, the term "analyte-specific receptor" as referred to herein includes an analyte-specific reactant that is capable of binding or complexing with an analyte. This includes, but is not limited to, antibodies, specifically monoclonal antibodies or antibody fragments. Such a receptor can act as a trap for the analyte, for example to immobilize the analyte. The epitope recognized by the binding antibody is then 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.

[0090] "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 or a hepatitis B virus or an HIV antigen.

[0091] In the context of the present disclosure, the term "antibody" relates to complete 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-regions. 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 complete antibodies as well as 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.

[0092] "Detectable labels" include moieties that are detectable or can become detectable. The skilled artisan knows that labels are compounds or compositions that are capable of providing a detectable signal in conjunction with a physical activation (or excitation) or chemical agent, and can be modified to reduce or increase a specific signal.

[0093] 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 terpyridine ruthenium (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. Commonly used 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 a diagnostic procedure (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 an ECL co-reactant). 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.

[0094] 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 species of partner, or a first and a second member of the pair, or a first and a second species. Under non-denaturing conditions, the partners are able to specifically recognize the other species of partner at the molecular level. After recognition, the partners of the binding pair form a stable non-covalent intermolecular bond connecting 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 a bond 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.

[0095] In this document, the punctuation mark (":") between the first and second members of a binding pair may be used to indicate a specific connection of the first and second members of the binding pair or the ability to form such a specific connection, thus 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. Therefore, depending on the context, "Member 1: Member 2" may mean that Member 1 and Member 2 can form a binding pair, and that Member 1 is able to specifically recognize and bind to Member 2; or, depending on the context, "Member 1: Member 2" may mean that Member 1 and Member 2 are a connected 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 connected to another entity, e.g., form part of another entity. For example, a "(strept)avidin: biotin" (="biotin: (strept)avidin") binding pair is fully 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 two members of this exemplary binding pair.

[0096] Single-stranded "nucleic acids" are polymers composed of nucleotide monomer units. Each nucleotide that makes up a nucleic acid is composed of a phosphate, a sugar, and a nucleobase. The nucleotide chains in nucleic acids are connected by 3', 5' phosphodiester bonds. This means that the 5'-phosphate group of one nucleotide is esterified by the 3'-hydroxyl group of the adjacent nucleotide.

[0097] Single-stranded "oligonucleotide" is a short nucleic acid, usually composed of up to about 15 nucleotide monomers, which are connected by a phosphodiester bond between the 3' carbon atom of a sugar molecule and the 5' carbon atom of another sugar molecule. The monomers contained in the oligonucleotide (in a general sense) can not only be 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 a methylene bridge connecting 2'-O atoms and 4'-C atoms. For the purposes of this disclosure, the term nucleotide includes natural and non-natural nucleotides as monomers in oligonucleotides. Therefore, an oligonucleotide according to this definition can be completely composed of natural or non-natural monomers, or it can be composed of a mixture thereof. In addition, it should be understood that different classes of non-natural monomers (e.g., PNA, D-LNA, L-LNA, homologous DNA (containing hexose), HNA (containing hexitol, hexitol nucleic acid), L-DNA, etc.) can be included in an oligonucleotide if not otherwise specified.

[0098] Non-naturally occurring monomers can include nucleobases, which themselves can be naturally occurring nucleobases or their non-naturally occurring analogs."Nucleobases" are nitrogen-containing 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 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.

[0099] Nucleoside is a component of a nucleotide that further includes a phosphate moiety or a derivative or functional analog thereof. Nucleotide is the monomer unit of single-stranded nucleic acid. In double-stranded nucleic acids such as DNA, the nucleobases are paired. The two complementary nucleobases are connected by hydrogen bonds.

[0100] 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, wherein 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, thiopurine, 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine, aminophylline, N 6 -Benzyl adenine, N 6-benzoyl adenine, 6-benzyloxypurine, 8-bromotheophylline, 8-bromo-3-methylxanthine, 8-bromo-7-(2-butyn-1-yl)-3-methylxanthine, 6-chloropurine, 8-chlorotheophylline, 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.

[0101] Complementary single-stranded oligonucleotides or polynucleotides can form double-stranded ("duplex") nucleic acids. Duplex formation is also referred to as the term "hybridization" and 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.) by sequence-specific interaction of two at least partially complementary single-stranded nucleic acids as an example of a binding pair. Association or separation (denaturation) of complementary single-stranded nucleic acids and renaturation of duplexes are often used to describe hybridization between fully complementary chains.

[0102] Hybridization in aqueous solution, also known as "annealing", is an integral part of the present disclosure. With respect to duplex molecules of hybridized oligonucleotides or polynucleotides (including analogs thereof), the skilled artisan understands that melting temperature, hybridization rate, and dissociation rate and temperature are interrelated.

[0103] According to common sense, a "Watson-Crick base pair" is a single non-covalent crosslink in a double-stranded nucleic acid helix (= duplex), wherein each single strand of the duplex is an oligonucleotide. Thus, in an exemplary embodiment of a duplex, the two oligonucleotide chains are crosslinked by purine and pyrimidine base pairs protruding inward from the oligonucleotide backbone sugars and linked by hydrogen bonds with, for example, adenine paired with thymine and cytosine paired with guanine. In line with the above, the nucleobases may be naturally occurring nucleobases or analogs thereof, as long as the pair of nucleobases can interact complementarily to form a single non-covalent crosslink (duplex) in a double-stranded nucleic acid helix.

[0104] It is well known that secondary structural motifs in single-stranded nucleic acids often impair the intended hybridization reaction (e.g., 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 structures can be generated by internal folding of single-stranded molecules, driven by intramolecular interactions (e.g., hydrogen bonds or hydrophobic interactions). In the specific case of the first single-stranded oligonucleotide, a certain folded structure is thermodynamically favorable, where the folded structure then interferes with the unimpeded presentation of the nucleobase sequence to the complementary second oligonucleotide. Therefore, it is necessary to make efforts to predict and avoid such structures. Vice versa, the secondary structure of the targeted binding site may also impair hybridization. Therefore, it is necessary to evaluate the secondary structure of the two partners of the binding pair composed of oligonucleotides. Several challenges confound this goal, including imperfect empirical rules and prediction parameters, and the poor scalability of folding algorithms in terms of sequence length.

[0105] In addition, among members of the same oligonucleotide species, i.e., oligonucleotides sharing the same core base sequence, there may be one or more parts that can be partially complementary, thereby potentially causing intermolecular interactions through Watson-Crick base pairing of one or more core bases. Otherwise, there may also be one or more parts in the sequence that can cause intermolecular interactions through non-Watson-Crick (e.g., Hoogsteen) base pairing or other forms of intermolecular interactions. In the case of intramolecular folding (see above), the intramolecular interactions between members of the first single-stranded oligonucleotide can lead to a thermodynamically favorable structure, wherein such a structure then interferes with the unhindered presentation of the core base sequence to the complementary second oligonucleotide.

[0106] If unwanted intermolecular or intramolecular structures occur, 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 usually preceded by a heating step, whereby the oligonucleotides are denatured in the heating step, i.e., the intramolecular secondary structure is destroyed. After the heating step, the temperature is usually gradually and controlled to reduce, aiming 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, triphosphate nucleosides, salts, buffers, and thermostable polymerases. However, other processes in which oligonucleotide annealing can play a role prohibit heating or other types of denaturation treatments, because these processes may include denaturation-sensitive components that may be irreversibly degraded. This is particularly true (but not exclusive) for analyte detection assays, in which protein analyte-specific receptors (e.g., antibodies) play a key functional role. Therefore, the disclosure and the report of surprising findings that specifically involve the use of heat in technical settings, especially incubation at temperatures above 68° C. (e.g., Childs JLPNAS 99 (2002) 11091-11096), are impossible. For practical reasons, in most assays, such as but not limited to immunoassays, temperatures above 40° C. are undesirable. That is, for practical applications of binding pairs consisting of complementary oligonucleotides, particularly desirable conditions are between 0° C. and 40° C. Under these conditions, any technical application must not be affected by intra- or intermolecular structure, as may be the case with isolated binding partners of an oligonucleotide binding pair.

[0107] Other options for the denaturation of nucleic acids, including oligonucleotides, are known to the skilled person from reports on DNA. Several methods for the denaturation of DNA are known in the art, including heating, incubation under alkaline conditions equivalent to more than 0.01 mol / L NaOH aqueous solution (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, incubation in the presence of ultrasonic treatment. 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 the detection of analytes, in which protein analyte-specific receptors such as antibodies play a key functional role.

[0108] Therefore, "denaturing conditions" on the one hand may be able to counteract unwanted intramolecular and intermolecular structures in the all-LNA ss-oligonucleotide species in aqueous solution, but on the other hand should be avoided in any aspect and embodiment of the technical method proposed in this report, which is 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 that can destroy the intramolecular and intermolecular structure, incubating in the presence of formamide at a concentration that can destroy the intramolecular and intermolecular structure, incubating in the presence of a chaotropic compound at a concentration that can destroy the intramolecular and intermolecular structure, and mixtures thereof. For the purpose 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 treatment, 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 a chaotropic compound at a concentration capable of destroying intra- and intermolecular structures, and mixtures thereof.

[0109] Importantly, under non-denaturing 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 non-denaturing conditions to inhibit or prevent the desired intramolecular bonding of the two different species of a binding pair.

[0110] All-LNA ss-oligonucleotides comprising 5 or more monomers have characteristics that cannot be reliably predicted by the tools available to the skilled person, taking into account non-denaturing conditions and specifically excluding any application of the denaturing treatment detailed herein. For practical reasons, the present study was limited to ss-oligonucleotides consisting of up to 15 LNA monomers, in order to identify those ss-oligonucleotides that are able to form a hybrid duplex from two separate single-stranded species in the absence of denaturing conditions. That is, each member of the binding pair must be fully free of any inter- or intra-molecular structure. The present report shows that for complementary all-LNA oligonucleotides this situation cannot be taken for granted.

[0111] 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 consisting of 5 to 15 LNA monomers, which are 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 model of uncertain reliability, such pairs can be unexpectedly identified and provided by the method of the second aspect as follows. To the best of the knowledge of the authors of this report, such a method has not been previously demonstrated or even suggested.

[0112] 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 following steps:

[0113] (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;

[0114] (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 complementary to the first nucleobase sequence in an antiparallel orientation, and the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other is predicted by complementarity, the predicted duplex comprising or consisting of 5 to 15 consecutive base pairs, wherein the two bases of each base pair are bound to each other by hydrogen bonds;

[0115] (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 specifically at a temperature of 0° C. to 40° C.;

[0116] (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 duplexes;

[0117] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0118] (f) 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;

[0119] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0120] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0121] In one embodiment, specifically, steps (c) and (d) are performed in the absence of conditions designated as "denaturing conditions" as described above.

[0122] 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 following steps:

[0123] (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;

[0124] (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 complementary to the first nucleobase sequence in an antiparallel orientation, and the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other is predicted by complementarity, the predicted duplex comprising or consisting of 5 to 7 consecutive base pairs, wherein the two bases of each base pair are bound to each other by hydrogen bonds;

[0125] (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 specifically at a temperature of 0° C. to 40° C.;

[0126] (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 duplexes;

[0127] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0128] (f) 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;

[0129] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0130] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0131] In one embodiment, specifically, steps (c) and (d) are performed in the absence of conditions designated as "denaturing conditions" as described above.

[0132] Each single-stranded oligonucleotide is composed of monomers, wherein each monomer is a ribonucleoside analog, wherein in the ribose moiety of the ribonucleoside analog, a methylene group is linked to the 2'-oxygen and 4'-carbon atoms, thereby locking the ribose into a C3-endo conformation. The all-LNA ss-oligonucleotides according to the present disclosure can be synthesized using standard techniques using protected phosphoramidite chemistry of building blocks such as single LNA nucleosides.

[0133] 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, thiopurine, 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine, aminophylline, N 6 -Benzyl adenine, N 6 -benzoyl adenine, 6-benzyloxypurine, 8-bromotheophylline, 8-bromo-3-methylxanthine, 8-bromo-7-(2-butyn-1-yl)-3-methylxanthine, 6-chloropurine, 8-chlorotheophylline, 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 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.

[0134] 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 as 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 of the present disclosure, the first ss-oligonucleotide consists of 9 monomers.

[0135] Initial experiments used these oligonucleotide sizes to provide the following combination: first, high binding specificity of the binding partner, second, a favorable speed at which the two partners of the binding pair hybridize and form a duplex, and third, the formation of a stable duplex with essentially no detectable tendency to dissociate into single strands again. It was surprisingly found that even binding pairs of all-LNA ss-oligonucleotides with complementary nucleobase sequences consisting of 7, 6 or even 5 consecutive LNA monomers can meet the criteria of sufficient specificity, pairing speed and duplex stability. Even more surprisingly, these criteria are met under specific environmental conditions, which is a prerequisite for using binding pairs of all-LNA ss-oligonucleotides as a molecular recognition means 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 a binding pair of single-stranded all-LNA oligonucleotides capable of forming an antiparallel duplex with 5, 6 or 7 consecutive base pairs in aqueous solution at a temperature of 0°C to 40°C, which method is a described specific embodiment.

[0136] Another specific embodiment related to all other aspects and embodiments disclosed herein is a pair of isolated ss-oligonucleotides, each 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 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 binding pair being capable of forming all-LNA duplexes in aqueous solution and 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 denaturation treatment part of the incubation after the members of the binding pair are in contact with each other in aqueous solution.

[0137] 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.

[0138] In a specific embodiment related to all aspects of this report, the member of the binding pair is an all-LNAss-oligonucleotide, wherein the all-LNA ss-oligonucleotide is a fragment of a larger all-LNA ss-oligonucleotide, the larger ss-oligonucleotide is a member, partner or species of the binding pair, which is 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 continuous nucleobase subsequence of the larger fragment, wherein the fragment comprises 5 to 7 LNA monomers. Therefore, the binding pair in one embodiment consists of a first and a second all-LNAss-oligonucleotide, each comprising 5, 6 or 7 LNA monomers, the nucleobase sequences of the first and second all-LNAss-oligonucleotides are complementary, thereby being able to form an antiparallel duplex with 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.

[0139] Thus, the present disclosure, in a third aspect, particularly relating to the second aspect but also relating 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 following steps:

[0140] (a) providing a first single-stranded (=ss-) oligonucleotide consisting of 8 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0141] (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 complementary to the first nucleobase sequence in an antiparallel orientation, and the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other is predicted by complementarity, the predicted duplex comprising or consisting of 8 to 15 consecutive base pairs, wherein the two bases of each base pair are bound to each other by hydrogen bonds;

[0142] (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 specifically at a temperature of 0° C. to 40° C.;

[0143] (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 duplexes;

[0144] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0145] (f) 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;

[0146] (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;

[0147] (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, a fragment consisting of 5 to 7 LNA monomers;

[0148] (i) synthesizing the ss-oligonucleotide fragment of step (g) and the ss-oligonucleotide fragment of step (h), respectively;

[0149] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0150] In one embodiment, specifically, steps (c) and (d) are performed in the absence of conditions designated as "denaturing conditions" as described above.

[0151] The property of the selected fragments, i.e. their ability to form antiparallel duplexes in aqueous solution at a temperature between 0° C. and 40° C., can be easily verified. Therefore, in a particular embodiment, the method comprises the following additional steps:

[0152] (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 specifically at a temperature of 0° C. to 40° C.;

[0153] (l) 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;

[0154] (m) If a duplex is detected in step (1) but no ss-oligonucleotide fragment is detectable, then the binding pair is selected.

[0155] 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, consisting of LNA monomers and capable of forming duplexes by the second and / or third aspects set out above, in particular under non-denaturing conditions. Thus, each such ss-oligonucleotide provided and optionally stored under non-denaturing conditions is capable of hybridizing with its binding partner under non-denaturing conditions and retaining this quality under non-denaturing conditions.

[0156] 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.

[0157] Denaturing conditions do not include conditions that must be performed in assays that use protein analyte-specific receptors to detect target analytes in order to maintain the desired capabilities and functions of these compounds. That is, under denaturing conditions, the desired capabilities and functions of these compounds may be lost. At the same time, these non-denaturing conditions do not include any of the following: temperatures above 68°C, application of ultrasonic treatment, incubation under alkaline conditions equivalent to an aqueous solution of more than 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, is to the extent that the intra- and intermolecular structures of the single all-LNA ss-oligonucleotides can be destroyed, and wherein the intra- and intermolecular structures will be able to prevent hybridization and duplex formation of the oligonucleotide with a complementary single all-LNA ss-oligonucleotide. 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 binding pair, i.e. when one member is in contact with the other.

[0158] The first and second ss-oligonucleotides do not necessarily have equal sizes, ie do not necessarily consist 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.

[0159] The skilled person knows that two oligonucleotides are antiparallel if they are parallel to each other but arranged in opposite directions. A specific example given is two complementary strands of a duplex extending in opposite directions to each other. Thus, each end of the duplex includes the 5' end of the first strand adjacent to / aligned with the 3' end of the opposite second strand. Similar to DNA and RNA, LNA exhibits Watson-Crick base pairing (Koshkin, AA et al. J Am Chem Soc 120 (1998) 13252-13260).

[0160] The specific Watson-Crick base pairing involving hydrogen bridges forming bases on complementary opposite strands is a well-known feature to 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.

[0161] In order to join the two members of the binding pair, one must be contacted with the other. After the contacting step, the two members can interact and form a duplex. As mentioned above, no denaturing conditions are required for this. 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 time interval of 20 minutes or less. That is, duplex formation is very fast, and to the extent that duplexes can be formed, the process is essentially completed 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 able to bind to each other under conditions comparable to biotin and (strept)avidin, with specific reference to molecular recognition and binding. In specific embodiments of all aspects and embodiments disclosed herein, the time interval for duplex formation (i.e., after the contacting step) is selected from 1s to 20min, 1s to 15min, 1s to 10min, 1s to 5min, 1s to 1min, 1s to 30s, 1s to 20s, 1s to 10s, and 1s to 5s. A highly desirable and advantageous time interval is selected between 1s to 10s and 1s to 5s.

[0162] Importantly, prior to step (c), the first ss-oligonucleotide and the second ss-oligonucleotide do not need to be denatured, but can be stored or kept under non-denaturing conditions, in particular 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 may interfere with the ability of the complementary all-LNA oligonucleotide to form a duplex. In one embodiment of all aspects and embodiments as disclosed herein, the first ss-oligonucleotide and the second ss-oligonucleotide are stored and / or maintained at a temperature of -80°C to 40°C, in particular 0°C to 40°C, more particularly at a temperature of 25°C to 37°C. The non-denaturing temperature of a single-stranded all-LNA oligonucleotide comprising 5 to 15 monomers is a temperature below 68°C, in particular below 40°C.

[0163] In a specific embodiment of all (particularly the second and third) aspects and embodiments as disclosed herein in step (c) and / or step (d), the temperature is below 68°C, more particularly the temperature is below 40°C, and even more particularly the temperature is 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 as disclosed herein, the temperature in steps (c) and (d) differs by no more than 5°C, or the two steps are performed at the same temperature. In an even more specific embodiment of all aspects and embodiments as 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 as disclosed herein, before step (c), the first ss-oligonucleotide and the second ss-oligonucleotide are stored and / or maintained at a temperature of -80°C to 40°C, particularly between 0°C and 40°C, more particularly between 25°C and 37°C.

[0164] 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.

[0165] In another 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.

[0166] The conditions described herein applied during the mixing (step (c)) and incubation (step (d)) steps are also applicable to the conditions for maintaining the separated ss-oligonucleotides. Therefore, 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.

[0167] The incubation of step (d) provides conditions for duplex formation, provided that 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 a 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; such as Biacore) and electrophoresis.

[0168] 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 inhibition is complete, only the presence of ss-oligonucleotides will be detectable after step (d) (incubating 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 detectable ss-oligonucleotides, and only duplexes can be detected.

[0169] 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 K d is the k 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 mutual affinity K of the binding partners selected by the method of the present invention is d <1x10 -15 M to >1x10 -5 M, the binding partner consists of a first and a second single-stranded oligonucleotide, each of which consists of 5 to 15 locked nucleic acid monomers.

[0170] In further embodiments, 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 of the present invention 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.

[0171] In yet another embodiment, the affinity 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.

[0172] It is noteworthy that the K d is substantially affected 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.

[0173] In order to exclude any interference of naturally occurring oligonucleotides or polynucleotides with the molecular recognition (i.e. duplex formation) of the binding pair consisting 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 make it impossible for the single-stranded oligonucleotide to form a duplex with naturally occurring oligonucleotides or polynucleotides, in particular, to form a Watson-Crick duplex with naturally occurring oligonucleotides or polynucleotides.

[0174] In one embodiment of all aspects and embodiments disclosed herein, the first and second ss-oligonucleotides (and any fragments thereof) consist of β-D-LNA monomers. That is, the first ss-oligonucleotide consists entirely of β-D-LNA monomers, and the second ss-oligonucleotide consists 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) consist of β-L-LNA monomers. That is, the first ss-oligonucleotide consists entirely of β-L-LNA monomers, and the second ss-oligonucleotide consists entirely of β-L-LNA monomers. The use of β-L-LNA has been shown to be advantageous because it does not interfere with the formation of duplexes in the presence of naturally occurring nucleic acids. In this regard, it should be noted that the current extensive technical experience with hybridization conditions for all-D-LNA and all-L-LNA oligonucleotide pairs is quite limited.

[0175] This report discloses pairs of isolated ss-oligonucleotides, each consisting of 5 to 15 LNA monomers, which 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 the selection and provision of such single-stranded oligonucleotides, whose monomers are LNA monomers. To the best of the knowledge of the authors of this report, the method described herein represents the first successful solution to overcome the limitations of algorithms known in the art in predicting the 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 to allow the two ss-oligonucleotides to pair in an antiparallel direction to be able to form a duplex.

[0176] 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 with 5 to 15 consecutive base pairs in aqueous solution at a temperature of 0° C. to 40° C., the method comprising the following steps:

[0177] (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;

[0178] (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 complementary to the first nucleobase sequence in an antiparallel orientation, and the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other is predicted by complementarity, the predicted duplex comprising or consisting of 5 to 15 consecutive base pairs, wherein the two bases of each base pair are bound to each other by hydrogen bonds;

[0179] (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 specifically at a temperature of 0° C. to 40° C.;

[0180] (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 duplexes;

[0181] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0182] (f) 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;

[0183] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0184] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0185] 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 with 5 to 7 consecutive base pairs in aqueous solution at a temperature of 0° C. to 40° C., the method comprising the following steps:

[0186] (a) providing a first single-stranded (=ss-) oligonucleotide consisting of 8 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0187] (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 complementary to the first nucleobase sequence in an antiparallel orientation, and the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other is predicted by complementarity, the predicted duplex comprising or consisting of 8 to 15 consecutive base pairs, wherein the two bases of each base pair are bound to each other by hydrogen bonds;

[0188] (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 specifically at a temperature of 0° C. to 40° C.;

[0189] (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 duplexes;

[0190] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0191] (f) 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;

[0192] (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;

[0193] (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, a fragment consisting of 5 to 7 LNA monomers;

[0194] (i) synthesizing the ss-oligonucleotide fragment of step (g) and the ss-oligonucleotide fragment of step (h), respectively;

[0195] 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 a temperature of 0°C to 40°C, can be easily verified. Therefore, in a specific embodiment, the method comprises the following additional steps:

[0196] (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 specifically at a temperature of 0° C. to 40° C.;

[0197] (l) 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:

[0198] (m) If a duplex is detected in step (1) but no ss-oligonucleotide fragment is detectable, then the binding pair is selected.

[0199] Each single-stranded all-LNA oligonucleotide may 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 the latter embodiment, all nucleobases in the ss-oligonucleotide are identical.

[0200] In one embodiment of all aspects and embodiments disclosed herein, for the nucleobases in each ss-oligonucleotide, the G+C content 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 a further 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, 5-methylcytosine. In a more specific embodiment, in the nucleobases of each ss-oligonucleotide, each cytosine is substituted by 5-methylcytosine.

[0201] 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:

[0202] 5'tgctcctg 3' (SEQ ID NO: 1) and 5'caggagca 3' (SEQ ID NO: 2),

[0203] 5'tgctcctgt 3' (SEQ ID NO: 9) and 5'acaggagca3' (SEQ ID NO: 10),

[0204] 5'gtgcgtct 3' (SEQ ID NO: 11) and 5'agacgcac 3' (SEQ ID NO: 12), and

[0205] 5'gttggtgt 3' (SEQ ID NO: 13) and 5'acaccaac 3' (SEQ ID NO: 14).

[0206] 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.

[0207] Several pairs of such single-stranded all-LNA oligonucleotides have been discovered and are reported herein as exemplary embodiments, i.e., non-limiting examples of pairs of isolated ss-oligonucleotides that can bind to each other by hybridization and duplex formation under non-denaturing conditions. Table 1 provides a non-limiting compilation thereof. It should be understood that the listed sequences represent all-LNA nucleosides, i.e., oligonucleotides containing only LNA monomers. The sequences are given in the conventional direction, i.e., from the 5' end to the 3' end.

[0208] Table 1

[0209]

[0210]

[0211] The binding pairs given in Table 1 reflect specific embodiments. It should be understood that any reference to the "first" and "second" members of a binding pair is arbitrary, as the "second" member can be equally regarded as 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 be changed. Therefore and exemplarily, 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 following group:

[0212] (SEQ ID NO: 1): (SEQ ID NO: 2),

[0213] (SEQ ID NO: 9): (SEQ ID NO: 10),

[0214] (SEQ ID NO: 11): (SEQ ID NO: 12),

[0215] (SEQ ID NO: 13): (SEQ ID NO: 14),

[0216] (SEQ ID NO: 9): (SEQ ID NO: 15),

[0217] (SEQ ID NO: 16): (SEQ ID NO: 20),

[0218] (SEQ ID NO: 21): (SEQ ID NO: 18),

[0219] (SEQ ID NO: 21): (SEQ ID NO: 20),

[0220] (SEQ ID NO: 21): (SEQ ID NO: 19),

[0221] (SEQ ID NO: 23): (SEQ ID NO: 17),

[0222] (SEQ ID NO:25): (SEQ ID NO:28).

[0223] 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.

[0224] 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.

[0225] In contrast, pairs of single-stranded all-LNA oligonucleotides have been found to be incapable or insufficient to form duplexes under non-denaturing conditions. Table 2 provides a non-limiting compilation thereof.

[0226] Table 2

[0227]

[0228] The pairing given by SEQ ID NO: 29 and SEQ ID NO: 39 exemplifies the case of minimal sequence complexity and in which one member of the binding pair comprises only 4 monomers. The binding properties characterizing this particular binding pair were found to be insufficient. One possible explanation could be that the 4-mer is too short and therefore can only provide insufficient intramolecular interactions with the corresponding single strand. This finding is in sharp contrast to the case in which the 4-mer is replaced by a 6-mer (SEQ ID NO: 27 combined with SEQ ID NO: 39).

[0229] With respect to the other pairs of all-LNA oligonucleotides presented in Table 2, it was found that there was always one binding partner comprising or consisting of the sequence "gcctgacg" (SEQ ID NO: 3). Thus, this particular sequence and its complementary sequence appear to have a negative impact on the ability of such all-LNA oligonucleotides to form duplexes under non-denaturing conditions. This particularly surprising finding may indeed guide the skilled person to select favorable single-stranded oligonucleotide pairs having 8 or more monomers. Therefore, 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 before or during duplex formation, wherein the pair of ss-oligonucleotides is obtainable and / or obtained by performing a method for selecting and providing a binding pair 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:

[0230] (a) providing a first single-stranded (=ss-) oligonucleotide 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, 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 5′gcctgacg 3′ (SEQ ID No: 3) and 5′cgtcaggc 3′ (SEQ ID NO: 4);

[0231] (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 complementary to the first nucleobase sequence in an antiparallel orientation, and the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other is predicted by complementarity, the predicted duplex comprising or consisting of 8 to 15 consecutive base pairs, wherein the two bases of each base pair are bound to each other by hydrogen bonds, wherein the second nucleobase sequence does not comprise or consist of a sequence selected from 5'gcctgacg3' (SEQ ID NO: 3) and 5'cgtcaggc3' (SEQ ID NO: 4);

[0232] (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 specifically at a temperature of 0° C. to 40° C.;

[0233] (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 duplexes;

[0234] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0235] (f) 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;

[0236] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0237] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0238] By the method of the second aspect or the method of the third aspect disclosed herein, and also 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'cgtcaggc3' (SEQ ID NO: 4) when the number of monomers of one or both of the oligonucleotides is 8 to 15, the present disclosure provides an antiparallel all-LNA duplex formed, obtainable and / or obtained from a non-denatured complementary single-stranded all-LNA oligonucleotide pair under non-denatured conditions at a preselected temperature of 25° C. to 40° C. Such a duplex in aqueous solution can be considered as the fourth aspect of this report. Each oligonucleotide chain in the duplex comprises LNA monomers, and the number of LNA monomers is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, more specifically the number is 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 of 8 to 15 or any one of 5 to 7.

[0239] 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 so that the first oligonucleotide and the second oligonucleotide can form 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.

[0240] 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 so that the first oligonucleotide and the second oligonucleotide can form 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 can be obtained by a method according to the second or third aspect of the present disclosure.

[0241] 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 so 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.

[0242] As a different embodiment, it was further surprisingly found that there are even longer complementary all-LNA ss-oligonucleotides that are able to form antiparallel duplexes under non-denaturing conditions. Therefore, the present disclosure provides a method for selecting and providing a binding pair of single-stranded all-LNA oligonucleotides that are able to form antiparallel duplexes with 16 to 20 consecutive base pairs in aqueous solution at a temperature of 0° C. to 40° C., the method comprising the following steps:

[0243] (a) providing a first single-stranded (=ss-) oligonucleotide consisting of 16 to 20 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence;

[0244] (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 complementary to the first nucleobase sequence in an antiparallel orientation, and the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other is predicted by complementarity, the predicted duplex comprising or consisting of 16 to 20 consecutive base pairs, wherein the two bases of each base pair are bound to each other by hydrogen bonds;

[0245] (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 specifically at a temperature of 0° C. to 40° C.;

[0246] (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 duplexes;

[0247] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0248] (f) 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;

[0249] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0250] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0251] In one embodiment, specifically, steps (c) and (d) are performed in the absence of conditions designated as "denaturing conditions" as described above.

[0252] 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:

[0253] (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;

[0254] (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 complementary to the first nucleobase sequence in an antiparallel orientation, and the ability of the first and second ss-oligonucleotides to form an antiparallel duplex with each other is predicted by complementarity, the predicted duplex comprising or consisting of 5 to 15 consecutive base pairs, wherein the two bases of each base pair are bound to each other by hydrogen bonds;

[0255] (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 specifically at a temperature of 0° C. to 40° C.;

[0256] (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 duplexes;

[0257] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0258] (f) 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;

[0259] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0260] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0261] In one embodiment, specifically, steps (c) and (d) are performed in the absence of conditions designated as "denaturing conditions" as described above.

[0262] 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.

[0263] Table 3

[0264]

[0265] The results show that none of the sequences shown in Table 3 contain the subsequence gcctgacg (SEQ ID NO: 3) or its complementary sequence, discussed further above.

[0266] 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 chambers, cuvettes, membranes, scaffold molecules, quartz crystals, thin films, filter papers, 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 a peptide, a polypeptide, an oligonucleotide, a polynucleotide, a sugar, a glycan, a hapten, and a dye. 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 a receptor-based assay (e.g., but not limited to, an immunoassay).

[0267] In general terms, an immunoassay provides one or more receptors that are capable of specifically binding to a target analyte. Such receptors may 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.

[0268] Therefore, the 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 by an embodiment according to any of the second or third aspects of this report.

[0269] In a receptor-based assay, the analyte binds to an analyte-specific receptor. The receptor with the bound analyte can be fixed to a solid phase by a duplex formed by a pair of single-stranded all-LNA oligonucleotides. Thus, a complex is formed as a result, which comprises a solid phase, a duplex, a receptor and an analyte. In a further step, the fixed analyte can be detected, for example using an additional analyte-specific binding agent, which itself is labeled or can be detected by other means for its presence. In a competitive assay format, 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.

[0270] 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, a plurality of 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.

[0271] About detecting the target analyte present in the liquid sample or thought to be present in the liquid sample, in the basic step, the analyte is specifically bound.Specific binding means that the analyte-specific receptor exists or is added, wherein the receptor has binding affinity and binding specificity to the analyte, and the binding affinity and binding specificity are high for the target analyte, and low or non-existent for other molecules also present in the sample. In a specific embodiment (and exemplifying many existing assays), a compound containing a receptor that can specifically bind to the analyte is added to the sample. Importantly, the sample and the mixture of the compound containing the receptor must provide conditions that allow the receptor to interact specifically with the target analyte in the sample. This includes that in the mixture, the conditions must allow the actual binding of the receptor to the analyte, and it is expected that the receptor is stabilized with the bound target analyte. At the same time, it is expected that the mixture of the sample and the compound does not promote or stabilize the non-specific binding of other molecules to the receptor or to the entire compound containing the receptor.

[0272] 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 of the sample. Immobilization requires a stationary phase to which the target analyte will attach. Once immobilized, the analyte can be separated from the mixture by phase separation. The analyte is then separated (i.e. purified) from the mixture and then detected.

[0273] Considering receptor based assays and immobilization steps, 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.

[0274] 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.

[0275] 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, the analyte-specific receptor being linked to a first member of a pair of isolated ss-oligonucleotides of the first aspect described herein, and with a solid phase being linked to 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, thereby determining 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.

[0276] Specific embodiments of "sandwich" immunoassays can be used for analytes with multiple recognition epitopes (i.e., more than one recognition epitope). Therefore, sandwich assays require 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 fixed to 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 called 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.

[0277] 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, a homogeneous assay generates an analyte-dependent detectable signal by a one-step incubation and does not require a washing step.

[0278] 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 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. In the case where the detection complex is fixed on the stationary phase, the amount of the label detectable on the stationary phase corresponds to the amount of the 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.

[0279] Any washing steps necessary in heterogeneous immunoassays require that the non-covalent connection of the first binding partner and the second binding partner must be sufficiently stable. However, the degree of desired stability of the connection depends on the intensity of the washing steps to be applied. Importantly and unexpectedly, the binding pairs demonstrated herein are very suitable for facilitating the immobilization step in the immunoassay. That is, in the immunoassay, the first binding partner of the binding pair of the full LNA oligonucleotide attached to the solid phase and the second binding partner of the binding pair bound to the analyte-specific capture receptor are very suitable for facilitating 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.

[0280] 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 analog of an artificially produced label that is capable of cross-reacting with the analyte-specific receptor. In the assay, the analyte and the analog compete for binding to a fixed or becoming fixed capture receptor. After the binding step, the higher the amount of fixed label, the less the amount of unlabeled analyte that can compete for the capture receptor. The fixed label is determined after a 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.

[0281] In all aspects and embodiments disclosed herein, the binding forces in the duplex formed by the pair of all-LNA oligonucleotides will exceed any binding forces that bind the analyte and any analyte-specific (capture and / or detection) receptors together in an assay for analyte detection (e.g., but not limited to, an immunoassay). In cases where the binding forces in the duplex need to be fine-tuned, the binding pairs can be selected so that Watson-Crick pairing regions of different lengths and / or with different base pair compositions can be provided. 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 specific technical needs.

[0282] Binding forces 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 that are 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.

[0283] However, an eighth aspect related 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 aspect or the 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.

[0284] 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 isolated ss-oligonucleotides 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, and the kit further comprises in a second container a second component to which is linked a second member of the pair.

[0285] 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 linked to first and second members of a complementary ss-oligonucleotide pair, respectively, capable of forming a duplex under non-denaturing conditions. Those skilled in the art are well aware of the numerous applications beyond the analyte detection assays described in more detail herein, but also extend to, for example, in situ analysis of target antigens in tissue samples.

[0286] 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.

[0287] Specific aspects and embodiments include the following more formal bulleted list. This numbered bulleted list forms part of the original disclosure of this report.

[0288] 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

[0289] (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;

[0290] (b) providing a second ss-oligonucleotide consisting of 5 to 15 LNA monomers, said second ss-oligonucleotide comprising at least the same number of monomers as said first ss-oligonucleotide, each monomer of said second ss-oligonucleotide comprising a nucleobase, the nucleobases of said second ss-oligonucleotide forming a second nucleobase sequence, said second nucleobase sequence comprising or consisting of a nucleobase sequence complementary to said first nucleobase sequence in an antiparallel orientation and predicting the ability of said first and second ss-oligonucleotides to form an antiparallel duplex with each other, said 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;

[0291] (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 specifically at a temperature of 0° C. to 40° C.;

[0292] (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 duplexes;

[0293] (e) detecting and quantifying ss-oligonucleotides and duplex oligonucleotides in the mixture obtained in step (d); followed by

[0294] (f) 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;

[0295] (g) optionally synthesizing the first and second ss-oligonucleotides of the binding pair selected in step (f), respectively;

[0296] Thereby, binding pairs of the single-stranded all-LNA oligonucleotides are selected and provided.

[0297] 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.

[0298] 3. The method according to any one of items 1 and 2, wherein when the monomer number 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.

[0299] 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.

[0300] 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.

[0301] 6. The method 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.

[0302] 7. A 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.

[0303] 8. A method according to item 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 an aqueous solution.

[0304] 9. A method according to item 7, wherein prior to step (c), each ss-oligonucleotide of any of steps (a) and (b) is maintained in an aqueous solution in the absence of a denaturant compound capable of reducing the melting temperature of a DNA duplex having a length of 20 base pairs and a G+C content of 50% by at least 15°C, specifically in the absence of either formamide and dimethyl sulfoxide.

[0305] 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.

[0306] 11. A method 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.

[0307] 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.

[0308] 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.

[0309] 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-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, thiopurine, 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine, aminophylline, N 6 -Benzyl adenine, N 6 -benzoyl adenine, 6-benzyloxypurine, 8-bromotheophylline, 8-bromo-3-methylxanthine, 8-bromo-7-(2-butyn-1-yl)-3-methylxanthine, 6-chloropurine, 8-chlorotheophylline, 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.

[0310] 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.

[0311] 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.

[0312] 17. A method according to item 16, wherein the incubation mixture of step (d) is subjected to column chromatography and / or electrophoresis.

[0313] 18. 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-D-LNA monomer.

[0314] 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.

[0315] 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.

[0316] 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).

[0317] 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.

[0318] 23. A pair of isolated complementary ss-oligonucleotides according to any one of items 20 to 22, wherein said pair of ss-oligonucleotides is obtained by performing a method according to any one of items 1 to 19.

[0319] 24. A pair of isolated complementary ss-oligonucleotides, wherein the pair is selected from the group consisting of

[0320] (SEQ ID NO: 1): (SEQ ID NO: 2),

[0321] (SEQ ID NO: 9): (SEQ ID NO: 10),

[0322] (SEQ ID NO: 11): (SEQ ID NO: 12),

[0323] (SEQ ID NO: 13): (SEQ ID NO: 14),

[0324] (SEQ ID NO: 9): (SEQ ID NO: 15),

[0325] (SEQ ID NO: 16): (SEQ ID NO: 20),

[0326] (SEQ ID NO: 21): (SEQ ID NO: 18),

[0327] (SEQ ID NO: 21): (SEQ ID NO: 20),

[0328] (SEQ ID NO: 21): (SEQ ID NO: 19),

[0329] (SEQ ID NO: 23): (SEQ ID NO: 17),

[0330] (SEQ ID NO:25): (SEQ ID NO:28).

[0331] 25. A pair of isolated complementary ss-oligonucleotides according to item 24, wherein the pair is selected from the group consisting of

[0332] (SEQ ID NO: 1): (SEQ ID NO: 2),

[0333] (SEQ ID NO: 28): (SEQ ID NO: 24),

[0334] (SEQ ID NO: 16): (SEQ ID NO: 20).

[0335] 26. A pair of isolated complementary ss-oligonucleotides according to any one of items 20 to 25, wherein a first ss-oligonucleotide of the pair is linked to a first target and a second ss-oligonucleotide is linked to a second target.

[0336] 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.

[0337] 28. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 and 27, wherein the targets are independently selected from the group consisting of a solid phase, a biomolecule and a chemically synthesized compound.

[0338] 29. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 to 28, wherein the target is independently selected from the group consisting of: amino acids or their analogs, peptides, polypeptides, proteins, nucleobases, nucleosides, oligonucleotides, nucleic acids, lipids and analyte-specific receptors, which include antibodies, antibody derivatives and antibody fragments.

[0339] 30. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 to 28, wherein the targets are independently selected from the group consisting of peptides, polypeptides, proteins, steroid or non-steroid hormones, haptens and conjugates thereof.

[0340] 31. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 to 28, wherein the target comprises a conjugate composed of a plurality of different molecules, wherein the different molecules are selected from the group consisting of peptides, polypeptides, proteins, steroid or non-steroid hormones, haptens, nucleobases, nucleosides, oligonucleotides, nucleic acids, lipids, analyte-specific receptors, analytes, cross-linking agents, and mixtures thereof.

[0341] 32. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 to 28, wherein the target comprises a solid phase.

[0342] 33. A pair of isolated complementary ss-oligonucleotides according to any one of items 26 to 28, wherein the target comprises a detectable label.

[0343] 34. A method for forming an antiparallel full LNA duplex in the absence of denaturing conditions, the method comprising the following steps

[0344] (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;

[0345] (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;

[0346] Thereby an antiparallel all-LNA duplex is formed.

[0347] 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.

[0348] 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 a first member of the pair and the second component is linked to a second member of the pair.

[0349] 37. Use of a pair of single-stranded all-LNA oligonucleotides of 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 fixing 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.

[0350] 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 of 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.

[0351] 39. A method for performing a receptor-based assay for determining an analyte, the method comprising the following steps: 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, the 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.

[0352] 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.

[0353] The following examples and drawings are provided to aid the understanding of the present invention, the true scope of the invention being set forth in the appended claims.It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention.

[0354] Example 1

[0355] Synthesis of LNA oligonucleotides

[0356] LNA oligonucleotides were synthesized on an ABI 394 DNA synthesizer at a 1 μmol scale using standard automated stationary phase DNA synthesis procedures 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) were used as building blocks. All phosphoramidites were at a concentration of 0.1 M in DNA grade acetonitrile. Standard DNA cycles with extended coupling time (180 sec), extended oxidation time (45 sec) and detritylation time (85 sec) as well as standard synthesis reagents and solvents were used to assemble the LNA oligonucleotides. 5'-biotinylated LNA oligonucleotides were synthesized as DMToff, while unmodified LNA oligonucleotides were synthesized as DMTon. Then, the LNA oligonucleotides were cleaved from the support by concentrated ammonia applying 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, 10x250 mm (Waters product number 186008167)) was eluted with a 0.1 M triethylammonium acetate / acetonitrile gradient at pH 7. The product fractions were combined 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.

[0357] The yields ranged from 85 nmol to 360 nmol.

[0358] The LNA oligonucleotides were analyzed by RP18 HPLC (Chromolith RP18e, Merck Product No. 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.

[0359] Each species of oligonucleotide was synthesized and stored separately.

[0360] Example 2

[0361] Identification of LNA oligonucleotide sequences capable of duplex formation without prior denaturation using RP-HPLC analysis

[0362] a) General method:

[0363] The LNA oligonucleotides from Example 1 were dissolved in buffer (0.01 M Hepes pH 7.4, 0.15 M NaCl) and 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% acetonitrile in 10 min; detection at 260 nm).

[0364] 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).

[0365] 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 h 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 min; detection at 260 nm).

[0366] 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 min) and, after reaching room temperature, analyzed again 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% acetonitrile in 10 min; detection at 260 nm).

[0367] If a new peak is formed at a different retention time compared to the individual single-stranded LNA oligonucleotides, duplex formation can be detected. In a positive control, the mixed strand and counterstrand are heat denatured before injection to generate duplexes. The kinetics of duplex formation can be monitored by mixing the strand and counterstrand LNA at room temperature without prior denaturation and then injecting them in a time-dependent manner.

[0368] An LNA sequence is determined to be able to rapidly duplex form if, without prior denaturation, after annealing at room temperature for 5 to 60 minutes, the HPLC% ratio of the duplex formed to one of the two single-stranded LNAs (corrected by the extinction coefficient; higher ratio values ​​are taken into account if the two strands are not completely equimolar) is ≥ 0.9 (HPLC% corrected by the extinction coefficient; the hyperchromaticity of the duplex is not taken into account).

[0369] b) Identification of rapidly duplex-forming sequences

[0370] LNA 1: 5'-tgctcctg-3' (SEQ ID NO 1)

[0371] LNA 2: 5′-Bi-Heg-caggagca-3′ (5′-modified SEQ ID NO 2)

[0372] Heg is hexaethylene glycol

[0373] Bi is a biotin label attached via the carboxyl functional group of the biotin pentanoic acid moiety

[0374] The results are as follows Figure 2-10 shown.

[0375] c) Identification of slowly duplex-forming sequences

[0376] For the 10-bp hybridization experiment, the following ratio calculations were performed

[0377] LNA 3: 5′-CTGCCTGACG-3′

[0378] LNA 4 (conjugate): 5'-Bi-Heg-cgtcaggcag-3'

[0379]

[0380] HPLC%*ε -1 *1000(LNA 3 / LNA 4 double chain) / HPLC%*ε -1 *1000(LNA 3 single chain)=0.023 / 0.456=0.05

[0381] HPLC%*ε -1 *1000(LNA 3 / LNA 4 double chain) / HPLC%*ε -1 *1000(LNA 4 single chain) = 0.023 / 0.457 = 0.05

[0382] Example 3

[0383] Identification of LNA oligonucleotide sequences capable of duplex formation without prior denaturation using RP-HPLC analysis

[0384] a) General method:

[0385] The LNA oligonucleotides from Example 1 were dissolved in buffer (0.01 M Hepes pH 7.4, 0.15 M NaCl) and 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% acetonitrile in 10 min; detection at 260 nm).

[0386] The strand and the corresponding reverse 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, Merckpart no. 1.02129.0001) using a 0.1M triethylammonium acetate / acetonitrile gradient at pH 7 (8-24% B in 10 min; detection at 260 nm).

[0387] In one type of experiment, the strand and the corresponding reverse strand LNA oligonucleotides were mixed at equimolar concentrations at rt and incubated at rt for 1 h. They were then 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 (8-25% acetonitrile in 10 min; detection at 260 nm). Other experiments were performed at different temperatures. The temperature was selected from 0°C to 70°C, more specifically from 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.

[0388] 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 analyzed again after cooling to room temperature 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).

[0389] If a new peak appears at a retention time different from that corresponding to a single single-stranded LNA oligonucleotide, duplex formation is detected. In a positive control (control experiment, see above), the mixed strand and reverse strand were heat denatured before injection, thereby destroying any structure that could have holes and could prevent duplex formation. Thus, after heat denaturation, duplexes are obtained. Without prior denaturation, the strand and reverse strand LNA were mixed at room temperature and injected time-dependently, and the kinetics of duplex formation were monitored.

[0390] The ability of the LNA sequences to rapidly form duplexes was analyzed. In an exemplary but non-limiting case, a positive result is obtained if the HPLC% ratio of the duplex formed and one of the two single-stranded LNAs after 5-60 min of warming at rt (corrected by the extinction coefficient; in the case where the two chains are not completely equimolar, higher ratio values ​​are taken into account) is ≥ 0.9. No prior denaturation (HPLC% corrected by the extinction coefficient; the hyperchromicity of the duplex is not taken into account).

[0391] b) Identification of exemplary sequence pairs that can rapidly form duplexes under non-denaturing conditions

[0392] 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).

[0393] Heg is hexaethylene glycol

[0394] Bi = biotin label attached via the carboxyl functionality of the biotin pentanoic acid moiety These results and others are reflected in the figures.

[0395] 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 the nucleobase sequence and hybridization properties.

[0396] The following list 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.

[0397] 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.

[0398] Number of Watson-Crick complementary base pairs: 5

[0399] It was found that 5′ggaag 3′ / 5′cttcc 3′ has rapid duplex formation;

[0400] (SEQ ID NO: 34 and 33, respectively).

[0401] It was found that 5′ggacc 3′ / 5′gctcc 3′ had rapid duplex formation;

[0402] (SEQ ID NOs: 43 and 42, respectively).

[0403] Number of Watson-Crick complementary base pairs: 6

[0404] It was found that 5′ACCAAC 3′ / 5′GTTGGT 3′ has rapid duplex formation;

[0405] (SEQ ID NOs: 20 and 16, respectively).

[0406] It was found that 5′tttttt 3′ / 5′aaaaaa 3′ had rapid duplex formation;

[0407] (SEQ ID NOs: 27 and 39, respectively).

[0408] It was found that 5′ggagca 3′ / 5′tgctcc 3′ had rapid duplex formation;

[0409] (SEQ ID NOs: 45 and 44, respectively).

[0410] It was found that 5′ctgtca 3′ / 5′tgacag 3′ has rapid duplex formation;

[0411] (SEQ ID NOs: 40 and 41, respectively).

[0412] It was found that 5′ggaaga 3′ / 5′tcttcc 3′ has rapid duplex formation;

[0413] (SEQ ID NO: 36 and 35, respectively).

[0414] Number of Watson-Crick complementary base pairs: 8

[0415] It was found that 5′caggagca 3′ / 5′tgctcctg 3′ had rapid duplex formation;

[0416] (SEQ ID NO: 2 and 1, respectively).

[0417] Number of Watson-Crick complementary base pairs: 9

[0418] It was found that 5′ggaagagaa 3′ / 5′ttctcttcc3′ had rapid duplex formation;

[0419] (SEQ ID NOs: 38 and 37, respectively).

[0420] Number of Watson-Crick complementary base pairs: 15

[0421] It was found that 5′caccaacacaccaac 3′ / 5′gttggtgtgttggtg 3′ had rapid duplex formation;

[0422] (SEQ ID NO: 32 and 31, respectively).

[0423] Typically, the first oligonucleotide of the binding pair described above was used as a Bi-Heg conjugate as described in the initial experiments.

[0424] For instructions, see Figure 11-17 .

[0425] c) Identification of slowly duplex-forming sequences

[0426] Number of possible Watson-Crick complementary base pairs: 10

[0427] It was found that 5′cgtcaggcag 3′ / 5′ctgcctgacg 3′ had slow duplex formation;

[0428] (SEQ ID NO: 6 and 5, respectively).

[0429] Number of possible Watson-Crick complementary base pairs: 15

[0430] 5'cgtcaggcagttcag 3' / 5'ctgaactgcctgacg 3' was found to have slow duplex formation; (SEQ ID NOs: 47 and 48, respectively).

[0431] For instructions, see Figure 18-20 and Figure 21-23 .

[0432] Fig. 20 Identification of binding pairs showing slow hybridization is illustrated. The ratio of the mixed LNA 5'-Bi-Heg-cgtcaggcagttcag-3' (5'-modified SEQ ID NO: 47) and 5'-ctgaactgcctgacg-3' (SEQ ID NO: 48) combination was calculated.

[0433]

[0434] HPLC%*ε -1 *1000(LNA duplex) / HPLC%*ε -1 *1000(LNA single chain)=0.104 / 0.237=0.44

[0435] Example 4

[0436] Biospecific interaction analysis, immobilized first LNA oligonucleotide in contact with second LNA oligonucleotide, three different motifs; kinetic characterization at 25°C and 37°C

[0437] a) Project Overview

[0438] ·12 different oligonucleotide LNA sequences were designed and terminally biotinylated (Bi-LNA sequences)

[0439] · Binding of 7 LNA oligonucleotide sequences to 12 immobilized Bi-LNA sequences was analyzed at 25°C / 37°C

[0440] · 3 min association time and 30 min dissociation time, respectively, flow rate 60 μl / min

[0441] Pre-incubate LNA samples overnight at RT (room temperature) in slightly alkaline buffer (chemical inactivation) as recommended by the manufacturer

[0442] Test setup such as Fig.24 Shown

[0443] b) Technical procedures

[0444] Kinetic studies were performed on a GE Healthcare Biacore 8k instrument.

[0445] The Biacore Biotin Capture Kit S Series Sensor (Catalog No. 28-9202-34) was installed into the instrument and hydrodynamics 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. The reference cells were not fixed 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.

[0446] c) Results

[0447] 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.

[0448] 0.05% Tween 20 was used as detergent in SPR measurements.

[0449] Initial molar ratios MR = 1.0-0.7 (data not shown) indicated 1:1 binding, and MR decreased during the assay period (MR = 0.5-0.3), most likely due to inactivation of the streptavidin surface by the harsh alkaline pH used during the regeneration step.

[0450] Motif "Sequence 1"

[0451] 9-mer LNA shows binding to complementary Bi-LNA 7-9-mer with motif 1

[0452] 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 similarity with and without (HEG) 4 -High complex stability of the complementary oligonucleotide 5'tgctcctgt 3' (SEQ ID NO: 9)' of the MH-5'-tag.

[0453] t / 2 diss(Bi-LNA 7&8mer / 9mer)=>247 / 228min,

[0454] t / 2 diss (Bi-LNA 7&8mer / 9mer with Heg4-MH5')=>734 / 800 min, resulting in high affinity (K D =6-9pM):

[0455] Hybridization with "sequence 2"-LNA showed weaker binding to sequence 1 Bi-LNA 7-9mer

[0456] For including (HEG) 4 -MH-5'-tag 9-mer A sequence '5'aaaaaaaaa'3' (SEQ ID NO: 28), no binding was detected.

[0457] Motif "Sequence 2"

[0458] 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.

[0459] Motif 2-LNA binding of different lengths showed comparable complex formation for 6-8mers, whereas complex formation for 12mers was only slightly slower.

[0460] Complex stability changes: Bi-LNA 7-mer showed the highest complex stability in these experiments, followed by Bi-LNA 8-mer

[0461] t / 2 diss(Bi-LNA 7mer / 6mer) = 238 minutes,

[0462] t / 2 diss(Bi-LNA 7mer / 7mer) = 720 minutes,

[0463] t / 2 diss(Bi-LNA 7mer / 8mer)=644 minutes,

[0464] t / 2 diss(Bi-LNA 8mer / 7mer)=545 minutes,

[0465] t / 2 diss(Bi-LNA 8mer / 8mer) = 433 min, resulting in high affinity (K D =1-5pM)

[0466] Motif 2 is estimated to be better than motif 1

[0467] Motif "Sequence 3"

[0468] 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".

[0469] At 37°C, as LNA length increased 1000-fold from 6-9mers (t / 2 diss = 1 to >1160 min), 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 length increased from 6mers to 9mers, and complex stability remained high; overhangs with >2 unpaired nucleotides significantly reduced complex stability for poly A / poly T pairing.

[0470] d) Conclusion

[0471] Considering the goal of providing an alternative to the streptavidin:biotin binding pair, it was very 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.

[0472] 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.

[0473] 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) combined 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 / 2 diss = 720 and 644 min, respectively, resulting in high affinity (K D =2pM). 'Bi-(HEG) 4 -5'acaccaac 3' (8-mer, 5'-modified SEQ ID NO: 14) binding (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)' showed sufficient complex formation and complex stability with t / 2 diss = 545 and 433 min, respectively, and high affinity at 37°C (KD = 2 pM).

[0474] LNAs with different lengths and the motif "sequence 1" did not bind. As negative controls, the following were tested: 4 - The sequence of the MH-5′-tag is 5′aaaaaaaaa 3′ (SEQ ID NO: 28), no measurable intermolecular interaction was observed.

[0475] Example 5

[0476] Biospecific interaction analysis

[0477] a) Overview of the protocol and assay setup

[0478] Reversible capture of streptavidin conjugates by CAP-Kit

[0479] Streptavidin conjugated to complementary ss-LNA

[0480] Reversible binding assay of oligonucleotides to pre-immobilized ss-LNA oligonucleotides on SCM:

[0481] Capture level (CL), association rate constant k a ,

[0482] Dissociation rate constant k d ,

[0483] Dissociation equilibrium constant K D

[0484] Molar ratio (MR)

[0485] Test setup such as Fig.51 As shown in A

[0486] 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.

[0487] The Bi-LNA sequences are captured as ligands on the CAP chip via a reversible biotin capture kit;

[0488] Free LNA or LNA-Fab <tsh>-Conjugates used as analytes in solution

[0489] Analyze hybridization using an association time of 3 minutes and a dissociation time of 30 minutes,

[0490] Flow rate 60μl / min

[0491] C(free LNA) = 9-0.1 nM, c(LNA-Fab <tsh>-conjugate) = 45-0.6 nM, c (12-mer LNA / Fab <tsh>-conjugate) = 45-0.6 nM

[0492] As suggested by the customer, LNA samples were pre-incubated overnight at RT in a weakly alkaline buffer (chemical inactivation)

[0493] B) Reagent: Sequence "Motif 2"

[0494] Biotinylated ligand

[0495] 'Bi-(HEG) 4 -5'accaac 3' (SEQ ID NO: 20)

[0496] BMO 28.542740, GO4094, ID 6681, 6-mer, MW 3.8 kDa

[0497] 'Bi-(HEG) 4 -5'caccaac 3' (SEQ ID NO: 19)

[0498] BMO 28.542739, GO4093, ID 6681, 7-mer, MW 4.1 kDa

[0499] 'Bi-(HEG) 4 -5'acaccaac 3' (SEQ ID NO: 14)

[0500] BMO 28.542738, GO4092, ID 6680, 8-mer, MW 4.4 kDa

[0501] 'Bi-(HEG) 4 -5'caacacaccaac 3' (SEQ ID NO: 52)

[0502] BMO 28.542742, GO4096, ID 6684, 12-mer, MW 5.8 kDa

[0503] Analytes

[0504] 2300 / 103(HEG) 4 -MH-5'gttggt 3'(SEQ ID NO: 16)'

[0505] BMO 28.170333, AO581, ID 6719, 6-mer, MW 3.8 kDa

[0506] 2300 / 104(HEG) 4 -MH-5'gttggtg 3'(SEQ ID NO: 21)'

[0507] BMO 28.170334, AO582, ID 6720, 7-mer, MW 4.1 kDa

[0508] 2300 / 105(HEG) 4 -MH-5'gttggtgt 3'(SEQ ID NO: 13)'

[0509] BMO 28.170335, AO583, ID 6721, 8-mer, MW 4.4 kDa

[0510] 2300 / 102(HEG) 4 -MH-5'gttggtgtgttg 3'(SEQ ID NO: 53)'

[0511] BMO 28.542727, GO4073, ID 6653, 12-mer, MW 5.8 kDa

[0512] 'mAb <tsh>M-Tu1.20-F(ab′)2-SATP-D-LNA-conjugate

[0513] 2331 / 111 mAb <tsh>M-Tu1.20-F(ab′) 2 -SATP-D-LNA-5'gttggt 3' (SEQ ID NO: 16), 6-mer, MW 104 kDa

[0514] 2331 / 112mAb <tsh>M-Tu1.20-F(ab′) 2 -SATP-D-LNA-5'gttggtg 3' (SEQ ID NO: 21), 7-mer, MW 104 kDa

[0515] 2331 / 113mAb <tsh>M-Tu1.20-F(ab′) 2 -SATP-D-LNA-5'gttggtgt 3' (SEQ ID NO: 13), 8-mer, MW 104 kDa

[0516] 2331 / 114mAb <tsh>M-Tu1.20-F(ab′) 2 -SATP-D-LNA-5'gttggtgtgttg 3'(SEQ IDNO:53),12 fragments,MW 106kDa

[0517] mAb <tsh>M-Tu1.20-F(ab′) 2 F(ab') represents a monoclonal antibody specific for TSH 2 fragment, TSH is human thyroid stimulating hormone. D-LNA means that the oligonucleotide with the subsequent nucleobase sequence is composed of D-LNA monomers. Use of D-LNA oligonucleotides

[0518] c) Results

[0519] The four different 5′-modified LNA oligonucleotides given above, as well as oligonucleotides having the same corresponding sequence but containing a F(ab′) 2 <tsh>The oligonucleotides in the conjugates represent the sequence "Motif 2" with 6-mer, 7-mer, 8-mer and 12-mer lengths. All assays were bound to their complementary Bi-LNA sequences at 25° / 37°C.

[0520] We also analyzed the hybridization of LNA-Fab <tsh>TSH binding of the conjugate (TSH is the analyte).

[0521] Sequence "motif 2"

[0522] Bi-LNAs of different lengths showed comparable complex formation, with complex stabilities ranging from t / 2diss154 to >232 min at 25°C and pM affinities (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 in red), corrected for MTL using 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.

[0523] 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-conjugates. Complex stability t / 2 diss > 232 min, resulting in pM affinity range (K D =22-11pM). At 37°C, FAb <tsh>-LNA conjugates (7-mer, 8-mer and 12-mer) showed no significant difference in complex formation compared to free LNA of the same length, with complex stability t / 2 diss > 232 min, ranging in the pM affinity range (K D <12-14pM).

[0524] LNA-Fab <tsh>The molar ratio of the conjugates MR 0.1-0.6 indicated substoichiometric 1:1 binding. TSH was analyzed with hybridized LNA-FAbs of different lengths. <tsh>Binding of conjugates. TSH and LNA-Fab <tsh>The binding of the conjugates (6-8mer and 12mer) showed comparable kinetics with rapid complex formation and sufficient complex formation, t / 2 diss 31-33 min, resulting in an affinity K D =0.7nM

[0525] The binding constant is within the range of known affinities for this interaction.

[0526] The molar ratio (MR) of 1.8 / 1.9 showed a fully functional stoichiometric 2:1 binding, indicating binding of a functional conjugate.

[0527] It was found that the hybridized Fab conjugate exhibited the full antigen binding activity of the antibody portion of the conjugate. The results are shown in Figure 52.

[0528] For the following data, see also Fig.51 C

[0529]

[0530] Table: Molar epitope accessibility matrix showing hTK sandwich formation by four anti-hTK antibodies. EA =1, completely independent epitope, MR EA <1 overlapping epitope.

[0531] Antibody A is able to form immune complexes with 23C11, 6C6 and 4H4. 23C11, 6C6 and 4H4 share the same epitope.

[0532] d) Alternatives (see Fig.51 B, result Figure 53)

[0533] TSH was analyzed at 37°C with pre-hybridized LNA-FAbs of different lengths (6-mer, 7-mer, 8-mer, and 12-mer). <tsh>Binding of conjugates

[0534] · See slide 10 for assay format and slide 11 for binding curves

[0535] ·3Bi-LNA sequence binds irreversibly to the SA chip on Fc2-4

[0536] ·LNA-Fab <tsh>Conjugates (6-mer, 7-mer and 8-mer) were pre-hybridized with their complementary Bi-LNA at 37°C

[0537] TSH was used as the analyte solution with an association time of 3 minutes and a dissociation time of 5 minutes.

[0538] Flow rate 60 μl / min, c TSH =270nM

[0539] The results are shown in Figure 53.

[0540] Example 6

[0541] Biospecific interaction analysis

[0542] a) Overview of the protocol and assay setup

[0543] A schematic overview of the experiment is as follows Fig.54 Shown

[0544] 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

[0545] The Bi-LNA sequences are captured as ligands on the CAP chip via a reversible biotin capture kit;

[0546] Free LNA used as analyte in solution

[0547] Hybridization was analyzed with an association time of 3 minutes and a dissociation time of 30 minutes, and a flow rate of 60 μl / min

[0548] C (free LNA) = optimized for each interaction

[0549] Measurements: Capture level (CL), association rate constant k a , dissociation rate constant k d , dissociation equilibrium constant K D , molar ratio (MR).

[0550] By means of the CAP-kit for reversible capture of SA-conjugates, streptavidin (=SA) is conjugated to a complementary ss-LNA oligonucleotide which reversibly binds to the pre-immobilized ss-LNA oligonucleotide.

[0551] b) Reagents

[0552] Biotinylated ligand

[0553] 2387 / L01 Bi-(HEG)-5'accaac 3' (SEQ ID NO: 20)

[0554] BMO 28.170341, AO591, ID 6730, 6-mer, MW 2.71 kDa

[0555] 2387 / L02 Bi-(HEG)-5'cacaccaac 3' (SEQ ID NO: 30)

[0556] BMO 28.170342, AO592, ID 6731, 9-mer, MW 3.71 kDa

[0557] 2387 / L03 Bi-(HEG)-5'caccaacaccaac 3' (SEQ ID NO: 54)

[0558] BMO 28.170343, AO593, ID6732, 15-mer, MW 5.73 kDa

[0559] 2387 / L04 Bi-(HEG)-5'ggaag 3' (SEQ ID NO: 34)

[0560] BMO 28.170347, AO597, ID6736, 5-mer, MW 2.44 kDa

[0561] 2387 / L05 Bi-(HEG)-5'ggaaga 3' (SEQ ID NO: 36)

[0562] BMO 28.170348, AO598, ID 6737, 6-mer, MW 2.78 kDa

[0563] 2387 / L06 Bi-(HEG)-5'ggaagagaa 3' (SEQ ID NO: 38)

[0564] BMO 28.170349, AO599, ID 6738, 9-mer, MW 3.82 kDa

[0565] 2300 / 12 Bi-(HEG) 4 -5'tttttt 3' (SEQ ID NO: 27)

[0566] BMO 28.170336, AO584, ID 6722, 6-mer, MW 3.71 kDa

[0567] 2387 / L08 Bi-(HEG)-5'ctgtca 3' (SEQ ID NO: 40)

[0568] BMO 28.170354, AO604, ID 6743, 6-mer, MW 2.71 kDa

[0569] 2387 / L09 Bi-(HEG)-5'cgtcaggcagttcag 3' (SEQ ID NO: 55)

[0570] BMO 28.170356, AO606, ID 6745, 15-mer, MW 5.12 kDa

[0571] 2387 / L10 Bi-(HEG)-5'ggagc 3' (SEQ ID NO: 43)

[0572] BMO 28.170358, AO608, ID 6747, 5-mer, MW 2.43 kDa

[0573] 2387 / L11 Bi-(HEG)-5'ggagca 3' (SEQ ID NO: 45)

[0574] BMO 28.170360, AO610, ID 6749, 6-mer, MW 2.77 kDa

[0575] 2387 / L12 Bi-(HEG) 4 -5'-ccaac 3' (SEQ ID NO: 46)

[0576] BMO 28.542748, GO4105, ID 6764, 5-mer, MW 3.40 kDa

[0577] 2387 / L13 Bi-(HEG) 4 -5'caac 3' (SEQ ID NO: 56)

[0578] BMO 28.542749, GO4106, ID 6765, 4-mer, MW 3.07 kDa

[0579] 2387 / L14 Bi-(HEG) 4 -5'ttttt 3' (SEQ ID NO: 57)

[0580] BMO 28.542750, GO4107, ID 67665mer, MW 3.38 kDa

[0581] 2387 / L15 Bi-(HEG) 4 -5'tttt 3' (SEQ ID NO: 58)

[0582] BMO 28.542751, GO4108, ID 6768 4-mer, MW 3.05 kDa

[0583] Analytes

[0584] 2387 / A01 3'-TGG TTG-5'

[0585] BMO 28.170344, AO594, ID, 6733, 6-mer, MW 2.01 kDa

[0586] 2387 / A02 3'-GTG TGG TTG-5'

[0587] BMO 28.170345, AO595 / ID 6734, 9-mer, MW 3.05 kDa

[0588] 2387 / A03 3'-GTG GTT GTG TGG GTT-5'

[0589] BMO 28.170346, AO596, ID 6735, 15-mer, MW 5.12 kDa

[0590] 2387 / A04 3'-CCT TC-5'

[0591] BMO 28.170350, AO600, ID 6739, 5-mer, MW 1.60 kDa

[0592] 2387 / A05 3-'CCT-TCT-5'

[0593] BMO 28.170351, AO601, ID 6740, 6-mer, MW 1.93 kDa

[0594] 2387 / A06 3'-CCT TCT CTT-5'

[0595] BMO 28.170352, AO602, ID 6741, 9-mer, MW 2.92 kDa

[0596] 2387 / A07 3'-AAA AAA--5'

[0597] BMO 28.170353, AO603, ID 6742, 6-mer, MW 1.99 kDa

[0598] 2387 / A08 3'-GAC AGT-5'

[0599] BMO 28.170355, AO605, ID 6744, 6-mer, MW 2.00 kDa

[0600] 2387 / A09 3'-GCA GTC CGT CAA GTC-5'

[0601] BMO 28.170357, AO607, ID 6746, 15-mer, MW 5.04 kDa

[0602] 2387 / A10 3'-CCT CG-5'

[0603] BMO 28.170359, AO609, ID 6748, 5-mer, MW 1.62 kDa

[0604] 2387 / A11 3'-CCT CGT-5'

[0605] BMO 28.170361, AO611, ID 6750, 6-mer, MW 1.95 kDa

[0606] c) Results

[0607] 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 paired with free LNA 6-mers at 25° / 37°C.

[0608] 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 kinetic profile was characterized by rapid hybridization and sustained high complex stability, with t / 2 diss = 160 to > 232 minutes at 25° and 37°C, and 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.

[0609] The molar ratio (MR) of 1.1 / 1.2 indicates that the stoichiometry of the 6-mer pair is 1:1 hybridization at both temperatures; MR 1.3 / 1.5 indicates that the stoichiometry of the 9-mer pair is too large. Compared with the 6-mer and 9-mer, the Bi-LNA 15-mer shows slowed hybridization kinetics, resulting in slightly lower affinity. K D = 24 / 53 pM at both temperatures; MR 1.3 indicating slightly superstoichiometric binding.

[0610] Free 6-mer-LNA3'-TGG TTG-5' showed reduced complex stability when bound to 5-mer (2387 / L12)Bi-LNA 5'Bi-4x(HEG)-CCA AC-3' with t / 2 diss = 50 min, 2-digit pM affinity range, and when bound to 4-mer (2387 / L13)5'Bi-4x(HEG)-CAA C-3', the complex stability dropped to t / 2 diss < 1 min, resulting in 2-digit nM affinity. MR 1.1-1.3 indicates slightly above stoichiometric binding. Thus, overhangs with 1 or 2 mismatched nucleotides can be explained in this case to reduce the stability of the complex to some extent.

[0611] "Motif 3" Poly-T control "short" (2300 / 12 and 2387 / L14 & L15); 3 poly-T-control Bi-LNAs of different lengths (5- & -6-mers) showed binding with poly A-6-mers with typical fast association / dissociation curves, with complex half-lives t / 2 diss < 2 minutes at 25° and 37°C, and Bi-LNA 4-mers showed only weak / no binding with poly A-6-mers.

[0612] "Motif 4" (2387 / L04-L06) 5-mer, 6-mer or 9-mer 5′-Bi-Heg-GGA AG-3′, 5′-Bi-Heg-GGA AGA-3′ or 5′-Bi-Heg-GGA AGA GAA-3′ were inferior to "motif 2" when binding to their complementary LNA, with complex half-lives t / 2 diss ranging from 20-65 minutes at 25°C, resulting in 2-3 digit pM affinities.

[0613] "Motif 5" Binding of (2387 / L08) 5′-Bi-Heg-CTG TCA-3′ to the complementary LNA 3′-GAC AGT-5′ showed slightly slower hybridization than the 6-mer of "Motif 2", with complex stability in the 2-digit pM affinity at 25° and 37°C. A molar ratio of 0.3 / 0.4 indicates substoichiometric binding.

[0614] "Motif 6" (2387 / L09) 15-mer 5′-Bi-Heg-CGT CAG GCA GTT CAG-3′ combined with 3′-GCA GTCCGT CAA GTC-5′ showed 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.

[0615] "Motif 7" (2387 / L10 and L11)

[0616] The 5-mer 5′-Bi-Heg-GGA GC-3′ binds to their complementary LNA better than the 6-mer 5′-Bi-Heg-GGA GCA-3′; at 25°C, the complex half-life t / 2 diss is between 174 and 62 min, the affinity range is 32 / 186 pM, the 5-mer shows 29 pM interaction at 37°C, the molar ratio MR 0.5 / 0.3 indicates substoichiometric binding at 25°C, and increases at 37°C (MR 1.2 / 0.6).

[0617] d) Conclusion

[0618] Both Bi-LNA 5′-Bi-Heg-ACC AAC-3′ and 5′-Bi-Heg-CAC ACC AAC-3′ (“Motif 2”) showed high affinity to their complementary LNA 6-mer 3′-TGG TTG-5′ and 9-mer 3′-GTG TGG TTG-5′, respectively. The molar ratios indicated fully functional 1:1 LNA hybridization. 5′-Bi-Heg-ACC AAC-3′ / 3′-TGGTTG-5′ showed 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.

[0619] According to the supplier information 1000RU = 1ng / 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. < / tsh> < / 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 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 7 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, said second ss-oligonucleotide comprising at least the same number of monomers as said first ss-oligonucleotide, each monomer of said second ss-oligonucleotide comprising a nucleobase, said nucleobases of said second ss-oligonucleotide forming a second nucleobase sequence, said second nucleobase sequence comprising or consisting of a nucleobase sequence complementary to said first nucleobase sequence in an antiparallel orientation and predicting by complementarity the ability of said first and second ss-oligonucleotides to form an antiparallel duplex with each other, said predicted duplex comprising or consisting of 5 consecutive base pairs, wherein the two bases of each base pair are bound to each other by hydrogen bonds; (c) mixing said first and second ss-oligonucleotides in aqueous solution, wherein the molar amount of either of said first and second ss-oligonucleotides is within the interval given by the molar amount of the other ss-oligonucleotide ± 5% of said molar amount, and wherein the step is performed at a non-denaturing temperature; (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 duplexes; (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 greater 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 selecting and providing a binding pair of said single-stranded all-LNA oligonucleotides, wherein prior to step (c), each ss-oligonucleotide in any of steps (a) and (b) is maintained at a temperature of -80°C to 40°C in aqueous solution.

2. The method according to claim 1, wherein step (c) is carried out at a temperature of 0°C to 40°C.

3. The method according to any one of claims 1 and 2, wherein before step (e), the mixture obtained in step (d) is subjected to an additional step of separating ss-oligonucleotides and duplex oligonucleotides.

4. The method according to any one of claims 1 and 2, wherein steps (c) and (d) are performed at a non-denaturing temperature.

5. The method of claim 4, wherein steps (c) and (d) are performed 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.

6. The method of claim 1, wherein each ss-oligonucleotide of any of steps (a) and (b) is maintained at a temperature of 0°C to 40°C in aqueous solution prior to step (c).

7. The method according to claim 6, wherein each ss-oligonucleotide of any of steps (a) and (b) is maintained at a temperature of 25°C to 37°C in aqueous solution prior to step (c).

8. The method according to any one of claims 1, 2 and 5 to 7, wherein in step (d), the time interval is from 1 second to 20 minutes.

9. The method according to any one of claims 1, 2 and 5 to 7, wherein in step (d), the time interval is from 1 second to 5 minutes.

10. The method according to any one of claims 1, 2 and 5 to 7, wherein in step (d), the time interval is 1 second to 60 seconds.

11. The method according to any one of claims 1, 2 and 5 to 7, wherein in step (d), the time interval is 1 second to 30 seconds.

12. The method according to any one of claims 1, 2 and 5 to 7, 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 of 20 base pairs in length and 50% G+C content by at least 15°C.

13. The method of claim 12, wherein steps (c) and (d) are performed in the absence of either formamide or dimethyl sulfoxide.

14. The method according to any one of claims 1, 2, 5 to 7 and 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-methyluracil, 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, azathiopurine, 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine, aminophylline, N 6 -Benzyl adenine, N 6 -Benzoyl adenine, 6-benzyloxypurine, 8-bromotheophylline, 8-bromo-3-methylxanthine, 8-bromo-7-(2-butyn-1-yl)-3-methylxanthine, 6-chloropurine, 8-chlorotheophylline, 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.

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

16. The method of claim 14, wherein one or more cytosines, where present, are replaced with 5-methylcytosine.

17. The method of claim 16, wherein each cytosine is replaced by 5-methylcytosine.

18. The method according to any one of claims 1, 2, 5 to 7, 13 and 15 to 17, wherein the monomer of the ss-oligonucleotide of any of steps (a) and (b) is a β-L-LNA monomer.

19. 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 having 5 consecutive Watson-Crick base pairs in aqueous solution in the absence of denaturing conditions prior to or during duplex formation, wherein the pair is selected from the group consisting of: SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 42 and SEQ ID NO: 43, and SEQ ID NO: 16 and SEQ ID NO:

46.

20. The pair of isolated complementary ss-oligonucleotides according to claim 19, wherein the pair of ss-oligonucleotides is obtained by performing the method according to any one of claims 1 to 18.

21. The pair of isolated complementary ss-oligonucleotides according to any one of claims 19 to 20, wherein a first ss-oligonucleotide of the pair is ligated to a first target and a second ss-oligonucleotide is ligated to a second target.

22. The pair of isolated complementary ss-oligonucleotides according to claim 21, wherein the targets are independently selected from the group consisting of a solid phase, a biomolecule and a chemically synthesized compound.

23. A method for forming an antiparallel full LNA duplex in the absence of denaturing conditions, the method comprising the following steps (a) providing a first and a second member of a pair of single-stranded all-LNA oligonucleotides according to any one of claims 19 to 20, respectively, wherein each single-stranded all-LNA oligonucleotide is separately dissolved in an aqueous solution in the absence of a denaturing agent and maintained at a temperature of 0°C to 40°C; (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; Thereby the antiparallel full LNA duplex is formed.

24. An antiparallel duplex formed by a first and a second member of a pair of single-stranded all-LNA oligonucleotides, wherein the antiparallel duplex is obtained by the method according to claim 23.

25. Use of a pair of isolated ss-oligonucleotides according to any one of claims 19 to 22 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.

26. 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 claims 19 to 22, the kit further comprising in a second container a solid phase to which is linked a second member of the pair.

27. 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 claims 19 to 20, and with a solid phase to which is linked a second member of the pair; incubating to form a complex, the 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.

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