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. immunoassays.

Claims

1. A method for providing a binding pair, the binding pair consisting of a first single-stranded (ss) locked nucleic acid (LNA) oligonucleotide and a second single-stranded LNA oligonucleotide, the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide being capable of forming an antiparallel duplex of 8 to 15 consecutive Watson-Crick base pairs at a temperature from about 20° C. to about 40° C., the method comprising the steps of: (a) providing a first ss-LNA oligonucleotide consisting of 8 to 15 LNA monomers, each LNA monomer comprising a nucleobase, the nucleobases of the first ss-LNA oligonucleotide forming a first nucleobase sequence of the first ss-LNA oligonucleotide; (b) providing a second ss-LNA oligonucleotide consisting of 8 to 15 LNA monomers, the second ss-LNA oligonucleotide consisting of at least the same number of LNA monomers as the first ss-LNA oligonucleotide, each LNA monomer of the second ss-LNA oligonucleotide comprising a nucleobase, the nucleobases of the second ss-LNA oligonucleotide forming a second nucleobase sequence of the second ss-LNA oligonucleotide, the second nucleobase sequence comprising a nucleobase sequence complementary to the first nucleobase sequence in antiparallel orientation, wherein the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide have the capability to form an antiparallel duplex with each other, the antiparallel duplex consisting of 8 to 15 consecutive Watson-Crick base pairs; (c) mixing equal molar amounts of the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide in an aqueous solution to obtain a mixture and incubating the mixture for a time interval of 20 minutes or less at a temperature ranging from about 20° C. to about 40° C. to form the antiparallel duplex; (d) separating the antiparallel duplex, if present, the first ss-LNA oligonucleotides and the second ss-LNA oligonucleotides from the mixture in step (c) at a temperature ranging from about 20° C. to about 40° C., followed by detecting and quantifying the separated antiparallel duplex, the separated first ss-LNA oligonucleotides and the separated second ss-LNA oligonucleotides; (e) selecting the separated antiparallel duplex as the binding pair if in step (d) the antiparallel duplex is detectably present, and if the molar amount of the antiparallel duplex is higher than the molar amounts of the separated first ss-LNA oligonucleotides and the separated second ss-LNA oligonucleotides; thereby providing the binding pair.

2. The method according to claim 1, wherein the first ss-LNA oligonucleotide consists of 8 to 12 LNA monomers.

3. The method according to claim 2, wherein the first ss-LNA oligonucleotide consists of 9 LNA monomers.

4. The method according to claim 1, 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 claim 1, wherein in step (c) the temperature ranges from about 20° C. to about 37° C.

6. The method according to claim 1, wherein prior to step (c) the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide are kept at a temperature ranging from about −80° C. to about 40° C.

7. The method according to claim 1, wherein in step (c) the incubation is performed for 1 minute or less.

8. The method according to claim 1, wherein in step (c) the aqueous solution contains a buffer maintaining the pH of the solution from about pH 6 to about pH 8.

9. The method according to claim 1, wherein in step (c) the aqueous solution contains an aggregate amount of dissolved substances from 10 mmol/L to 500 mmol/L.

10. The method according to claim 1, wherein step (d) comprises subjecting the incubated mixture of step (c) to column chromatography with an aqueous solvent as mobile phase.

11. The method according to claim 1, wherein the first ss-LNA oligonucleotides and the second ss-LNA oligonucleotides of (a) and (b) consist of beta-D-LNA monomers.

12. The method according to claim 1, wherein the first ss-LNA oligonucleotides and the second ss-LNA oligonucleotides of (a) and (b) consist of beta-L-LNA monomers.

13. A liquid composition comprising an aqueous solvent and a binding pair, the binding pair comprising a first single-stranded (ss-) locked nucleic acid (LNA) oligonucleotide and a second ss-LNA oligonucleotide, wherein each of the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide consists of 8 to 15 LNA monomers, each LNA monomer comprising a nucleobase, the nucleobases of the LNA monomers forming a first nucleobase sequence of the first ss-LNA oligonucleotide and a second nucleobase sequence of the second ss-LNA oligonucleotide, and wherein the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide form an antiparallel duplex of 8 to 15 consecutive Watson-Crick base pairs at a temperature from 20° C. to 40° C.

14. The composition according to item 13, wherein each of the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide consists of 8 to 15 LNA monomers, and wherein the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide form an antiparallel duplex of 8 to 12 consecutive Watson-Crick base pairs at a temperature from 20° C. to 40° C.

15. The composition according to claim 14, wherein each of the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide consists of 8 to 15 LNA monomers, and wherein the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide form an antiparallel duplex of 9 consecutive Watson-Crick base pairs at a temperature from 20° C. to 40° C.

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

17. The composition according to claim 13, wherein each of the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide contains two or three different nucleobases.

18. The composition according to claim 17, wherein among the nucleobases in each of the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide the G+C content is lower than 75%.

19. The composition according to claim 17, wherein among the nucleobases in each of the first ss-LNA oligonucleotide and the second ss-LNA oligonucleotide each cytosine is replaced by a 5-methylcytosine.

20. A kit for performing a heterogeneous immunoassay for detecting an analyte, the kit containing in separate containers a solid phase having attached thereto the first ss-LNA oligonucleotide of the binding pair according to claim 13, and an analyte-specific receptor having attached thereto the second ss-LNA oligonucleotide of the binding pair according to claim 13.

Description

DESCRIPTION OF THE FIGURES

[0065] FIG. 1 HPLC analysis of single-stranded LNA 1 (Example 2)

[0066] FIG. 2 HPLC analysis of single-stranded LNA 2 (Example 2)

[0067] FIG. 3 HPLC analysis of mixed LNA 1 and LNA 2, immediate injection into HPLC system (Example 2)

[0068] FIG. 4 HPLC analysis of mixed LNA 1 and LNA 2 after thermal denaturation prior to injection (Example 2) positive control: duplex formation

[0069] FIG. 5 HPLC analysis of single-stranded LNA 3 (Example 2)

[0070] FIG. 6 HPLC analysis of single-stranded LNA 4 (Example 2)

[0071] FIG. 7 HPLC analysis of mixed LNA 3 and LNA 4, immediate injection into HPLC system (Example 2) slow duplex formation (ratio<0.05)

[0072] FIG. 8 HPLC analysis of mixed LNA 3 and LNA 4, injection after 50 min (Example 2) slow duplex formation (ratio=0.05)

[0073] FIG. 9 HPLC analysis of mixed LNA 3 and LNA 4 after thermal denaturation prior to injection (Example 2) positive control: duplex formation

EXAMPLE 1

[0074] Synthesis of LNA Oligonucleotides

[0075] 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); ß-L-LNA analogues were synthesized analogously to ß-D-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.

[0076] Yields ranged from 85 to 360 nmoles.

[0077] 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.

EXAMPLE 2

[0078] Identification of LNA Oligonucleotide Sequences Capable of Forming Duplex without Prior Denaturation Applying RP-HPLC Analysis

[0079] a) General Method:

[0080] 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).

[0081] 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).

[0082] 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.

[0083] 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).

[0084] 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).

[0085] 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.

[0086] 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).

[0087] b) Identification of Sequence which Forms Duplex Fast

TABLE-US-00002 LNA 1: 5′-tgctcctg-3' LNA 2: 5′-Bi-Heg-caggagca-3′

[0088] Heg=hexaethyleneglycol

[0089] Bi=biotin label attached via the carboxy function of the valeric acid moiety of biotin

[0090] The results are displayed in Figures.

[0091] c) Identification of Sequence which Forms Duplex Slowly

TABLE-US-00003 LNA 3: (SEQ ID NO: 1) 5′-ctgcctgacg-3′ LNA 4: (SEQ ID NO: 2) 5'-Bi-Heg-cgtcaggcag-3′

[0092] The results are displayed in Figures.

[0093] calculation of ratio:

TABLE-US-00004 retention extinction time coefficient (ε) HPLC % LNA [min] HPLC % [l*mol.sup.−1*cm.sup.−1] * ε.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