Hybridizing all-LNA oligonucleotides

Abstract

To identify and provide the binding pairs of single-stranded all-LNA oligonucleotides which are capable of hybridizing, thereby forming duplex molecules with Watson-Crick base pairing.SOLUTION: The present disclosure shows hybridization experiments with pairs of entirely complementary ss-oligonucleotides, which fail to form a duplex within a given time interval. The present report provides methods to identify such incompatible oligonucleotide pairs. In another aspect, the present report provides pairs of complementary ss-oligonucleotides which are capable of rapid duplex formation. The present report also provides methods to identify and select compatible oligonucleotide pairs. In yet another aspect, the present report provides use of compatible oligonucleotide pairs as binding partners in binding assays, e.g. immunoassays.SELECTED DRAWING: None

Description

[Technical field]

[0001] This report relates to hybridization of single-stranded (ss-) oligonucleotides composed entirely of locked nucleic acid (LNA) monomers. This report shows hybridization experiments with fully complementary ss-oligonucleotide pairs that unexpectedly fail to form duplexes within a given time interval. This report provides an efficient method for identifying such incompatible oligonucleotide pairs. In another aspect, this report provides complementary single-stranded oligonucleotide pairs composed entirely of locked nucleic acid (LNA) monomers that are surprisingly capable of rapid duplex formation in the absence of prior denaturation. This report also provides a method for identifying and selecting such compatible all-LNAss oligonucleotide pairs. In yet another aspect, this report provides the use of compatible oligonucleotide pairs as binding partners in biochemical assays such as binding assays, immunoassays, etc. In certain embodiments, it is discussed that compatible LNA oligonucleotide pairs are used to immobilize different target molecules, such as capture molecules specific for the analyte, in assays for detecting or quantifying an analyte in a sample. [Background technology]

[0002] Particular focus is on common biochemical applications where the specific interaction of the two partners of a binding pair and their eventual interconnection plays a functional role. Very frequently, heterogeneous immunoassays use a biotin:(strept)avidin binding pair to immobilize an analyte-specific capture receptor to a solid phase. This report conceptualizes, describes and demonstrates an alternative binding pair suitable for immunoassays, among other applications. Specifically, an alternative binding pair consisting of two single-stranded all-LNA oligonucleotides capable of forming a duplex by hybridization offers a technical alternative to the biotin:(strept)avidin binding pair.

[0003] An important focus of this disclosure is the means by which the capture receptor is anchored to the solid phase during the course of an immunoassay. In particular, this disclosure focuses on binding pairs that facilitate the immobilization of the capture receptor in the presence of a sample containing the analyte and / or that can anchor the detection complex after the complex has formed. It is technically necessary that the binding pair in an immunoassay has specific characteristics. Thus, the interaction of the two binding partners must be specific. Furthermore, it is desirable that the kinetics of linkage formation, i.e., the rate at which the two separate partners of the binding pair interact and ultimately associate, i.e., bind to each other, is high. Furthermore, it is desirable that the linkage of the two binding partners, once formed, is stable. Furthermore, the binding partner must be suitable for chemical conjugation with other molecules, such as analyte-specific receptors or solid-phase surfaces, in order to be applied to the immunoassay. It is important to understand that in an immunoassay, the receptor and usually also the analyte to be detected, retain their conformation and function only under certain conditions, which may vary depending on the particular receptor or the particular analyte under consideration. Thus, the receptor molecule or analyte may only tolerate limited deviations from these conditions. Such conditions may include, but are not limited to, a buffered aqueous solution having a pH in the range of about pH 6 to about pH 8, one or more dissolved salts, one or more helper substances, a total amount of solute of about 250 to about 400 mosm / kg, at a preselected temperature in the range of 20°C to 40°C, just to name a few.

[0004] The distinct partners of the binding pair must be suitable for conjugation, particularly conjugation with a capture molecule or receptor, and conjugation with a solid surface, without losing their ability to specifically associate and bind with one another. For conjugation in an immunoassay, each distinct binding partner of the surrogate binding pair must be , must be functional under the assay conditions. The same reasoning applies to all other materials required for conjugation with a binding partner, including, but not limited to, the analyte, the support material, the solid phase, and other substances or compounds that may be present during the course of the assay.

[0005] Single-stranded oligonucleotides with complementary sequences, i.e. oligonucleotides capable of forming a duplex by hybridization, have previously been proposed as binding pair means for linking macromolecules or attaching molecules to a solid phase. EP0488152 discloses a heterogeneous immunoassay using a solid phase in which an analyte-specific capture antibody is immobilized by a nucleic acid duplex linking the antibody and the solid phase. In one embodiment, it is shown that one hybridized oligonucleotide is attached to the antibody and the complementary oligonucleotide is attached to the solid phase, thereby forming a linked duplex. Similar disclosures are provided in documents EP0698792, WO1995 / 024649, WO1998 / 029736, and EP0905517. WO2013 / 188756 discloses a method of flow cytometry and a composition comprising an antibody bound to a first oligonucleotide, an oligosphere bound to a second oligonucleotide having the same sequence as the first oligonucleotide, and an oligonucleotide probe having a label and a third sequence complementary to the first and second oligonucleotides. In a particular embodiment, the oligosphere is magnetic. This document reports the particular use of the oligosphere as a reference for standardization procedures.

[0006] Modified oligonucleotides such as peptide nucleic acid (PNA) and locked nucleic acid (LNA) have been explored for physiological applications. LNA has a methylene linker between the 2'-oxygen and 4'-carbon of the ribose moiety, resulting in the sugar being locked in a C3-endo conformation, hence the name "locked nucleic acid". This chemical modification confers nuclease resistance, as well as higher affinity and higher specificity for oligonucleotide targets in technical applications involving duplex formation by hybridization. WO1998 / 39352 discloses locked nucleic acid (LNA) structures. WO2000 / 056746 discloses the synthesis of LNA monomers, including intermediate products of specific stereoisomers of LNA. By chemical synthesis, single strands consisting only of LNA nucleoside analogue monomers ("all-LNA") can be synthesized.

[0007] As mentioned above, locked nucleic acid (LNA) monomers are conformationally restricted nucleotide analogues with an extra 2'-O, 4'-C-methylene bridge added to the ribose ring. LNA monomers are provided as 2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleoside monomers (Singh SK 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). WO2000 / 066604 and WO2000 / 056746 disclose certain stereoisomers of LNA nucleoside monomers. Mixed DNA-LNA oligonucleotides containing DNA and LNA monomers show stability against 3'-exonuclease degradation and have significantly improved thermal stability when hybridized to complementary DNA and RNA. In fact, compared to other synthesized high affinity nucleic acid mimics, such as peptide nucleic acid (PNA), hexitol nucleic acid (HNA), and 2'-fluoro N3'-phosphoramidates, LNA shows exceptional binding affinity. Hybridization kinetics of LNA-DNA mixed oligonucleotides, also known as "mixmers", was reported by Christensen U. et al. (Biochem J 354 (2001) 481-484). The crystal structure of a duplex from two complementary ss-oligonucleotides, each consisting of seven LNA monomers, was reported by Eichert et al. Reported by A. et al. (Nucleic Acids Research 38(2010)6729-6736).

[0008] WO1999 / 14226 proposes the use of LNA in the construction of affinity pairs for attaching to target molecules and solid supports.However, it is also known in the art that the hybridization of complementary all-LNA single strands causes technical problems.The thermodynamic analysis of all-LNA hybridization can be mainly verified by experiment, and it seems that the sequence prediction of hybridizing monomers without a prior denaturation step (such as heating before hybridization) is not possible so far.

[0009] So far, in most cases, mixed LNA-DNA oligonucleotides (also called "miximer single strands" or "miximers") have been analyzed. So far, there are few reports on the characterization of hybridizing single stranded oligonucleotides made only from LNA monomers (i.e., "all-LNA" single stranded oligonucleotides), especially by Koshkin AA et al. (J Am Chem So 120 (1998) 13252-13253) and Mohrle BP et al. (Analyst 130 (2005) 1634-1638). Eze NA et al. (Biomacromolecules 18 (2017) 1086-1096) reported that the binding rate from DNA-LNA miximers and DNA probes was 10 5 M -1 s -1 According to these authors, the hybridization rate in solution does not appear to be affected by the replacement of one or more DNA monomers by LNA monomers, taking into account one-third of the monomers available for replacement.

[0010] Predictions regarding the thermodynamic behavior of LNA-containing oligonucleotides are aided by dedicated computer programs referenced by Tolstrup N et al. (Nucleic Acids Research 31(2003)3758-3762). However, the report explicitly mentions that the prediction error is higher for LNA oligonucleotides, not due to a lack of experimental data, but due to the more complex properties of these LNA oligonucleotides. Specifically, the report shows that complementary ss-oligonucleotides consisting of eight or more LNA monomers cannot be predicted with respect to their ability to form double-stranded molecules with Watson-Crick base pairs.

[0011] Thus, the general objective of this report is the identification and provision of binding pairs of single-stranded all-LNA oligonucleotides that can hybridize and thereby form a duplex molecule with Watson-Crick base pairs. More specifically, we seek binding pairs capable of duplex formation under non-denaturing conditions, more specifically under conditions compatible with the function of analyte-specific receptors in analyte detection assays (such as, but not limited to, immunoassays). Importantly, we seek single-stranded all-LNA oligonucleotides that can be stored and hybridized to each other in aqueous solution at ambient temperatures, such as, but not limited to, room temperature, without intermittent heating steps to remove intramolecular secondary structures that cause hybridization incompatibility of complementary oligonucleotides. Summary of the Invention

[0012] The present disclosure provides in a first aspect, which relates to all other aspects and embodiments disclosed herein, a method for providing a binding pair, the binding pair consisting of a first single stranded LNA oligonucleotide and a second single stranded LNA oligonucleotide, the two oligonucleotides being capable of forming an antiparallel duplex of 8 to 15 consecutive Watson-Crick base pairs at a temperature between 20°C and 40°C, (a) A first single strand (=ss-) consisting of 8 to 15 locked nucleic acid (=LNA) monomers. ) providing oligonucleotides, each monomer comprising a nucleobase, wherein the nucleobases of the first ss-oligonucleotide form a first nucleobase sequence; (b) providing a second ss-oligonucleotide consisting of 8 to 15 LNA monomers; The second ss-oligonucleotide is at least a second ss-oligonucleotide comprising a first nucleobase and a second nucleobase comprising a second nucleobase sequence of said second ss-oligonucleotide, each monomer of said second ss-oligonucleotide comprising a nucleobase, said nucleobases of said second ss-oligonucleotide forming a second nucleobase sequence of said second ss-oligonucleotide, said second nucleobase sequence comprising or consisting of a nucleobase sequence complementary to said first nucleobase sequence in an antiparallel orientation; and theoretically predicting the ability of said first and second ss-oligonucleotides to form a duplex with each other, said duplex consisting of 8 to 15 consecutive Watson-Crick base pairs; (c) mixing and incubating equimolar amounts of the first and second ss-oligonucleotides in an aqueous solution at a temperature between 20° C. and 40° C. for a time interval of not more than 20 minutes, thereby obtaining a mixture of ss-oligonucleotides or a double-stranded mixture; followed by (d) separating the mixture obtained in step (c) at a temperature between 20°C and 40°C, followed by detecting and quantifying the separated duplexes and detecting and quantifying the separated ss oligonucleotides; followed by (e) selecting said binding pair if in step (d) a duplex is detectably present and if the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide; Thereby, providing said binding pair The present invention provides a method comprising:

[0013] The present disclosure, in a second aspect, related to all other aspects and embodiments disclosed herein, 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, each oligonucleotide consists of 8 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising nucleobases of said monomers forming a first nucleobase sequence of said first oligonucleotide and a second nucleobase sequence of said second oligonucleotide, the first nucleobase sequence and the second nucleobase sequence are selected such that the first oligonucleotide and the second oligonucleotide can form an antiparallel duplex of 8 to 15 consecutive Watson-Crick base pairs at a temperature of 20° C. to 40° C.; and said binding pair provides a composition obtainable by the method according to the first aspect disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] A binding pair is understood to be a set of two different binding partners that can form specific non-covalent intermolecular bonds with each other under non-denaturing conditions. In the context of the present disclosure, the broadest understanding of non-denaturing conditions means the absence of externally applied influences, such as heating or adding a certain amount of denaturing compound, to molecularly unfold the target material and thereby destroy its secondary or higher structure. In this regard, heating is exemplified by increasing the temperature substantially above 40°C, 50°C, 60°C or even higher for a desired period of time, and denaturing compounds are exemplified by detergents, chaotropes, or compounds that can lower the melting temperature of nucleic acid duplexes, such as formamide.

[0015] Importantly, each of the first and second binding partners does not form a specific bond with a partner of the same species. That is, no specific intramolecular binding occurs between two first partners or two second partners. At the same time, under non-denaturing conditions, each separate partner exhibits the ability to bind other partners. Specifically, under non-denaturing conditions, the separate partners do not form any intramolecular bonds that render them unable to form bonds with other species partners. For example, intramolecular folding may result in a secondary structure that is stable enough under non-denaturing conditions to inhibit or prevent the desired intermolecular binding of two different species of binding partners.

[0016] However, intramolecular folding affecting one or both binding partners may be It does not necessarily completely inhibit the desired intermolecular binding of two different species. The kinetics of intermolecular binding is expected to be slower compared to unhindered binding partners without intramolecular folding. Especially considering the mechanism of standardized high-throughput assays such as (but not limited to) automated immunoassays, such a setup usually requires rapid formation of intermolecular linked forms of binding pairs from previously separate binding partners. Therefore, it is a desirable technical feature that each binding partner has no or greatly minimized intramolecular folding.

[0017] In the context of the present disclosure, and particularly with respect to immunoassays and the interaction of a receptor with its target substance (analyte), non-denaturing conditions are more specifically understood as the collective characteristics of an environment that permits a receptor (e.g., an antibody) to achieve and / or maintain a conformation that allows the receptor to interact with and bind to its target substance (analyte). At the same time, the environment provided by non-denaturing conditions permits the target substance to achieve and / or maintain a conformation that allows it to become and / or remain bound to the receptor.

[0018] In particular, all LNA oligonucleotides have characteristics that cannot be reliably predicted with the tools available to those skilled in the art.For practical reasons, the present study was limited to ss-oligonucleotides consisting of a maximum of 15 LNA monomers.

[0019] The present disclosure thus provides in a first aspect, which relates to all other aspects and embodiments disclosed herein, a method for providing a binding pair, the binding pair consisting of a first single stranded LNA oligonucleotide and a second single stranded LNA oligonucleotide, the two oligonucleotides being capable of forming an antiparallel duplex of 8 to 15 consecutive Watson-Crick base pairs at a temperature between 20°C and 40°C, (a) providing a first single-stranded (=ss-) oligonucleotide consisting of 8 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, said nucleobases of said first ss-oligonucleotide forming a first nucleobase sequence; (b) providing a second ss-oligonucleotide consisting of 8 to 15 LNA monomers, said second ss-oligonucleotide consisting of at least the number of monomers of 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 a duplex with each other, said duplex consisting of 8 to 15 consecutive Watson-Crick base pairs; (c) mixing and incubating equimolar amounts of the first and second ss-oligonucleotides in an aqueous solution at a temperature between 20° C. and 40° C. for a time interval of not more than 20 minutes, thereby obtaining a mixture of ss-oligonucleotides or a double-stranded mixture; followed by (d) separating the mixture obtained in step (c) at a temperature between 20°C and 40°C, followed by detecting and quantifying the separated duplexes, if present, and the separated ss oligonucleotides; followed by (e) selecting said binding pair if in step (d) a duplex is detectably present and if the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide; Thereby, providing said binding pair The present invention provides a method comprising:

[0020] All LNA ss-oligonucleotides specified herein can contain a number of monomers selected from the group consisting of 8, 9, 10, 11, 12, 13, 14, and 15. In one embodiment of all aspects and embodiments disclosed herein, the first ss-oligonucleotide contains between 8 and 12 monomers (i.e., 8, 9, 10, 11, and 12 monomers). In another embodiment of all aspects and embodiments disclosed herein, the first ss-oligonucleotide consists of 8 to 10 monomers (i.e., a number selected from 8, 9, and 10 monomers), and in a more specific embodiment of all aspects and embodiments disclosed herein, the first ss-oligonucleotide consists of 9 monomers. The first and second ss-oligonucleotides do not have to be the same size, i.e., they do not have to consist of the same number of monomers. However, the same number of monomers making up the first and second ss-oligonucleotides is a specific embodiment of all aspects and embodiments disclosed herein.

[0021] Two oligonucleotides are antiparallel when they run parallel to each other but in opposite sequences. A specific example is the two complementary strands of a nucleic acid duplex, which run in opposite directions alongside each other. As a result, each end of the duplex contains the 5' end of the first strand adjacent / aligned with the 3' end of the second strand on the opposite side. Like DNA and RNA, LNA exhibits Watson-Crick base pairing (Koshkin, AA et al., J Am Chem Soc 120 (1998) 13252-13260). In one embodiment of all aspects and embodiments disclosed herein, each LNA monomer comprises a nucleobase selected from the group consisting of adenine, thymine, uracil, guanine, cytosine, 5-methylcytosine, 5-hydroxymethylcytosine, 7-deazaguanine and 7-deazaadenine. The specific Watson-Crick base pairing involving these bases on complementary opposite strands is well known to those skilled in the art and is an accepted feature widely published in the art. Apart from the described nucleobases, others that can be incorporated into the all-LNAss-oligonucleotide are also known to those skilled in the art. Generally, these include pyrimidines derivatized at the C-5 atom.

[0022] Importantly, after the two different (i.e., first and second) ss-oligonucleotides have been contacted, step (c) of the method specifies incubation for a time interval of 20 minutes or less. In certain embodiments of all aspects and embodiments disclosed herein, the time interval is selected from the group consisting of 1 second to 20 minutes, 1 second to 15 minutes, 1 second to 10 minutes, 1 second to 5 minutes, 1 second to 1 minute, 1 second to 30 seconds, 1 second to 20 seconds, 1 second to 10 seconds, and 1 second to 5 seconds. Highly advantageous time intervals are selected from 1 second to 10 seconds and 1 second to 5 seconds.

[0023] In step (c), the temperature is selected independently of the temperature in step (d), and vice versa. In certain embodiments of all aspects and embodiments disclosed herein, the temperatures in steps (c) and (d) do not differ by more than 5° C. In certain embodiments of all aspects and embodiments disclosed herein, in step (c) and / or step (d), the temperature is between 20° C. and 25° C. In certain embodiments of all aspects and embodiments disclosed herein, in step (c) and / or step (d), the temperature is between 25° C. and 37° C. In another specific embodiment of all aspects and embodiments disclosed herein, prior to step (c), the first ss-oligonucleotide and the second ss-oligonucleotide are kept at a temperature between 80° C. and 40° C., specifically between 20° C. and 40° C., more specifically between 20° C. and 25° C., and even more specifically between 25° C. and 37° C. In another embodiment of all 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 specifically pH 6.5 to pH 7.5. In another embodiment of all aspects and embodiments disclosed herein, in step (c), the aqueous solution comprises a salt. In another embodiment of all aspects and embodiments disclosed herein, in step (c), the aqueous solution comprises a salt. In another embodiment of all aspects and embodiments disclosed herein, in step (c), the aqueous solution comprises a salt. Contains the total amount of dissolved substances in mol / L.

[0024] In another embodiment of all aspects and embodiments disclosed herein, step (d) comprises subjecting the incubated mixture of step (c) to column chromatography using an aqueous solvent as a mobile phase. Thus, column chromatography is used to separate the separate double-stranded molecules from the ss-oligonucleotides. Suitable chromatographic methods, such as HPLC, are well known to those skilled in the art in this regard.

[0025] In another embodiment of all aspects and embodiments disclosed herein, the ss-oligonucleotides (a) and (b) are composed of beta-D-LNA monomers, i.e., the first ss-oligonucleotide is composed entirely of beta-D-LNA monomers and the second ss-oligonucleotide is composed entirely of beta-D-LNA monomers. In yet another embodiment of all aspects and embodiments disclosed herein, the ss-oligonucleotides (a) and (b) are composed of beta-L-LNA monomers, i.e., the first ss-oligonucleotide is composed entirely of beta-L-LNA monomers and the second ss-oligonucleotide is composed entirely of beta-L-LNA monomers.

[0026] By the methods disclosed therein, and by any of its embodiments, the present disclosure provides all-LNA duplexes formed at a preselected temperature of 25°C to 40°C from non-denaturing pairs of complementary single-stranded all-LNA oligomers, each containing 8 to 15 LNA monomers.

[0027] The present disclosure in a second aspect related to all other aspects and embodiments disclosed herein 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, each oligonucleotide consisting of 8 to 15 Locked Nucleic Acid (=LNA) monomers, each monomer comprising a nucleobase of a monomer forming a first nucleobase sequence of the first oligonucleotide and a second nucleobase sequence of the second oligonucleotide, the first nucleobase sequence and the second nucleobase sequence being capable of forming an antiparallel duplex of 8 to 15 consecutive Watson-Crick base pairs at a temperature of 20°C to 40°C, and the binding pair being obtainable by a method according to the first aspect disclosed herein.

[0028] In a particular aspect relevant to all aspects and embodiments, there is provided a liquid composition comprising an aqueous solvent and a binding pair consisting of a first single-stranded oligonucleotide and a second single-stranded oligonucleotide, each oligonucleotide consisting of 8 to 15 Locked Nucleic Acid (=LNA) monomers, each monomer comprising a nucleobase of a monomer forming a first nucleobase sequence of the first oligonucleotide and a second nucleobase sequence of the second oligonucleotide, the first nucleobase sequence and the second nucleobase sequence being capable of forming an antiparallel duplex of 8 to 15 consecutive Watson-Crick base pairs at a temperature of 20°C to 40°C, and the binding pair being obtainable by a method according to the first aspect disclosed herein.

[0029] In a particular aspect relating to all aspects and embodiments, there is provided a liquid composition comprising an aqueous solvent and a binding pair consisting of a first single-stranded oligonucleotide and a second single-stranded oligonucleotide, each oligonucleotide consisting of 8 to 15 Locked Nucleic Acid (=LNA) monomers, each monomer comprising a nucleobase of a monomer forming a first nucleobase sequence of the first oligonucleotide and a second nucleobase sequence of the second oligonucleotide, the first nucleobase sequence and the second nucleobase sequence being capable of forming an antiparallel duplex of 8 to 15 consecutive Watson-Crick base pairs at a temperature of 20°C to 40°C, the binding pair being a binding pair according to the first aspect disclosed herein. It is obtained by the method.

[0030] In one embodiment of all aspects and embodiments disclosed herein, each oligonucleotide consists of 9 to 15 LNA monomers, the first nucleobase sequence and the second nucleobase sequence are selected such that the first oligonucleotide and the second oligonucleotide form an antiparallel duplex of 9 consecutive Watson-Crick base pairs at a temperature of 20°C to 40°C, and the binding pair is obtainable or is obtained by the method of the first aspect and embodiments thereof.

[0031] In one embodiment of all aspects and embodiments disclosed herein, each ss-oligonucleotide comprises two or three different nucleobases. In an embodiment of all aspects and embodiments disclosed herein, the G+C (including G and C analogs) content of the nucleobases of each ss-oligonucleotide is less than 75%. In a particular embodiment, the G+C content is lower than a value selected from 74%, 73%, 72%, 71%, and 70%. In yet another embodiment of all aspects and embodiments disclosed herein, each LNA monomer in the binding pair comprises a nucleobase selected from the group consisting of adenine, thymine, uracil, guanine, cytosine, 5-methylcytosine, 5-hydroxymethylcytosine, 7-deazaguanine, and 7-deazaadenine. In a more particular embodiment, each cytosine of the nucleobases in each ss-oligonucleotide is replaced by a 5-methylcytosine.

[0032] In one embodiment of all aspects and embodiments disclosed herein, the binding pair of two distinct compatible binding partners is an all-LNAss-oligonucleotide pair selected from the group consisting of:

[0033] 5'tgctcctg3' and 5'caggagca3', 5'gcctgacg3' and 5'cgtcaggc3', 5'ctgcctgacg3' and 5'cgtcaggcag3', 5'gactgcctgacg3' and 5'cgtcaggcagtc3', 5'tgctcctgt3' and 5'acaggagca3', 5'gtgcgtct3' and 5'agacgcac3', 5'gttggtgt3' and 5'acaccaac3', 5'caacacaccaac3' and 5'gttggtgtgttg3', 5'acacaccaac3' and 5'gttggtgtgt3', 5'acaccaac3' and 5'gttggtgt3'

[0034] In a particular embodiment, the monomer of the ss-oligonucleotide in any selected pair of the aforementioned group is a beta-D-LNA monomer. In yet another particular embodiment, the monomer of the ss-oligonucleotide in any selected pair of the aforementioned group is a beta-L-LNA monomer.

[0035] In an embodiment of all aspects and embodiments disclosed herein, one ss-oligonucleotide of the binding pair is attached to a solid phase selected from the group consisting of magnetic beads, paramagnetic beads, synthetic organic polymer (latex) beads, polysaccharide beads, test tubes, microwell plate cavities, cuvettes, membranes, scaffold molecules, quartz crystals, films, filter paper, disks, and chips. In another embodiment of all aspects and embodiments disclosed herein, one ss-oligonucleotide of the binding pair is linked to a molecule selected from the group consisting of peptides, polypeptides, oligonucleotides, polynucleotides, sugars, glycans, haptens, and dyes. In yet another embodiment of all aspects and embodiments disclosed herein, the ss-oligonucleotide is covalently attached to a linker. In 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 described herein, the ss-oligonucleotides are covalently attached to analyte-specific receptors useful in receptor-based analyte detection assays, such as, but not limited to, immunoassays.

[0036] In its broadest sense, and in line with the commonly accepted understanding in biochemistry, a receptor is a structure that has affinity for a specific target molecule as a whole, or for a specific molecular region and / or three-dimensional aspect of the target molecule. For the purposes of this disclosure, a receptor is understood to interact with and bind to a target molecule. In a biochemical assay, the receptor can be used to capture its target molecule, separate the target from a complex mixture, and quantify the target molecule as an analyte. As an example, immunoassays usually use antibodies or antibody-derived molecules as receptors. A capture receptor is a receptor that is provided in immobilized form (i.e. attached to a solid phase) or, preferably, in a form that can be immobilized. Immobilization can be performed by a binding pair that links or can link the solid phase and the receptor.

[0037] Very generally speaking, immunoassays provide one or more receptors that can specifically bind to target analytes. Such receptors can be exemplified by analyte-specific immunoglobulins. Hence the name immunoassay. However, for the purposes of this disclosure, other types of analyte-specific receptors are also considered. Therefore, the more general term receptor-based analyte detection assay is appropriate.

[0038] Typically, the target analyte is contained in a sample, which is a complex mixture of different molecules. For the purposes of this disclosure, liquid samples are considered. A liquid sample comprises a liquid phase, i.e. a liquid solvent, which is usually an aqueous solvent. In the aqueous solvent, a number of molecules are present in a dissolved state. Thus, in certain embodiments, the sample is in a liquid aggregate state, which is a single-phase homogeneous mixture. In certain embodiments, the analyte is contained in the mixture in dissolved form, and one or more additional molecules are present in the mixture in dissolved form.

[0039] For the detection of a target analyte present or suspected to be present in a liquid sample by a receptor-based analyte detection assay, in the first essential step, the analyte specifically binds. Specific binding means that a receptor is present or becomes present, which has a high binding affinity and specificity for the target analyte and low or absent for further molecules also present in the sample. In certain embodiments (and many existing assays are exemplified), a compound containing a receptor that can specifically bind to the analyte is added to the sample. Importantly, the mixture of the sample and the compound containing the receptor must provide conditions that allow for a specific interaction of the receptor in the sample with the target analyte. This includes that the conditions in the mixture must be permissive for the actual binding of the analyte by the receptor, and it is also desirable that the conditions stabilize the receptor with the bound target analyte. At the same time, it is desirable that the mixture of the sample and the compound does not support or stabilize the non-specific binding of further molecules to the receptor, or to the compound containing the receptor as a whole.

[0040] The analyte is then immobilized. Immobilization is a key step in the detection process, allowing the analyte to be separated from the surrounding complex mixture, particularly from further molecules of the sample. Immobilization requires a solid phase to which the target analyte becomes attached. Once immobilized, the analyte can be separated from the mixture by phase separation. The analyte is then detected, having been separated from the mixture (i.e. purified).

[0041] Considering a receptor-based analyte detection assay and the immobilization step, it is necessary to provide a solid phase and establish a link between the solid phase and the target analyte. The linkage consists of a self-assembly process. It is desirable to build such a system.

[0042] Immunoassays are well-established bioanalytical methods that detect or quantify analytes and rely on the reaction of the analyte with at least one analyte-specific receptor, resulting in the formation of an analyte:receptor complex. A non-limiting example is the reaction between an antigen and an antibody, respectively. Certain embodiments of "sandwich" immunoassays can be used for analytes with multiple recognition epitopes. Thus, a sandwich assay requires at least two receptors that attach to non-overlapping epitopes on the analyte. In a "heterogeneous sandwich immunoassay", one of the receptors plays the functional role of an analyte-specific capture receptor. This receptor is immobilized or becomes immobilized (during the course of the assay) on a solid phase. A second analyte-specific receptor is provided in dissolved form in the liquid phase. When the respective analyte is bound by the first and second receptors, a sandwich-like complex is formed (receptor-1:analyte:receptor-2). The sandwich-like complex is also called a "detection complex". Within the detection complex, the analyte is sandwiched between the receptors. That is, within such a complex, the analyte represents the link between the first receptor and the second receptor.

[0043] The term "heterogeneous" (as opposed to "homogeneous") denotes two essential and separate steps in the assay procedure. In the first step, a detection complex containing a label is formed and immobilized, while unbound label still surrounds the complex. Before determining the label-dependent signal, the unbound label is washed away from the immobilized detection complex, thus representing the second step. In contrast, homogeneous assays generate an analyte-dependent detectable signal by a single-step incubation and do not require a wash step.

[0044] In a heterogeneous assay, the solid phase is functionalized such that it may bind a functional capture receptor (first receptor) on its surface before contacting the analyte. Alternatively, the surface of the solid phase is functionalized such that it can tether the first receptor after reacting with the analyte. In the latter case, the tethering process must not interfere with the ability of the receptor to specifically capture and bind the analyte. A second receptor present in the liquid phase is used to detect the bound analyte. Thus, in a heterogeneous immunoassay, the analyte can bind to the first (capture) and second (detection) receptors. Thereby, a "detection complex" is formed in which the analyte is sandwiched between the capture receptor and the detector receptor. In a typical embodiment, the detector receptor is labeled before contacting the analyte. Alternatively, the label is specifically attached to the detector receptor after the analyte is bound. If the detection complex is immobilized on the solid phase, the amount of label detectable on the solid phase corresponds to the amount of sandwiched analyte. After a washing step to remove the unbound label, the immobilized label can be detected, indicating the presence and amount of analyte.

[0045] Another well-known embodiment is the competitive heterogeneous immunoassay, which in its simplest form differs from the sandwich-type format by the lack of a second detector receptor. In contrast, a sample containing the analyte is mixed with an artificially generated, labeled analogue that is capable of cross-reacting with the analyte-specific receptor. In the assay, the analyte and the analogue compete for binding to a capture receptor that is immobilized or becomes immobilized. Following this binding step, the greater the amount of immobilized label, the less the amount of unlabeled analyte that was able to compete for the capture receptor. The immobilized label is quantified after a washing step. The amount of label detectable on the solid phase thus corresponds inversely to the amount of analyte initially present in the sample.

[0046] In heterogeneous immunoassays, the washing steps require the non-covalent linkage of the first binding partner to the second binding partner to be sufficiently stable, although the degree of stability required for the linkage varies depending on the strength of the washing step applied. Importantly and unexpectedly, the present invention Binding pairs as described herein are exceptionally well suited to facilitating the immobilization step in immunoassays, i.e., in immunoassays where, for example, a first binding partner of the binding pair attached to a solid phase and a second binding partner of the binding pair attached to an analyte-specific (capture) receptor is highly suited to facilitate immobilization of the receptor to the solid phase.

[0047] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. [Brief description of the drawings]

[0048] [Figure 1] HPLC analysis of single-stranded LNA1 (Example 2) [Diagram 2] HPLC analysis of single-stranded LNA2 (Example 2) [Diagram 3] HPLC analysis of mixed LNA1 and LNA2, immediate injection into the HPLC system (Example 2) [Figure 4] HPLC analysis of mixed LNA1 and LNA2 after heat denaturation before injection (Example 2) Positive control: duplex formation [Diagram 5] HPLC analysis of single-stranded LNA3 (Example 2) [Figure 6] HPLC analysis of single-stranded LNA4 (Example 2) [Figure 7] HPLC analysis of mixed LNA3 and LNA4, slower duplex formation (ratio <0.05) than immediate injection into the HPLC system (Example 2) [Figure 8] HPLC analysis of mixed LNA3 and LNA4, slower duplex formation (ratio=0.05) after 50 min injection (Example 2) [Figure 9] HPLC analysis of mixed LNA3 and LNA4 after heat denaturation prior to injection (Example 2) Positive control: duplex formation

[0049] Example 1 Synthesis of LNA oligonucleotides

[0050] LNA oligonucleotides were synthesized in 1 μmol scale synthesis on an ABI 394 DNA synthesizer using standard automated solid-phase DNA synthesis procedures, applying phosphoramidite chemistry. Glen UnySupport PS (Glen Research cat. no. 26-5040) and LNA phosphoramidites (Qiagen / Exiqon cat. Nos. 33970 (LNA-A(Bz), 339702 (LNA-T), 339705 (LNA-mC(Bz) and 339706 (LNA-G(dmf); β-L-LNA analogs were synthesized analogously to D-β-LNA phosphoramidites starting from L-glucose (Carbosynth, cat. No. MG05247) according to AA Koshkin et al., J. Org. Chem 2001, 66, 8504-8512). Spacer phosphoramidite 18 (Glen Research cat. No. 10-1918) and 5′-biotin phosphoramidite (Glen Research cat.No.10-5950) was used as a component. All phosphoramidites were applied at a concentration of 0.1M in DNA grade acetonitrile. Standard DNA cycles with extended coupling times (180 s), extended oxidation (45 s) and detritylation times (85 s) and standard synthesis reagents and solvents were used for the assembly of LNA oligonucleotides. 5'-biotinylated LNA oligonucleotides were synthesized DMToff, whereas unmodified LNA oligonucleotides were synthesized as DMTon. Cleavage of the LNA oligonucleotides from the support was then performed by concentrated ammonia applying a standard cleavage program. 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, 250 × 21.5 mm (Hamilton, part no. 79352) or XBridge BEH C18 OBD, 5 The oligonucleotides were purified using a 0.1M triethylammonium acetate pH 7 / acetonitrile gradient on a 10μm, 10x250mm (Waters part no. 186008167). The product fractions were combined and desalted by dialysis against water (MWCO 1000, SpectraPor 6, part no. 132638) for 3 days, which also cleaved the DMT group of the DMTon purified oligonucleotides. Finally, the LNA oligonucleotides were lyophilized.

[0051] The yields ranged from 85 to 360 nmol.

[0052] LNA oligonucleotides were analyzed by RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate pH 7 / acetonitrile gradient. Typical purity was ≧90%. The identity of the LNA oligonucleotides was confirmed by LC-MS analysis.

[0053] Example 2

[0054] Identification of LNA oligonucleotide sequences capable of forming duplexes without prior denaturation using RP-HPLC analysis a) General method:

[0055] The LNA oligonucleotides of Example 1 were dissolved in buffer (0.01M Hepes pH 7.4, 0.15M NaCl) and analyzed on a RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1M triethylammonium acetate pH 7 / acetonitrile gradient (8-24% acetonitrile in 10 min, detection at 260 nm).

[0056] The strand and the corresponding counterstrand LNA oligonucleotide were mixed in equimolar concentrations at room temperature and then immediately analyzed on a RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate pH 7 / acetonitrile gradient (8-24% B in 10 min, detection at 260 nm).

[0057] In the first control experiment, the strand and the corresponding counterstrand LNA oligonucleotide were mixed in equimolar concentrations at room temperature and incubated for 1 h at room temperature, then analyzed on a RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate pH 7 / acetonitrile gradient (8-25% acetonitrile in 10 min, detection at 260 nm).

[0058] In a second control experiment showing duplex-forming (positive control) strands and the corresponding opposing strand LNA oligonucleotides were mixed in equimolar concentrations at room temperature, heat denatured at 95°C (10 min), then allowed to reach room temperature and reanalyzed on a RP18 HPLC (Chromolith RP18e, Merck part no. 1.02129.0001) using a 0.1 M triethylammonium acetate pH 7 / acetonitrile gradient (8-24% acetonitrile in 10 min, detection at 260 nm).

[0059] Duplex formation can be detected if a new peak is formed at a different retention time compared to the individual single-stranded LNA oligonucleotides. In a positive control, the mixed and counter strands are heat denatured prior to injection to generate duplexes. The kinetics of duplex formation can be monitored by time-dependent injections after mixing of the strands and counter strand LNAs at room temperature, without prior denaturation.

[0060] The LNA sequences were annealed for 5-60 min at room temperature without prior denaturation and then the HPLC% ratio of the formed duplex to both single-stranded LNAs (corrected for extinction coefficients, taking into account the hyperchromaticity of the duplex) was calculated (HPLC% corrected for extinction coefficients, taking into account higher ratio values ​​if both strands are not exactly equimolar). ≧ When the value is 0.9, it is judged that the double strand can be rapidly formed.

[0061] b) Identification of sequences that rapidly form double strands LNA1: 5'-tgctcctg-3' LNA2: 5'-Bi-Heg-caggagca-3' Heg = hexaethylene glycol Bi = biotin label attached via the carboxy function of the valeric acid moiety of biotin The results are shown in the figure.

[0062] c) Identification of sequences that slowly form double strands LNA3: 5'-ctgcctgacg-3' LNA4: 5'-Bi-Heg-cgtcaggcag-3' The results are shown in the figure.

[0063] Ratio calculation: [Table 1] HPLC%*ε -1 *1000(LNA3 / LNA4 double strand) / HPLC%*ε -1 *1000(LNA3 single strand)=0.023 / 0.456=0.05 HPLC%*ε -1 *1000(LNA3 / LNA4 double strand) / HPLC%*ε -1 *1000(LNA4 single strand)=0.023 / 0.457=0.05

[0064] [Table 2-1] [Table 2-2]

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Claims

1. A method for providing a binding pair, said binding pair consisting of a first single stranded LNA oligonucleotide and a second single stranded LNA oligonucleotide, said two oligonucleotides capable of forming an antiparallel duplex of 8 to 15 consecutive Watson-Crick base pairs at a temperature between 20°C and 40°C, (a) providing a first single-stranded (=ss-) oligonucleotide consisting of 8 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, said nucleobases of said first ss-oligonucleotide forming a first nucleobase sequence; (b) providing a second ss-oligonucleotide consisting of 8 to 15 LNA monomers, said second ss-oligonucleotide consisting of at least the number of monomers of 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 of said second ss-oligonucleotide, said second nucleobase sequence comprising or consisting of a nucleobase sequence complementary to said first nucleobase sequence in an antiparallel orientation, and theoretically said first and second ss-oligonucleotides exhibiting the ability to form a duplex with each other, said duplex consisting of 8 to 15 consecutive Watson-Crick base pairs; (c) mixing and incubating equimolar amounts of the first and second ss-oligonucleotides in an aqueous solution at a temperature between 20° C. and 40° C. for a time interval of not more than 20 minutes, thereby obtaining a mixture of ss-oligonucleotides or a double-stranded mixture; followed by (d) separating the mixture obtained in step (c) at a temperature between 20°C and 40°C, followed by detecting and quantifying the separated duplexes, if present, and the separated ss oligonucleotides; followed by (e) selecting said binding pair if in step (d) a duplex is detectably present and if the molar amount of the duplex is higher than the molar amount of the ss-oligonucleotide; Thereby, providing said binding pair The method includes:

2. The method of claim 1, wherein the first ss-oligonucleotide consists of 5 to 9 monomers.

3. The method of claim 2, wherein the first ss-oligonucleotide consists of 6 monomers.

4. The method of any one of claims 1 to 3, wherein each LNA monomer comprises a nucleobase selected from the group consisting of adenine, thymine, uracil, guanine, cytosine, 5-methylcytosine, 5-hydroxymethylcytosine, 7-deazaguanine, and 7-deazaadenine.

5. The method according to any one of claims 1 to 4, wherein in step (c), the temperature is between 20°C and 37°C.

6. The method according to any of claims 1 to 5, wherein prior to step (c), the first ss-oligonucleotide and the second ss-oligonucleotide are kept at a temperature between -80°C and 40°C, particularly between 20°C and 40°C, more particularly between 20°C and 37°C.

7. The method according to any one of claims 1 to 6, wherein in step (c), the incubation is carried out for 1 minute or less.

8. In step (c), the aqueous solution is added to the aqueous solution, the pH of the solution being adjusted to a value between pH 6 and pH 8, more particularly The method according to any one of claims 1 to 7, further comprising a buffer solution for maintaining the pH at 6.5 to 7.

5.

9. 9. The method according to any of claims 1 to 8, wherein in step (c), the aqueous solution comprises a total amount of dissolved substances of from 10 mmol / L to 500 mmol / L, more particularly from 200 mmol / L to 300 mmol / L.

10. The method of any of claims 1 to 9, wherein step (d) comprises subjecting the incubated mixture of step (c) to column chromatography using an aqueous solvent as a mobile phase.

11. The method according to any one of claims 1 to 10, wherein said ss-oligonucleotides of (a) and (b) consist of beta-D-LNA monomers.

12. The method according to any one of claims 1 to 10, wherein said ss-oligonucleotides of (a) and (b) consist of beta-L-LNA monomers.

13. A liquid composition comprising an aqueous solvent and a binding pair consisting of a first single-stranded oligonucleotide and a second single-stranded oligonucleotide, each oligonucleotide consists of 8 to 15 Locked Nucleic Acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of said monomers forming a first nucleobase sequence of said first oligonucleotide and a second nucleobase sequence of said second oligonucleotide, the first nucleobase sequence and the second nucleobase sequence are selected such that the first oligonucleotide and the second oligonucleotide are capable of forming an antiparallel duplex of 8 to 15 consecutive Watson-Crick base pairs at a temperature of 20° C. to 40° C.; 13. A composition, wherein the binding pair is obtainable by the method according to any one of items 1 to 12.

14. each oligonucleotide consists of 8 to 15 LNA monomers, the first nucleobase sequence and the second nucleobase sequence are selected such that the first oligonucleotide and the second oligonucleotide are capable of forming an antiparallel duplex of 5 to 9 consecutive Watson-Crick base pairs at a temperature of 20° C. to 40° C.; The binding pair can be obtained by the method according to any one of items 2 to 12. Item 14. The composition according to item 13.

15. each oligonucleotide consists of 9 to 15 LNA monomers, the first nucleobase sequence and the second nucleobase sequence are selected such that the first oligonucleotide and the second oligonucleotide are capable of forming an antiparallel duplex of nine consecutive Watson-Crick base pairs at a temperature between 20° C. and 40° C.; The binding pair can be obtained by the method according to any one of items 3 to 12. Item 15. The composition according to item 14.

16. 16. The composition according to any of items 13 to 15, wherein each LNA monomer comprises a nucleobase selected from the group consisting of adenine, thymine, uracil, guanine, cytosine, and 5-methylcytosine.

17. 17. The composition according to any of items 13 to 16, wherein each ss-oligonucleotide comprises two or three different nucleobases.

18. Among the nucleobases of each ss-oligonucleotide, the G+C content is less than 75%. Item 18. The composition according to item 17.

19. 19. The composition according to any one of items 17 and 18, wherein each cytosine of the nucleobases of each ss-oligonucleotide is substituted with a 5-methylcytosine.

20. Use of a composition having a binding pair in a heterogeneous immunoassay comprising an analyte-specific receptor and a solid phase, said composition being a composition according to any one of claims 13 to 19, said binding pair immobilizing the analyte-specific receptor on the solid phase.

21. 20. A kit for performing a heterogeneous immunoassay for detecting an analyte, the kit comprising in separate containers a solid phase to which is attached the first single-stranded oligonucleotide of the binding pair according to any one of claims 13 to 19, and an analyte-specific receptor to which is attached the second single-stranded oligonucleotide of the binding pair according to any one of claims 13 to 19.

Images