Labelling Strategies for the Sensitive Detection of Analytes

• US 20120190838A1
• Filed: 03/02/2012
• Published: 07/26/2012
• Est. Priority Date: 05/02/2005
• Status: Abandoned Application

• Current Assignee: Baseclick GmbH (BASF SE)
• Original Assignee: Baseclick GmbH (BASF SE)
• Inventors: CARELL, Thomas, SCHWOEGLER, Anja, BURLEY, Glenn A., GIERLICH, Johannes, MOFID, Mohammad Reza
• Application Number:
  US13/410,955
• Publication Number:
  US 20120190838A1
• Time in Patent Office: Days
• US Class Current: 536/27.2
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to methods and reagents for detecting analytes, e.g. nucleic acids. The new methods and reagents allow a simple and sensitive detection even in complex biological samples.

Claims

1. A nucleic acid or a nucleic acid analogue molecule having incorporated therein at least one compound of formula (II):

C—

S—

N 

 

(II)

wherein

C is a Click functional group selected from alkyne and azide groups which is a first reaction partner for a Click reaction, said Click reaction being a (3+2) cycloaddition reaction between azide and alkyne groups

forming a 1,2,3-triazole ring

S is a spacer, and

N is a nucleic acid or nucleic acid analogue building block such as a nucleosidic or nucleotidic compound, wherein the Click functional group is attached to a nucleobase,

wherein the functional group is an alkyne group attached to position 5 and/or 6 of a pyrimidine nucleobase, or to positions 7 and/or 8 of a purine nucleobase, and wherein the nucleic acid or nucleic acid analogue molecule is capable of hybridising with a complementary nucleic acid.

2. The nucleic acid or a nucleic acid analogue molecule of claim 1, wherein the compound of formula (II) further comprises marker groups or marker precursor groups.

3. The nucleic acid or a nucleic acid analogue molecule of claim 1, wherein the nucleobase is selected from the group consisting of naturally occurring and non-naturally occurring purine and pyrimidine bases.

4. The nucleic acid or a nucleic acid analogue molecule of claim 1, wherein the nucleobase is selected from the group consisting of adenine, 7-deazaguanine, guanine, 7-deazaguanine, cytosine, thymidine, uracil, inosine, and xanthine.

5. The nucleic acid or a nucleic acid analogue molecule of claim 1, wherein the functional group is attached to positions 5 and 6 of a pyrimidine nucleobase, or to positions 7 and 8 of a purine nucleobase.

6. The nucleic acid or a nucleic acid analogue molecule of claim 1, wherein the spacer is an at least partially rigid spacer.

7. The nucleic acid or a nucleic acid analogue molecule of claim 1, wherein the spacer comprises at least one group selected from the group consisting of alkene groups, alkyne groups, cyclic groups, aromatic groups, heteroaromatic groups, and combinations thereof.

8. The nucleic acid or a nucleic acid analogue molecule of claim 1, wherein the molecule is a building block for the chemical or enzymatic synthesis of nucleic acids or nucleic acid analogues or a precursor thereof.

9. The nucleic acid or a nucleic acid analogue molecule of claim 1, wherein the molecule is a nucleoside triphosphate, a ribose, 2′

-deoxyribose, a 2′

,3′

-dideoxyribose nucleoside triphosphate, a nucleoside phosphoramidite, H-phosphonate or phosphotriester.

10. The nucleic acid or a nucleic acid analogue molecule of claim 1, wherein the molecule is a building block for the synthesis of peptide nucleic acids, morpholino nucleic acids, or locked nucleic acids.

11. The nucleic acid or a nucleic acid analogue molecule of claim 1, wherein the functional group is attached to the nucleobase via a spacer having a chain length of up to 10 atoms.

12. The nucleic acid or a nucleic acid analogue molecule of claims 1, wherein the chain length is of up to 3 atoms.

13. The nucleic acid or a nucleic acid analogue molecule of claim 1 which is a nucleic acid selected from the group consisting of DNA and RNA, or a nucleic acid analogue molecule selected from the group consisting of peptidic nucleic acids, morpholino nucleic acids and locked nucleic acids.

14. An association product of the nucleic acid or nucleic acid analogue molecule of claim 1 with an analyte.

15. An association product of the nucleic acid or nucleic acid analogue molecule of claim 13 with an analyte.

16. The product of claim 14 which is a hybridization product with a complementary nucleic acid.

17. The product of claim 15 which is a hybridization product with a complementary nucleic acid.

18. A compound obtained by synthesizing a nucleic acid or nucleic acid analogue molecule that is capable of hybridizing with a complementary nucleic acid, comprising incorporating a nucleotide or nucleotide analogue building block comprising a compound (II):

C—

S—

N 

 

(II)

wherein

C is a Click functional group selected from alkyne and azide groups which is a first reaction partner for a Click reaction, said Click reaction being a (3+2) cycloaddition reaction between azide and alkyne groups forming a 1,2,3-triazole ring,

S is a spacer, and

N is a nucleic acid or nucleic acid analogue building block such as a nucleosidic or nucleotidic compound, wherein the Click functional group is attached to a nucleobase; and

contacting the nucleic acid or nucleic acid analogue molecule with a second reaction partner of compound (II) and performing a Click reaction between the first and second reaction partners, said Click reaction being a (3+2) cycloaddition reaction between azide and alkyne groups forming a 1,2,3-triazole ring,

wherein the functional group is an alkyne group attached to positions 5 or/and 6 of a pyrimidine nucleobase, or to positions 7 or/and 8 of a purine nucleobase into a nucleic acid or nucleic acid analogue molecule.

19. An association product of the compound of claim 18 with a complementary nucleic acid.