Base analogues

• US 6,605,611 B2
• Filed: 09/03/2002
• Issued: 08/12/2003
• Est. Priority Date: 07/31/1997
• Status: Expired due to Fees

• Current Assignee: Medical Research Council (Government of United Kingdom)
• Original Assignee: Nycomed Amersham Inc.
• Inventors: McDougall, Mark, Kumar, Shiv, Nampalli, Satyam, Smith, Clifford, Hill, Fergal, Brown, Daniel, Simmonds, Adrian Christopher, Loakes, David, Hamilton, Alan
• Primary Examiner(s): Raymond, Richard L.
• Assistant Examiner(s): Patel, Sudhaker B.
• Application Number:
  US10/233,993
• Publication Number:
  US 20030060431A1
• Time in Patent Office: 343 Days
• Field of Search: 514/274, 514/248, 514/262.1, 514/264.1, 514/209, 514/461, 514/23, 544/236, 544/256, 544/279, 544/298, 544/315, 546/118, 546/121, 546/123, 536/22.1
• US Class Current: 514/274
• CPC Class Codes: A61P 31/12 Antivirals
• 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: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

Nucleoside analogues or base analogues having structure (I), where X=O or NH or S, Y=N or CHR⁶ or CR⁶, W=N or NR⁶ or CHR⁶ or CR⁶ or S, n=1 or 2; each R⁶ is independently H or O or alkyl or alkenyl or alkoxy or aryl or a reporter moiety; where necessary (i.e. when Y and/or W is N or CR⁶ where R⁶ is not O) a double bond is present between Y and W or W and W, and Q is H or a sugar or a sugar analogue or a nucleic acid backbone or backbone analogue.

Claims

1. A compound having the structure

where X=O or NH or S

Y=N or CHR⁶ or CR⁶ or CO

W=N or NR⁶ or CHR⁶ or CR⁶ or S or CO

n=2

each R⁶ is independently H or alkyl or alkenyl or alkoxy or aryl or a reporter moiety,

if Y or W=N or CR⁶, then a double bond is present between the Y and W or between the W and W, and

Q is selected from the group consisting of H, a sugar, a sugar analogue, a nucleic acid backbone, and backbone analogue provided that

(i) when X is NH and W is CHR⁶ or CR⁶, and Y is CO, then at least one R⁶ is a reporter moiety,

(ii) when W is S, then W_n is —

CHR⁶—

S—

or ═

CR⁶—

S—

,

(iii) when X is NH and Y is CHR⁶ or CR⁶, then at least one R⁶ is a reporter moiety that is selected from the group consisting of a reactive group, signal moiety, and solid surface joined to the remainder of the molecule by a linker of at least 3 chain atoms, and

(iv) when X is O and Y is CHR⁶ and W is CHR⁶ or S, then at least one R⁶ is a reporter moiety.

2. The compound of claim 1 wherein the compound is

(a) a nucleoside or nucleoside analogue wherein Q is

  where Z is O

R¹, R², R³ and R⁴ are the same or different and each is H or OH

R⁵ is mono-, di-, or tri-phosphate,

or one of R² and R⁵ is a phosphoramidite or other group for incorporation in a polynucleotide chain;

or

(b) a polynucleotide wherein Q is a nucleic acid backbone selected from the group consisting of sugar-phosphate repeats, modified sugar-phosphate repeats (LNA), or a backbone analogue such as peptide or polyamide nucleic acid (PNA).

3. A compound of claim 1 wherein the structure is selected from the group consisting of

4. The compound of claim 3, wherein a reporter moiety is present.

5. The compound of claim 4, wherein the reporter moiety is a signal moiety.

6. The compound of claim 4, wherein the reporter moiety is a reactive group or signal moiety or solid surface joined to the remainder of the molecule by a linker of at least 3 chain atoms.

7. The compound of claim 3, wherein R⁵ is triphosphate.

8. The compound of claim 3, wherein one of R² and R⁵ is selected from phosphoramidite and H-phosphonate.

9. A polynucleotide comprising at least one residue of the compound of claim 1.

10. The polynucleotide of claim 9, wherein the polynucleotide is DNA or RNA.

11. A chain extension method which comprises reacting a polynucleotide with a nucleoside triphosphate analogue of claim 1 in the presence of a polymerase or a terminal deoxynucleotidyl transferase enzyme.

12. A method of detecting the polynucleotide of claim 9, which method comprises using for detection an antibody which binds to a base component selected from the group consisting of structure (1), structure (2), structure (3), and structure (4).

13. A method of making cDNA which comprises incubating an RNA template with a monomer mixture including a nucleotide analogue as claimed in claim 1 in the presence of a reverse transcriptase.

14. A method of amplifying a polynucleotide by PCR which method comprises using a monomer mixture including a nucleotide analogue as claimed in claim 1.

15. A method of reducing compression artefacts in nucleic acid sequencing, comprising

(a) incorporating at least one nucleoside analogue of claim 1 into polynucleotides using a polymerase;

and

(b) separating polynucleotides created by the polymerase by electrophoresis, wherein incorporation of the nucleoside analogue into one or more polynucleotides reduces detection of compression artefacts.

16. A method of detecting a residue of the compound of claim 1, the method comprising analyzing by mass spectrometry.