HYBRIDIZING all-LNA OLIGONUCLEOTIDES

Abstract

The present report relates to hybridizing single-stranded (ss-) oligonucleotides which entirely consist of locked nucleic acid (LNA) monomers. The present document 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. receptor-based assays.

Claims

1. A method for selecting and providing a binding pair of single-stranded all-LNA oligonucleotides capable of forming in aqueous solution at a temperature from 0° C. to 40° C. an antiparallel duplex with 5 to 15 consecutive base pairs, the method comprising the steps of (a) providing a first single-stranded (=ss-) oligonucleotide consisting of 5 to 15 locked nucleic acid (=LNA) monomers, each monomer comprising a nucleobase, the nucleobases of the first ss-oligonucleotide forming a first nucleobase sequence; (b) providing a second ss-oligonucleotide consisting of 5 to 15 LNA monomers, the second ss-oligonucleotide comprising at least the number of monomers as the first ss-oligonucleotide, each monomer of the second ss-oligonucleotide comprising a nucleobase, the nucleobases of the second ss-oligonucleotide forming a second nucleobase sequence, the second nucleobase sequence comprising or consisting of a nucleobase sequence complementary to the first nucleobase sequence in antiparallel orientation and predicting the capability of the first and second ss-oligonucleotide to form with each other an antiparallel duplex, 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; (c) mixing in an aqueous solution about equal molar amounts of the first and second ss-oligonucleotide, wherein this step is performed at a non-denaturing temperature, more specifically at a temperature from 0° C. to 40° C.; (d) incubating the mixture of (c) for a time interval of 20 min or less, thereby obtaining a mixture still comprising the first and second oligonucleotide as ss-oligonucleotides or a mixture containing or consisting of the first and second oligonucleotide as duplex; (e) detecting and quantifying in the mixture obtained in step (d) ss-oligonucleotides and duplex oligonucleotides; followed by (f) selecting the binding pair if in step (e) duplex is detectably present, and the molar amount of duplex is higher than the molar amount of ss-oligonucleotides; (g) optionally synthesizing separately the first and the second ss-oligonucleotide of a binding pair selected in step (f); thereby selecting and providing the binding pair of single-stranded all-LNA oligonucleotides.

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

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

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

5. The method of claim 4, wherein prior to step (c) each ss-oligonucleotide of any of the steps (a) and (b) is kept in aqueous solution at a temperature from −80° C. to 40° C., specifically from 0° C. to 40° C.

6. The method of claim 1, 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.

7. The method of claim 1, wherein steps (c) and (d) are performed in the absence of a denaturant compound capable of lowering the melting temperature of a DNA duplex of 20 base pairs in length and with a G+C content of 50% by at least 15° C., more specifically in the absence of any of formamide and dimethyl sulfoxide.

8. The method of claim 1, wherein each LNA monomer comprises a nucleobase selected from the group consisting of N.sup.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.sup.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-acetamido-6-hydroxypurine, 2-amino-6-chloropurine, 2-amino-6-iodopurine, azathioprine, 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine, aminophylline, N.sup.6-benzyladenine, N.sup.6-benzoyladenine, 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.sup.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 a derivative thereof.

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

10. The method of claim 8, wherein one or more cytosine(s), if present, is/are replaced by 5-methylcytosine.

11. The method of claim 10, wherein each cytosine is replaced by 5-methylcytosine.

12. The method of claim 1, wherein the monomers of the ss-oligonucleotides of any of the steps (a) and (b) are beta-L-LNA monomers.

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

14. (canceled)

15. The pair of separate complementary ss-oligonucleotides of claim 13, wherein the pair is selected from the group consisting of (SEQ ID NO:1):(SEQ ID NO:2), (SEQ ID NO:9):(SEQ ID NO:10), (SEQ ID NO:11):(SEQ ID NO:12), (SEQ ID NO:13):(SEQ ID NO:14), (SEQ ID NO:15):(SEQ ID NO:16), (SEQ ID NO:16):(SEQ ID NO:20), (SEQ ID NO:17):(SEQ ID NO:18), (SEQ ID NO:19):(SEQ ID NO:20), (SEQ ID NO:21):(SEQ ID NO:22), (SEQ ID NO:23):(SEQ ID NO:24), (SEQ ID NO:25):(SEQ ID NO:26).

16. The pair of separate complementary ss-oligonucleotides of claim 13, wherein the first ss-oligonucleotide of the pair is attached to a first target, and the second ss-oligonucleotide is attached to a second target.

17. The pair of separate complementary ss-oligonucleotides of claim 16, wherein a target is independently selected from the group consisting of a solid phase, a biomolecule, and a chemically synthesized compound.

18. A method of forming an antiparallel all-LNA duplex in the absence of denaturing conditions, the method comprising the steps of (a) providing separately the first and the second member of a pair of single-stranded all-LNA oligonucleotides of claim 13, wherein each single-stranded all-LNA oligonucleotide is separately dissolved in aqueous solution in the absence of a denaturant and kept at a temperature from 0° C. to 40° C.; (b) contacting the single-stranded all-LNA oligonucleotides of the pair with each other at a temperature from 0° C. to 40° C. in the absence of a denaturant; thereby forming the antiparallel all-LNA duplex.

19. (canceled)

20. (canceled)

21. A kit for performing a receptor-based assay for determining an analyte, the kit comprising in a first container an analyte-specific receptor having attached thereto a first member of a pair of separate ss-oligonucleotides of claim 13, the kit further comprising in a second container a solid phase having attached thereto a second member of the pair.

22. A method of performing an receptor-based assay for determining an analyte, the method comprising the steps of contacting the analyte with an analyte-specific receptor having attached thereto a first member of a pair of separate ss-oligonucleotides of claim 13, and with a solid phase having attached thereto a second member of the pair, incubating thereby forming 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 the second member of the pair, wherein the duplex connects the analyte-specific receptor and the solid phase in the complex, followed by detecting analyte bound in the complex, thereby determining the analyte.

23. The method of claim 1, wherein steps (c) and (d) are performed in the absence of a denaturant compound capable of lowering the melting temperature of a DNA duplex of 20 base pairs in length and with a G+C content of 50% by at least 15° C., in the absence of any of formamide and dimethyl sulfoxide.

Description

DESCRIPTION OF THE FIGURES

[0300] FIGS. 1A-1C Schematic view depicting the screening approach for compatible all-LNA oligonucleotide binding pairs (Example 2). Black bars represent quantities of the respective single-stranded molecules, duplex molecules or other complex molecules. [0301] The first (1) and a second (2) single-stranded oligonucleotides are contacted with each other. [0302] FIG. 1A: None of the two oligonucleotides is characterized by inter- or intramolecular secondary structures, both are capable of unencumbered molecular recognition of the respective partner; as a result, duplex is the main abundant resulting product, and single strands are either undetectable or present at insignificant amounts (desired outcome). [0303] FIG. 1B: At least one of the two oligonucleotides is characterized by inter- or intramolecular secondary structures; as a result, duplex is less abundant and the majority of single strands are still present; duplex is formed, however at a reduced rate (not desired outcome). [0304] FIG. 1C: Both oligonucleotides are characterized by inter- or intramolecular secondary structures; as a result, no duplex or an insignificant amount of duplex is formed and the single strands are still present, even after prolonged incubation (not desired outcome).

[0305] FIG. 2 HPLC analysis of single-stranded LNA 1 (SEQ ID NO:1; Example 2); the indicated retention time of the main peak is 3.353 min.

[0306] FIG. 3 HPLC analysis of single-stranded LNA 2 (SEQ ID NO:2; Example 2); the indicated retention time of the minor peak is 6.440 min, the indicated retention time of the main peak is 7.145 min.

[0307] FIG. 4 HPLC analysis of mixed LNA 1 and LNA 2, immediate injection into HPLC system (Example 2); the indicated retention time of the minor peak is 1.671 min, the indicated retention time of the main peak is 6.641 min.

[0308] FIG. 5 HPLC analysis of mixed LNA 1 and LNA 2 after thermal denaturation prior to injection (Example 2); positive control: duplex formation; the indicated retention time of the minor peak is 1.710 min, the indicated retention time of the main peak is 6.656 min.

[0309] FIG. 6 HPLC analysis of single-stranded LNA 3 (SEQ ID NO:5; Example 2); the indicated retention time of the main peak is 3.353 min.

[0310] FIG. 7 HPLC analysis of single-stranded LNA 4 (SEQ ID NO:6; Example 2); the indicated retention time of the minor peak is 6.440 min, the indicated retention time of the main peak is 7.145 min.

[0311] FIG. 8 HPLC analysis of mixed LNA 3 and LNA 4, immediate injection into HPLC system (Example 2); slow duplex formation (ratio <0.05); the indicated retention time of the first peak is 3.387 min, the indicated retention time of the main peak is 7.157 min.

[0312] FIG. 9 HPLC analysis of mixed LNA 3 and LNA 4, injection after 50 min (Example 2); slow duplex formation (ratio=0.05); the indicated retention time of the first peak is 3.365 min, the indicated retention time of the second peak is 6.871 min, the indicated retention time of the third peak is 7.148 min.

[0313] FIG. 10 HPLC analysis of mixed LNA 3 and LNA 4 after thermal denaturation prior to injection (Example 2); positive control: duplex formation; the indicated retention time of the main peak is 6.882 min.

[0314] FIG. 11 HPLC analysis of single stranded LNA 5′-Bi-Heg-accaac-3′ (5′ modified SEQ ID NO:20); the indicated retention time of the main peak is 6.184 min.

[0315] FIG. 12 HPLC analysis of single-stranded LNA 5′-gttggt-3′ (SEQ ID NO:16); the indicated retention time of the minor peak is 1.496, and the indicated retention time of the main peak is 1.865 min.

[0316] FIG. 13 HPLC analysis of mixed LNAs 5′-Bi-Heg-accaac-3′ (SEQ ID NO:20) and 5′-gttggt-3′ (SEQ ID NO:16) (mixing at r.t. and immediate injection); the indicated retention time of the main peak is 6.568 min. Fast duplex formation (100% duplex formation: ratio 1.0). [0317] 5′-gttggt-3′ (SEQ ID NO:14) used up for duplex formation, some residual single stranded LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO:20) detectable.

[0318] FIG. 14 HPLC analysis of mixed LNAs 5′-Bi-Heg-accaac-3′ (SEQ ID NO:20) and 5′-gttggt-3′ (SEQ ID NO:16) (storage and mixing at +4° C. to +6° C. and immediate injection); the indicated retention time of the main peak is 6.588 min, another retention time of 7.056 min is indicated. Fast duplex formation (100% duplex formation: ratio 1.0). 5′-gttggt-3′ (SEQ ID NO:16) used up for duplex formation, minor amount of residual single stranded LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO:20) detectable.

[0319] FIG. 15 HPLC analysis of mixed LNAs 5′-Bi-Heg-accaac-3′ (SEQ ID NO:20) and 5′-gttggt-3′ (SEQ ID NO:16) (storage and mixing at 0° C. (ice bath) and immediate injection); the indicated retention time of the main peak is 6.552 min. Fast duplex formation (100% duplex formation: ratio 1.0). 5′-gttggt-3′ (SEQ ID NO:16) used up for duplex formation, minor amount of residual single stranded LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO:20) detectable.

[0320] FIG. 16 HPLC analysis of mixed LNAs 5′-Bi-Heg-accaac-3′ (SEQ ID NO:20) and 5′-gttggt-3′ (SEQ ID NO:16) (storage and mixing at −10° C. (magnesium chloride/ice bath) and immediate injection); the indicated retention time of the main peak is 6.547 min. Fast duplex formation (100% duplex formation: ratio 1.0). 5′-gttggt-3′ (SEQ ID NO:16) used up for duplex formation, minor amount of residual single stranded LNA 5′-Bi-Heg-accaac-3′ (SEQ ID NO:20) detectable.

[0321] FIG. 17 HPLC analysis of mixed LNAs 5′-Bi-Heg-accaac-3′ (SEQ ID NO:20) and 5′-gttggt-3′ (SEQ ID NO:16) after thermal denaturation and annealing prior to injection; the indicated retention time of the main peak is 6.583 min, another retention time of 6.967 min is indicated. Positive control for duplex formation.

[0322] FIG. 18 HPLC analysis of single stranded LNA 5′-Bi-Heg-cgtcaggcagttcag-3′ (5′ modified SEQ ID NO:47); the indicated retention time of the main peak is 6.840 min.

[0323] FIG. 19 HPLC analysis of single-stranded LNA 5′-ctgaactgcctgacg-3′ (SEQ ID NO:48); the indicated retention time of the main peak is 3.488 min.

[0324] FIG. 20 HPLC analysis of mixed LNAs 5′-Bi-Heg-cgtcaggcagttcag-3′ (SEQ ID NO:47)/5′-ctgaactgcctgacg-3′ (SEQ ID NO:48) (mixing at room temperature and immediate injection); the indicated retention time of the first peak is 3.594 min, the indicated second retention time of the respective peak is 6.580 min. Identification of a sequence pair with slow duplex formation. Slow duplex formation (ratio <0.5).

[0325] FIG. 21 HPLC analysis of mixed LNAs 5′-Bi-Heg-cgtcaggcagttcag-3′ (SEQ ID NO:47)/5′-ctgaactgcctgacg-3′ (SEQ ID NO:48) (storage and mixing at 0° C. (ice bath) and immediate injection); the indicated retention time of the first peak is 3.541 min, the indicated second retention time of the respective peak is 6.853 min. Slow duplex formation (ratio <0.5).

[0326] FIG. 22 HPLC analysis of mixed LNAs 5′-Bi-Heg-cgtcaggcagttcag-3′ (SEQ ID NO:47)/5′-ctgaactgcctgacg-3′ (SEQ ID NO:48) (storage and mixing at −10° C. (magnesium chloride/ice bath) and immediate injection); the indicated retention time of the first peak is 3.516 min, the indicated second retention time of the respective peak is 6.848 min. Slow duplex formation (ratio <0.5).

[0327] FIG. 23 HPLC analysis of mixed LNAs 5′-Bi-Heg-cgtcaggcagttcag-3′ (SEQ ID NO:47)/5′-ctgaactgcctgacg-3′ (SEQ ID NO:48) after thermal denaturation and annealing prior to injection; the indicated retention time of the main peak is 6.580 min. Positive control: duplex formation.

[0328] FIGS. 24A(1)-24A(2) and 24B FIGS. 24A(1)-24A(2): Schematic depiction of the Biacore sensor used in Example 4. [0329] FIG. 24A(1): depicted is the situation of FIG. 24A when the second ss-oligonucleotide is contacted with the sensor having attached the first ss-oligonucleotide [0330] FIG. 24A(2): depicted is the outcome in FIG. 24A wherein the two ss-oligonucleotide are compatible and a duplex is formed under non-denaturing conditions; as a result, the bound second ss-oligonucleotide causes a change that can be detected by the Biacore instrument [0331] FIG. 24B: Individual components as depicted in FIGS. 24A(1)-24A(2). [0332] 1: Sensor surface [0333] 2: Streptavidin attached to the sensor surface [0334] 3: biotin [0335] 4: linker molecule attaching covalently the first ss-oligonucleotide [0336] to biotin. [0337] 5: first ss-oligonucleotide [0338] 6: second ss-oligonucleotide

[0339] FIGS. 25-50 Results of Example 4.

[0340] FIGS. 51A-51C Schematic depiction of the Biacore experiments in Example 5.

[0341] FIGS. 52-53 Results of Example 5.

[0342] FIG. 54 Schematic depiction of the Biacore experiments in Example 6.

[0343] FIG. 55 LNA-constructs binding to Bi-LNA-constructs; binding constants at 25° C.

[0344] FIG. 56 LNA-constructs binding to Bi-LNA-constructs; binding constants at 37° C.

EXAMPLE 1

[0345] Synthesis of LNA Oligonucleotides

[0346] LNA oligonucleotides were synthesized in a 1 μmole scale synthesis on an ABI 394 DNA synthesizer using standard automated solid phase DNA synthesis procedure and applying phosphoramidite chemistry. Glen UnySupport PS (Glen Research cat no. 26-5040) and LNA phosphoramidites (Qiagen/Exiqon cat. No. 33970 (LNA-A(Bz), 339702 (LNA-T), 339705 (LNA-mC(Bz) and 339706 (LNA-G(dmf); beta-L-LNA analogues were synthesized analogously to D-beta-LNA phosphoramidites starting from L-glucose (Carbosynth, cat. No. MG05247) according to A. A. Koshkin et al., J. Org. Chem 2001, 66, 8504-8512) as well as spacer phosphoramidte 18 (Glen Research cat. No. 10-1918) and 5′-Biotin phosphoramidte (Glen Research cat. No. 10-5950) were used as building blocks. All phosphoramidites were applied at a concentration of 0.1 M in DNA grade acetonitrile. Standard DNA cycles with extended coupling time (180 sec), extended oxidation (45 sec) and detritylation time (85 sec) and standard synthesis reagents and solvents were used for the assembly of the LNA oligonucleotides. 5′-biotinylated LNA oligonucleotides were synthesized DMToff, whereas unmodified LNA oligonucleotides were synthesized as DMTon. Then, a standard cleavage program was applied for the cleavage of the LNA oligonucleotides from the support by conc. ammonia. Residual protecting groups were cleaved by treatment with conc. ammonia (8 h at 56° C.). 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 μm, 10×250 mm (Waters part no. 186008167) using a 0.1 M triethylammonium acetate pH 7/acetonitrile gradient. Product fractions were combined and desalted by dialysis (MWCO 1000, SpectraPor 6, part no. 132638) against water for 3 days, thereby also cleaving DMT group of DMTon purified oligonucleotides. Finally, the LNA oligonucleotides were lyophilized.

[0347] Yields ranged from 85 to 360 nmoles.

[0348] 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 purities were ≥90%. Identity of LNA oligonucleotides were confirmed by LC-MS analysis.

[0349] Each species of oligonucleotide was synthesized and kept separately.

EXAMPLE 2

[0350] Identification of LNA Oligonucleotide Sequences Capable of Forming Duplex without Prior Denaturation Applying RP-HPLC Analysis

[0351] a) General Method:

[0352] LNA oligonucleotides from example 1 were dissolved in buffer (0.01 M Hepes pH 7.4, 0.15 M NaCl) and analyzed on 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).

[0353] Strand and corresponding counterstrand LNA oligonucleotides were mixed at equimolar concentration at r.t. (room temperature) and immediately analyzed on 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).

[0354] In a first control experiment strand and corresponding counterstrand LNA oligonucleotides were mixed at equimolar concentration at r.t., incubated 1 h at r.t. and thereafter analyzed on 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).

[0355] In a second control experiment to show duplex formation (positive control) strand and corresponding counterstrand LNA oligonucleotides were mixed at equimolar concentration at r.t., thermally denaturated at 95° C. (10 min), and after having reached r.t. again analyzed on 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).

[0356] Duplex formation can be detected if new peak at different retention time compared to the individual single stranded LNA oligonucleotides is formed. In the positive control mixed strand and counterstrand are thermally denaturated prior to injection yielding duplex. By time dependent injection after mixing strand and counterstrand LNA at r.t. without prior denaturation kinetics of duplex formation can be monitored.

[0357] LNA sequences are determined to be capable of quickly forming duplex if the HPLC % ratio of formed duplex and one of both single stranded LNA (corrected by extinction coefficient; in case both strands are not exactly equimolar higher ratio value is considered) is ≥0.9 after tempering 5-60 min at r.t. without prior denaturation (HPLC % corrected by extinction coefficients; hyperchromicity of duplex not considered).

[0358] b) Identification of Sequence which Forms Duplex Fast

TABLE-US-00005 LNA 1: (SEQ ID NO 1) 5′-tgctcctg-3′ LNA 2: (5′-modified SEQ ID NO 2) 5′-Bi-Heg-caggagca-3′

[0359] Heg=hexaethyleneglycol

[0360] Bi=biotin label attached via the carboxy function of the valeric acid moiety of biotin The results are displayed in FIGS. 2-10.

[0361] c) Identification of Sequence which Forms Duplex Slowly

[0362] For the 10-bp hybridization experiment the following calculation of ratio was made

TABLE-US-00006 LNA 3: 5′-ctgcctgacg-3′ LNA 4 (conjugate): 5′-Bi-Heg-cgtcaggcag-3′

TABLE-US-00007 extinction retention coefficient HPLC time HPLC (ε) [I * % * ε.sup.−1 * LNA [min] % mol.sup.−1 * cm.sup.−1] 1000 LNA 3 single 3.365 45.14 98900 0.456 strand LNA 4 single 7.148 49.98 109300 0.457 strand LNA 3/LNA 4 6.871 4.88 208200 0.023 double strand HPLC % * ε.sup.−1 * 1000 (LNA 3/LNA 4 double strand)/HPLC % * ε.sup.−1 * 1000 (LNA 3 single strand) = 0.023/0.456 = 0.05 HPLC % * ε.sup.−1 * 1000 (LNA 3/LNA 4 double strand)/HPLC % * ε.sup.−1 * 1000 (LNA 4 single strand) = 0.023/0.457 = 0.05

EXAMPLE 3

[0363] Identification of LNA Oligonucleotide Sequences Capable of Forming Duplex without Prior Denaturation Applying RP-HPLC Analysis

[0364] a) General Method:

[0365] LNA oligonucleotides from Example 1 were dissolved in buffer (0.01 M Hepes pH 7.4, 0.15 MNaCl) and analyzed on 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).

[0366] Strand and corresponding counterstrand LNA oligonucleotides (i.e. first and second oligonucleotides) were mixed at equimolar concentration at r.t. (room temperature) or at a temperature selected from 0° C. to 40° C., and immediately analyzed on 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).

[0367] In one type of experiment, strand and corresponding counterstrand LNA oligonucleotides were mixed at equimolar concentration at r.t., incubated 1 h at r.t. and thereafter analyzed on 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). Other experiments were made at different temperatures. Temperatures were selected from 0° C. to 70° C., and 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.

[0368] In a control experiment to show duplex formation (positive control), strand and corresponding counterstrand LNA oligonucleotides were mixed at equimolar concentration at r.t., thermally denaturated at 95° C. (10 min), and, after having cooled down again to room temperature, analyzed on 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).

[0369] Duplex formation is detected if a new peak appears, at a different retention time compared to a peak corresponding to individual single stranded LNA oligonucleotides. In the positive control (control experiment, see above), mixed strand and counterstrand were thermally denaturated prior to injection, thereby disrupting any structures that could be present and capable of preventing duplex formation. Thus, following thermal denaturation, duplex was obtained. By time dependent injection after mixing strand and counterstrand LNA at room temperature without prior denaturation kinetics of duplex formation was monitored.

[0370] LNA sequences were analyzed regarding their capability of quickly forming duplex. In an exemplary but non-limiting case, a positive result was obtained if the HPLC % ratio of formed duplex and one of both single stranded LNA (corrected by extinction coefficient; in case both strands are not exactly equimolar higher ratio value is considered) was ≥0.9 after tempering 5-60 min at r.t. without prior denaturation (HPLC % corrected by extinction coefficients; hyperchromicity of duplex not considered).

[0371] b) Identification of an Exemplary Sequence Pair which is Capable of Fast Duplex Formation Under Non-Denaturing Conditions

[0372] An initial experiment yielded 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).

[0373] Heg=hexaethyleneglycol

[0374] Bi=biotin label attached via the carboxy function of the valeric acid moiety of biotin. These results and others are reflected in Figures.

[0375] The presence of the HEG moiety was found to be without impact on the hybridization properties of the respective oligonucleotide. Regarding nucleobase sequences and hybridization properties, no differences were found with respect to beta-D-LNA oligonucleotide pairs and beta-L-LNA oligonucleotide pairs.

[0376] The following list presents further exemplary results of oligonucleotide pairs which can be kept separately under non-denaturing conditions, and when contacted with each other are capable of forming a duplex under non-denaturing conditions.

[0377] The 15-mer 5′ caccaacacaccaac 3′ (SEQ ID NO 32, tested as 5′-modified Bi-Heg molecule) and 15-mer 5′ gttggtgtgttggtg 3′ (SEQ ID NO 31) showed fast duplex formation upon being contacted with each other.

[0378] Number of Watson-Crick Complementary Base Pairs: 5

[0379] 5′ ggaag 3′/5′ cttcc 3′ were found with fast duplex formation;

[0380] (SEQ ID NOs: 34 and 33, respectively).

[0381] 5′ ggagc 3′/5′ gctcc 3′ were found with fast duplex formation;

[0382] (SEQ ID NOs: 43 and 42, respectively).

[0383] Number of Watson-Crick Complementary Base Pairs: 6

[0384] 5′ accaac 3′/5′ gttggt 3′ were found with fast duplex formation;

[0385] (SEQ ID NOs: 20 and 16, respectively).

[0386] 5′ tttttt 3′/5′ aaaaaa 3′ were found with fast duplex formation;

[0387] (SEQ ID NOs: 27 and 39, respectively).

[0388] 5′ ggagca 3′/5′ tgctcc 3′ were found with fast duplex formation;

[0389] (SEQ ID NOs: 45 and 44, respectively).

[0390] 5′ ctgtca 3′/5′ tgacag 3′ were found with fast duplex formation;

[0391] (SEQ ID NOs: 40 and 41, respectively).

[0392] 5′ ggaaga 3′/5′ tcttcc 3′ were found with fast duplex formation;

[0393] (SEQ ID NOs: 36 and 35, respectively).

[0394] Number of Watson-Crick Complementary Base Pairs: 8

[0395] 5′ caggagca 3′/5′ tgctcctg 3′ were found with fast duplex formation;

[0396] (SEQ ID NOs: 2 and 1, respectively).

[0397] Number of Watson-Crick Complementary Base Pairs: 9

[0398] 5′ ggaagagaa 3′/5′ ttctcttcc 3′ were found with fast duplex formation;

[0399] (SEQ ID NOs: 38 and 37, respectively).

[0400] Number of Watson-Crick Complementary Base Pairs: 15

[0401] 5′ caccaacacaccaac 3′/5′ gttggtgtgttggtg 3′ were found with fast duplex formation;

[0402] (SEQ ID NOs: 32 and 31, respectively).

[0403] Typically, the first oligo of a binding pair as mentioned in the foregoing was used as a Bi-Heg conjugate as described for the initial experiments.

[0404] See FIGS. 11-17 for illustration.

[0405] c) Identification of Sequence which Forms Duplex Slowly

[0406] Number of possible Watson-Crick complementary base pairs: 10

[0407] 5′ cgtcaggcag 3′/5′ ctgcctgacg 3′ were found with slow duplex formation;

[0408] (SEQ ID NOs: 6 and 5, respectively).

[0409] Number of possible Watson-Crick complementary base pairs: 15

[0410] 5′ cgtcaggcagttcag 3′/5′ ctgaactgcctgacg 3′ were found with slow duplex formation; (SEQ ID NOs: 47 and 48, respectively).

[0411] See FIGS. 18-20 and FIGS. 21-23 for illustration.

[0412] FIG. 20 illustrates identification of a binding pair showing slow hybridization. Calculation of ratio of mixed LNAs 5′-Bi-Heg-cgtcaggcagttcag-3′ (5′-modified SEQ ID NO:47) combined with 5′-ctgaactgcctgacg-3′ (SEQ ID NO:48.

TABLE-US-00008 extinction retention coefficient HPLC time HPLC (ε) [I * % * ε.sup.−1 * LNA oligo [min] % mol.sup.−1 * cm.sup.−1] 1000 LNA of SEQ 3.59 37.44 158000 0.237 ID NO:48 (single strand) LNAs of SEQ ID 6.58 33.29 320400 0.104 NO:47/SEQ ID NO:48 (duplex) HPLC % * ε.sup.−1 * 1000 (LNA duplex)/HPLC % * ε.sup.−1 * 1000 (LNA single strand) = 0.104/0.237 = 0.44

EXAMPLE 4

[0413] Biospecific Interaction Analysis, Immobilized First LNA Oligonucleotide Contacted with Second LNA Oligonucleotide, Three Different Motifs; Kinetic Characterization at 25° C. and 37° C.

[0414] a) Outline of the Approach [0415] 12 different oligonucleotide LNA sequences were designed and terminally biotinylated (Bi-LNA-sequences) [0416] 7 LNA oligonucleotide sequences were analyzed for binding to the 12 immobilized Bi-LNA-sequences at 25° C./37° C. [0417] 3 min association time and 5 respectively 30 min dissociation time with flow rate 60 μl/min [0418] LNA-samples were preincubated over night at RT (room temperature) in slightly basic buffer as recommended by the manufacturer (chemical deactivation) [0419] Test setup is depicted on FIGS. 24A(1) & 24A(2)

[0420] b) Technical Procedure

[0421] Kinetic investigations were performed on a GE Healthcare Biacore 8k instrument.

[0422] A Biacore Biotin Capture Kit, Series S sensor (Cat.-No. 28-9202-34) was mounted into the instrument and was hydrodynamically addressed and preconditioned 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, as provided by the manufacturer GE Healthcare, was diluted 1:50 in system buffer and was injected at 10 μl/min for 60 sec into all measurement flow cells. The reference cells were not immobilized and remained blanc controls. 10 nM of the respective biotinylated ligand was injected at 30 μl/min to obtain ligand capture levels between 4 RU to 30 RU. Concentration series of analytes in solution were injected at 60 μl/min for 3 min association time. Dissociation was monitored for 5 minutes. High affinity interactions were monitored for 30 minutes dissociation time. Analyte concentration series were 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 1 minute injection of 100 mM NaOH. Kinetic data was determined using the Biacore Evaluation software.

[0423] c) results

[0424] 7 different LNA oligonucleotides representing 3 different sequence motifs were analyzed for binding 12 different complementary biotinylated LNA oligonucleotides (Bi-LNA) having sequences of varying length, at two different temperatures: 25° C. and 37° C.

[0425] 0.05 Tween 20 was used as detergent in the SPR-measurements.

[0426] Initial Molar ratio MR=1.0−0.7 (data not shown) indicated 1:1 binding, MR decreased during analysis cycles (MR=0.5−0.3), most probably due to deactivation of streptavidin surface by the use of harsh basic pH during regeneration steps.

[0427] Motif “Sequence 1”

[0428] 9mer LNA showed binding to complementary Bi-LNA 7-9mers with motif 1

[0429] At 37° C. ′′ Bi-(HEG).sub.4-5′ caggagca 3′ (5′-modified SEQ ID NO:2)′ and ‘Bi-(HEG).sub.4-5′ caggagc 3′ (5′-modified SEQ ID NO:15) showed high complex stabilities with the complementary oligonucleotide 5′ tgctcctgt 3′ (SEQ ID NO:9) ‘with and without (HEG).sub.4-MH-5′-tag.

[0430] t.sub./2 diss (Bi-LNA 7&8mer/9mer)=>247/228 minutes,

[0431] t.sub./2 diss (Bi-LNA 7&8mer/9mer with Heg4-MH5′)=>734/800 minutes, resulting in high affinities (K.sub.D=6-9 pM):

[0432] Hybridization with “Sequence 2”-LNA showed weaker binding to Sequence 1 Bi-LNA 7-9mer

[0433] No binding detectable for 9-mer A sequence ‘5’ aaaaaaaaa ‘3’ (SEQ ID NO:28) including the (HEG).sub.4-MH-5′-tag.

[0434] Motif “Sequence 2”

[0435] Bi-LNAs representing motif 2 with varying length (12mer, 10 mer, 8mer, 7mer and 6mer) didn't show any binding to LNA-motifs 1 or 3.

[0436] Motif 2-LNA-binding with varying length showed comparable complex formation for 6-8mers, and only slightly slower complex formation for the 12mer.

[0437] Complex stability varied: Bi-LNA 7mer showed highest complex stabilities in these experiments, followed by the Bi-LNA 8mer

[0438] t.sub./2 diss (Bi-LNA 7mer/6mer)=238 minutes,

[0439] t.sub./2 diss (Bi-LNA 7mer/7mer)=720 minutes,

[0440] t.sub./2 diss (Bi-LNA 7mer/8mer)=644 minutes,

[0441] t.sub./2 diss (Bi-LNA 8mer/7mer)=545 minutes,

[0442] t.sub./2 diss (Bi-LNA 8mer/8mer)=433 minutes, resulting in high affinities (K.sub.D=1-5 pM)

[0443] Motif 2 was estimated to be superior to motif 1

[0444] Motif “Sequence 3”

[0445] The 5′ modified poly-A Sequence (HEG).sub.4-MH-5′ sequence 5′ aaaaaaaaa 3′ (SEQ ID NO:28) ‘ ’ was used as negative control. This control did n'ot show any binding to any of the biotinylated Sequences 1 and 2. However, specific binding was detected with complementary Poly-T-Sequences from “group 3”.

[0446] At 37° C. complex stability increased significantly with increasing LNA-length from 6-9mer by factor 1000 (t/2 diss=1 to >1160 minutes) & complex formation decreased by factor 55, resulting affinities were in a range KD=300 pM-10 pM-ssL-DNA hybridization is slower with increasing length from 6mer to 9mer, complex stabilities are persistently high; Overhangs with >2 unpaired nucleotides obviously decreased the complex stability for PolyA/PolyT-pairing.

[0447] d) Conclusion

[0448] With the goal in mind to provide a replacement for the streptavidin:biotin binding pair it is important to select a low affinity (desired to be in the pM range) binding pair with very fast association rate constant already at 25° C., and a persisting high complex stability at 37° C.

[0449] All-LNA oligonucleotide duplex with 4 to 5 complementary LNA nucleobase pairings representing motif “Sequence 2” meet the demands for a desired binding pair in that they show fast association into saturation and high complex stability.

[0450] In order to secure quick association, the binding pair is desired to be as short as possible to circumvent time consuming “mispriming” intermediates. ‘Bi-(HEG).sub.4-5’ caccaac 3′ (7mer oligonucleotide, SEQ ID NO:19) binding to 5′ gttggt 3′ and Bi-(HEG).sub.4-MH-5′ gttggtgt 3′ (5′-modified SEQ ID NO:16) showed complex formation and complex stability with t.sub./2 diss=720 respectively 644 minutes and a resulting high affinity (K.sub.D=2 pM) at 37° C. ‘Bi-(HEG).sub.4-5’ acaccaac 3′ (8mer, 5′-modified SEQ ID NO:14) binding to (HEG).sub.4-MH-5′ gttggtg 3′ (5′-modified SEQ ID NO:21) and to (HEG).sub.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 minutes, respectively, and a resulting high affinity (KD=2 pM) at 37° C.

[0451] LNA with varying length with motif “sequence 1” did not bind. As a negative control the sequence 5′ aaaaaaaaa 3′ (SEQ ID NO:28) including the (HEG).sub.4-MH-5′-tag was tested, ‘without any observation of measurable intermolecular interactions.

EXAMPLE 5

[0452] Biospecific Interaction Analysis

[0453] a) Outline of the Approach and Assay Set-Up [0454] reversible captured streptavidin-conjugate via CAP-Kit [0455] streptavidin is conjugated with complementary ss-LNA [0456] oligo binding reversible to pre-immobilized ss-LNA oligo on a SCM

[0457] Determination of: [0458] Capture Level (CL), association rate constant k.sub.a, [0459] dissociation rate constant k.sub.d, [0460] dissociation equilibrium constant K.sub.D [0461] Molar ratio (MR) [0462] Test setup is depicted on FIG. 51 A [0463] 4 free LNA constructs “motif 2” with varying length (6-8mer & 12mer) and

[0464] LNA-Fab<TSH>-conjugates (LNA-Fab<TSH>=antibody Fab fragments specific for the TSH antigen) were analyzed for binding to complementary Bi-LNA-Sequences at 25°/37° C. [0465] Bi-LNA-Sequences were captured as ligands on a CAP-Chip via reversible Biotin-Capture-Kit; [0466] free LNA or LNA-Fab<TSH>-conjugates were used as analytes in solution [0467] Hybridization was analyzed with 3 minutes association time and 30 minutes dissociation time, [0468] flow rate 60 μl/min [0469] c.sub.(free LNAs)=9-0.1 nM, c.sub.(LNA-Fab<TSH>-conjugates)=45−0.6 nM, c.sub.(12merLNA/Fab<TSH>-conjugates)=45−0.6 nM [0470] LNA-samples were pre-incubated over night at RT in slightly basic buffer as recommended from customer (chemically deactivation)

[0471] b) Reagents: Sequence “Motif 2”

[0472] Biotinylated Ligands

TABLE-US-00009 (SEQ ID NO: 20) ′Bi-(HEG).sub.4-5′ accaac 3′ BMO 28.542740, GO4094, ID 6681, 6mer, MW 3.8 kDa (SEQ ID NO: 19) ′Bi-(HEG).sub.4- 5′ caccaac 3′ BMO 28.542739, GO4093, ID 6681, 7mer, MW 4.1 kDa (SEQ ID NO: 14) ′Bi-(HEG).sub.4-5′ acaccaac 3′ BMO 28.542738, GO4092, ID 6680, 8mer, MW 4.4 kDa (SEQ ID NO: 52) ′Bi-(HEG).sub.4- 5′ caacacaccaac 3′ BMO 28.542742, GO4096, ID 6684, 12mer, MW 5.8 kDa

[0473] Analytes

TABLE-US-00010 (SEQ ID NO: 16)′ 2300/103 (HEG).sub.4-MH-5′ gttggt 3′ BMO 28.170333, AO581, ID 6719, 6mer, MW 3.8 kDa (SEQ ID NO: 21)′ 2300/104 (HEG).sub.4-MH-5′ gttggtg 3′ BMO 28.170334, AO582, ID 6720, 7mer, MW 4.1 kDa (SEQ ID NO: 13)′ 2300/105 (HEG).sub.4-MH-5′ gttggtgt 3′ BMO 28.170335, AO583, ID 6721, 8mer, MW 4.4 kDa (SEQ ID NO: 53)′ 2300/102 (HEG).sub.4-MH-5′ gttggtgtgttg 3′ BMO 28.542727, GO4073, ID 6653, 12mer, MW 5.8 kDa

[0474] ‘mAb<TSH>M-Tu1.20-F(ab′)2-SATP-D-LNA-conjugates

TABLE-US-00011 (SEQ ID NO: 16) 2331/111 mAb<TSH>M-Tu1.20-F(ab′).sub.2-SATP-D-LNA- 5′ gttggt 3′, 6mer, MW 104 kDa (SEQ ID NO: 21) 2331/112 mAb<TSH>M-Tu1.20-F(ab′).sub.2-SATP-D-LNA- 5′ gttggtg 3′, 7mer, MW 104 kDa (SEQ ID NO: 13) 2331/113 mAb<TSH>M-Tu1.20-F(ab′).sub.2-SATP-D-LNA- 5′ gttggtgt 3′, 8mer, MW 104 kDa (SEQ ID NO: 53) 2331/114 mAb<TSH>M-Tu1.20-F(ab′).sub.2-SATP-D-LNA- 5′ gttggtgtgttg 3′, 12mer, MW 106 kDa

[0475] mAb<TSH>M-Tu1.20-F(ab′).sub.2 signifies a F(ab′)2 fragment of a monoclonal antibody specific for TSH which is human Thyroid-stimulating hormone. D-LNA signifies that the oligonucleotide with the subsequent nucleobase sequence consists of D-LNA monomers. D-LNA oligonucleotides were used

[0476] c) Results

[0477] The four different 5′-modified LNA oligonucleotides as given above, and oligonucleotides with the same respective sequences but comprised in F(ab′).sub.2<TSH>conjugates represented sequence “motif 2” with 6-, 7-, 8- & 12-mer length. All were analyzed for binding their complementary Bi-LNA-Sequence at 25°/37° C.

[0478] TSH-binding (TSH being the analyte) to hybridized LNA-Fab<TSH>conjugates was analyzed, too.

[0479] Sequence “Motif 2”

[0480] Bi-LNAs of varying length showed comparable complex formation, the complex stabilities range from t.sub./2 diss 154 to >232 minutes with pM affinity range (K.sub.D 3-10 pM) at 25° C. At 37° C., the complex formations are in the pM affinity range (K.sub.D 11-26 pM). Hybridization kinetics of the free oligos are mass-transport limited (data depicted in red) at 25° C. & 37° C., correction for MTL was performed using SW Scrubber. Molar Ratios (MR) 0.8-1.1 indicated stoichiometric 1:1 hybridization at both temperatures. Oversaturation of 12-mer association phase at 25° C.

[0481] The LNA-Fab<TSH>conjugates showed a factor 2-4 slowed down complex formation in comparison to the unconjugated LNA oligonucleotides. No MTL during hybridization of the Fab<TSH>-LNA-conjugates. Complex stabilities t.sub./2 diss>232 minutes, resulting in pM affinity range (K.sub.D=22-11 pM) at 25° C. At 37° C. FAb<TSH>-LNA-conjugates (7-, 8- & 12mer) showed no significant differences in complex formation when compared to free LNA of same length, complex stabilities t.sub./2 diss>232 minutes, ranging in the pM affinity range (K.sub.D<12-14 pM).

[0482] Molar Ratios for LNA-Fab<TSH>conjugates MR 0.1-0.6 indicated sub-stoichiometric 1:1 binding. TSH binding to hybridized LNA-FAb<TSH>conjugates of varying length was analyzed. TSH-binding to LNA-Fab<TSH>conjugates (6-8mer & 12mer) showed comparable kinetic profiles Fast complex formation and sufficient complex formation with t.sub./2 diss 31-33 minutes, resulting affinities K.sub.D=0.7 nM

[0483] The binding constants are in the known affinity range for this interaction.

[0484] Molar Ratios (MR) 1.8/1.9 showed fully functional stoichiometric 2:1 binding indicating binding functional conjugates.

[0485] It was found that hybridized Fab conjugates show full antigen binding activity of the antibody moieties in the conjugates. Results see FIG. 52.

TABLE-US-00012 Also see FIG. 51 C for the following data primary secondary antibodies antibodies Antibody A 23C11-IgG 6C6-IgG 4H4-IgG Antibody A 0.1 0.1 0.1 0.1 23C11-IgG 0.2 0.0 0.0 0.0 6C6-IgG 0.4 0.0 0.0 0.0 4H4-IgG 0.2 0.0 0.0 0.0

[0486] Table: Molar Ratio Epitope Accessibility Matrix showing the hTK sandwich formation of 4 anti-hTK antibodies. MR.sub.EA=1, fully independent epitope, MR.sub.EA<1 overlapping epitopes.

[0487] Antibody A is able to form immunocomplexes with 23C11, 6C6 and 4H4. 23C11, 6C6 and 4H4 share the same epitope.

[0488] d) Alternative Approach (See FIG. 51 B, Results FIG. 53) [0489] TSH-binding to pre-hybridized LNA-FAb<TSH>conjugates of varying length (6-, 7-, 8- & 12mer) was analyzed at 37° C. [0490] Assay format see slide 10, binding profiles see slides 11 [0491] 3 Bi-LNA-Sequences were irreversibly bound to a SA-Chip on Fc2-4 [0492] LNA-Fab<TSH>conjugations (6-, 7- & 8mer) were pre-hybridized with their complementary Bi-LNA at 37° C. [0493] TSH was used as analyte in solution with 3 minutes association time and 5 minutes dissociation time, [0494] flow rate 60 μl/min, x.sub.TSH=270 nM

[0495] Results see FIG. 53.

EXAMPLE 6

[0496] Biospecific Interaction Analysis

[0497] a) Outline of the Approach and Assay Set-Up [0498] Schematic overview of the experiment is presented on FIG. 54 [0499] free LNA constructs of varying length (5-, -6-, 9- or 15mer) and sequences were analyzed for binding to complementary Bi-LNA-Sequences (4-6-, 9- or 15mer) at 25°/37° C. [0500] Bi-LNA-Sequences were captured as ligands on a CAP-Chip via reversible Biotin-Capture-Kit; [0501] free LNAs were used as analytes in solution [0502] Hybridization was analyzed with 3 minutes association time and 30 minutes dissociation time, [0503] flow rate 60 μl/min [0504] c.sub.(free LNAs)=optimized for each interaction

[0505] Determination of: Capture level (CL), association rate constant k.sub.a, dissociation rate constant k.sub.d, dissociation equilibrium constant K.sub.D, molar ratio (MR).

[0506] reversible captured SA-conjugate via CAP-Kit, streptavidin (=SA)-conjugated with complementary ss-LNA oligo binding reversible to pre-immobilized ss-LNA oligo.

[0507] b) Reagents

[0508] Biotinylated Ligands

TABLE-US-00013 (SEQ ID NO: 20) 2387/L01 Bi-(HEG)-5′ accaac 3′ BMO 28.170341, AO591, ID 6730, 6mer, MW 2.71 kDa (SEQ ID NO: 30) 2387/L02 Bi-(HEG)-5′ cacaccaac 3′ BMO 28.170342, AO592, ID 6731, 9mer, MW 3.71 kDa (SEQ ID NO: 54) 2387/L03 Bi-(HEG)-5′ caccaacacaccaac 3′ BMO 28.170343, AO593, ID6732, 15mer, MW 5.73 kDa (SEQ ID NO: 34) 2387/L04 Bi-(HEG)-5′ ggaag 3′ BMO 28.170347, AO597, ID6736, 5mer, MW 2.44 kDa (SEQ ID NO: 36) 2387/L05 Bi-(HEG)-5′ ggaaga 3′ BMO 28.170348, AO598, ID 6737, 6mer, MW 2.78 kDa (SEQ ID NO: 38) 2387/L06 Bi-(HEG)-5′ ggaagagaa 3′ BMO 28.170349, AO599, ID 6738, 9mer, MW 3.82 kDa (SEQ ID NO: 27) 2300/12 Bi-(HEG).sub.4-5′ tttttt 3′ BMO 28.170336, AO584, ID 6722, 6mer, MW 3.71 kDa (SEQ ID NO: 40) 2387/L08 Bi-(HEG)-5′ ctgtca 3′ BMO 28.170354, AO604, ID 6743, 6mer, MW 2.71 kDa (SEQ ID NO: 55) 2387/L09 Bi-(HEG)-5′ cgtcaggcagttcag 3′ BMO 28.170356, AO606, ID 6745, 15mer, MW 5.12 kDa (SEQ ID NO: 43) 2387/L10 Bi-(HEG)-5′ ggagc 3′ BMO 28.170358, AO608, ID 6747, 5mer, MW 2.43 kDa (SEQ ID NO: 45) 2387/L11 Bi-(HEG)-5′ ggagca 3′ BMO 28.170360, AO610, ID 6749, 6mer, MW 2.77 kDa (SEQ ID NO: 46) 2387/L12 Bi-(HEG).sub.4-5′-ccaac 3′ BMO 28.542748, GO4105, ID 6764, 5mer, MW 3.40 kDa (SEQ ID NO: 56) 2387/L13 Bi-(HEG).sub.4-5′ caac 3′ BMO 28.542749, GO4106, ID 6765, 4mer, MW 3.07 kDa (SEQ ID NO: 57) 2387/L14 Bi-(HEG).sub.4-5′ ttttt 3′ BMO 28.542750, GO4107, ID 6766 5mer, MW 3.38 kDa (SEQ ID NO: 58) 2387/L15 Bi-(HEG).sub.4-5′ tttt 3′ BMO 28.542751, GO4108, ID 6768 4mer, MW 3.05 kDa

[0509] Analytes

TABLE-US-00014 2387/A01 3′-TGG TTG-5′ BMO 28.170344, AO594, ID, 6733, 6mer, MW 2.01 kDa 2387/A02 3′-GTG TGG TTG-5′ BMO 28.170345, AO595, ID 6734, 9mer, MW 3.05 kDa 2387/A03 3′-GTG GTT GTG TGG GTT -5′ BMO 28.170346, AO596, ID 6735, 15mer, MW 5.12 kDa 2387/A04 3′-CCT TC-5′ BMO 28.170350, AO600, ID 6739, 5mer, MW 1.60 kDa 2387/A05 3′-CCT-TCT-5′ BMO 28.170351, AO601, ID 6740, 6mer, MW 1.93 kDa 2387/A06 3′-CCT TCT CTT-5′ BMO 28.170352, AO602, ID 6741, 9mer, MW 2.92 kDa 2387/A07 3′-AAA AAA-5′ BMO 28.170353, AO603, ID 6742, 6mer, MW 1.99 kDa 2387/A08 3′-GAC AGT-5′ BMO 28.170355, AO605, ID 6744, 6mer, MW 2.00 kDa 2387/A09 3′-GCA GTC CGT CAA GTC-5′ BMO 28.170357, AO607, ID 6746, 15mer, MW 5.04 kDa 2387/A10 3′-CCT CG-5′ BMO 28.170359, AO609, ID 6748, 5mer, MW 1.62 kDa 2387/A11 3′-CCT CGT-5′ BMO 28.170361, AO611, ID 6750, 6mer, MW 1.95 kDa

[0510] c) Results

[0511] 11 LNAs with different sequences were analyzed for binding to their complementary Bi-LNA-Sequences. Additionally 2 Bi-LNAs of different length (5mer & 4mer) pairing with free LNA 6mer were analyzed at 25°/37° C.

[0512] Sequence “Motif 2” (2387/L01-L03) & “Short Motif 2” (2387/L12& L13) 6mer & 9mer Bi-LNA 5′-Bi-Heg-ACC AAC-3′ and 5′-Bi-Heg-CAC ACC AAC-3′ show high affinity-binding to their complementary LNAs 6mer 3′-TGG TTG-5′ respectively 9mer 3′-GTG TGG TTG-5′. Kinetic signatures are characterized by fast hybridization, complex stabilities are persistently high with t.sub./2 diss=160 to >232 minutes and pM-affinity range (K.sub.D=1-9 pM) at 25° & 37° C.; hybridization kinetics are mass-transport limited at 25° C. & 37° C., correction for MTL was made, too.

[0513] Molar Ratios (MR) 1.1/1.2 indicate stoichiometric 1:1 hybridization at both temperatures for the 6mer pair; MR 1.3/1.5 indicate over-stoichiometric binding for the 9mer-pair. The Bi-LNA 15mer shows slowed down hybridization kinetics compared to 6 & 9mers, resulting in slightly lower affinities. K.sub.D=24/53 pM at both temperatures; MR 1.3 indicates slightly over-stoichiometric binding.

[0514] When binding to 5mer (2387/L12) Bi-LNA 5′Bi-4x(HEG)-CCA AC-3′ free 6mer-LNA 3′-TGG TTG-5′ shows reduced complex stability t.sub./2 diss=50 minutes with 2 digit pM-affinity range, when binding to 4mer (2387/L13) 5′Bi-4x(HEG)-CAA C-3′ the complex stability drops down to t.sub./2 diss<1 minute, resulting in 2 digit nM affinity. MR 1.1-1.3 indicate slightly over-stoichiometric binding Overhangs with 1 or 2 unmatched nucleotides may thus be interpreted in the present case to decrease complex stability to some extent.

[0515] “Motif 3” Poly-T controls “short” (2300/12 and 2387/L14 & L15); 3 PolyT-controls Bi-LNA of varying length (5- &-6mer) show binding to PolyA-6mer with typical fast on-/off-profiles, complex-half-lifes t.sub./2 diss<2 minutes at 25° & 37° C., the Bi-LNA 4mer shows only weak/no binding to PolyA-6mer.

[0516] “Motif 4” (2387/L04-L06) 5-, 6- or 9mer 5′-Bi-Heg-GGA AG-3′, 5′-Bi-Heg-GGA AGA-3′, or 5′-Bi-Heg-GGA AGA GAA-3′ are inferior to “motif 2” when binding to their complementary LNAs, complex half-lifes t.sub./2 diss between 20-65 minutes with resulting 2-3 digit pM affinities at 25° C.

[0517] “Motif 5” (2387/L08) 5′-Bi-Heg-CTG TCA-3′ binding to complementary LNA 3′-GAC AGT-5′ shows slightly slower hybridization than 6mer of “motif 2”, complex stabilities with 2-digit pM affinities for 25° & 37° C. The Molar Ratios 0.3/0.4 indicate sub-stoichiometric binding.

[0518] “Motif 6” (2387/L09) The 15mer 5′-Bi-Heg-CGT CAG GCA GTT CAG-3′ binding 3′-GCA GTC CGT CAA GTC-5′ shows a 1-digit nM-affinity interaction caused by slowed down hybridization and reduced complex stability; The Molar Ratios 0.1/0.4 indicate sub-stoichiometric binding.

[0519] “Motif 7” (2387/L10 & L11)

[0520] 5mer 5′-Bi-Heg-GGA GC-3′ outperformed the 6mer 5′-Bi-Heg-GGA GCA-3′ binding their complementary LNAs; complex half-lifes t.sub./2 diss between 174 and 62 minutes with 32/186 pM affinity-range at 25° C., 5 mer shows 29 pM interaction at 37° C., Molar Ratios MR 0.5/0.3 indicate sub-stoichiometric binding at 25° C. and increasing at 37° C. (MR 1.2/0.6).

[0521] d) Conclusion

[0522] Both Bi-LNA 5′-Bi-Heg-ACC AAC-3′ and 5′-Bi-Heg-CAC ACC AAC-3′ (“motif 2”) show high-affinity binding to their complementary LNAs 6mer 3′-TGG TTG-5′ resp. 9mer 3′-GTG TGG TTG-5′. The Molar Ratio indicates fully functional 1:1 LNA-hybridization. 5′-Bi-Heg-ACC AAC-3′/3′-TGG TTG-5′ shows slightly improved hybridization kinetics in comparison to 5′-Bi-(HEG)4-ACCAAC-3′/3′-TGG-TTG-5′-Heg4-MH-5′, due to 2-fold improved complex stability.

[0523] The absolute density for captured Bi-LNA 5′-Bi-Heg-ACC AAC-3′ on the sensor surface used in the experiments was 11 fmol/mm.sup.2 (30 pg/mm.sup.2), based on the vendor information 1000 RU=1 ng/mm.sup.2. In a hydrogel it is assumed to correspond to a concentration 0.3 mg/mL.