JPWO2021144587A5

JPWO2021144587A5 -

Info

Publication number: JPWO2021144587A5
Application number: JP2022543377A
Authority: JP
Publication date: 2024-01-17

Claims

From 5' to 3' direction:
capture array,
internal stretch blocker,
a capture oligomer comprising a complement of the capture sequence, and a target hybridizing sequence,
the complement of the capture sequence is configured to anneal to the capture sequence in the absence of the target hybridizing sequence and an extended target sequence annealed to the complement of the capture sequence; comprises a first additional sequence 5' of said capture sequence comprising a first and a second stabilizing sequence, respectively, and a third additional sequence 3' of the complement of said capture sequence, wherein said stabilizing sequence The capture oligomer as described above , wherein the sequence is for stabilizing the duplex region and/or controlling the alignment of hybridization of the duplex region .

The capture oligomer has the formula:
5'-A1-CL-B-A2-C'-A3-RB-A4-THS-X-3'
(where A1 is an optional first additional sequence;
C is the capture sequence;
L is an optional linker,
B is the internal extension blocker;
A2 is an optional second additional sequence;
C' is the complement of the capture sequence;
A3 is a third additional array;
RB is an optional reversible extension blocker;
A4 is an optional fourth additional sequence;
THS is said target hybridization sequence;
X is an optional blocking moiety)
2. A capture oligomer according to claim 1, having:

Capture oligomer according to claim 1 or 2, comprising a linker between the capture sequence and the internal extension blocker, optionally a nucleotide sequence or a non-nucleotide linker or a combination thereof.

Any one of claims 1 to 3, wherein the internal extension blocker comprises a non-nucleotide linker, or one or more of one or more abasic sites, non-natural nucleotides, or chemically modified natural nucleotides. Capture oligomers as described in Section.

5. A capture oligomer according to any one of claims 1 to 4, wherein the capture oligomer comprises a reversible extension blocker located 5' to the target hybridization sequence.

the capture oligomer comprises a third additional sequence located 3' of the complement of the capture sequence and 5' of the target hybridization sequence, and the reversible extension blocker is located 3' of the adapter sequence. 6. The capture oligomer of claim 5, wherein said third additional sequence optionally comprises an adapter sequence.

12. The capture oligomer comprises a fourth additional sequence located 3' of the reversible blocker and 5' of the target hybridization sequence, optionally said fourth additional sequence comprising an adapter sequence. 7. A capture oligomer according to any one of 1 to 6.

8. Any of claims 1 to 7 comprising a second additional sequence between the internal extension blocker and the complement of the capture sequence, optionally the second additional sequence comprising a mixed nucleotide segment. Capture oligomer according to paragraph 1.

The reversible extension blocker is Iso-dC or Iso-dG, xanthine or 5-(2,4 diaminopyrimidine), 2-amino-6-(N,N-dimethylamino)purine or pyridin-2-one, 4 - 4-Methylbenzimidizole or 2,4-difluorotoluene, 7-azaindole or isocarbostyryl, dMMO2 or d5SICS, dF or dQ; one or more chemically modified nucleotides comprising: one or more chemical modifications, wherein the modification is attached via a reversible linkage, said linkage being reversible by supplying any one or more of a chemical, an enzyme, a change in temperature, a change in reagent composition; a nucleotide; a structural feature of a reversible nucleic acid; or a molecule reversibly bound to said capture oligomer, optionally said reversibly bound molecule being a protein, enzyme, lipid, carbohydrate, or chemical moiety. 9. The capture oligomer of claim 8, comprising a molecule reversibly bound to the capture oligomer.

Capture oligomer according to any one of claims 1 to 9, wherein the first and second stabilizing sequences are clamp sequences, mixed nucleotide regions, GC-rich sequences or sequences containing affinity-enhancing modifications.

Capture oligomer according to any one of claims 1 to 9, wherein the first and second stabilizing sequences are GC clamp sequences.

The first and second stabilizing sequences are each (GC) _３3 Array or (CG) _３3 A capture oligomer according to any one of claims 1 to 9, which is a GC clamp sequence comprising a GC clamp sequence.

13. A combination comprising a capture oligomer or combination according to any one of claims 1 to 12 , wherein the capture oligomer comprises an adapter sequence 5' of the target hybridizing sequence, and the combination is non-extendable. further comprising a blocker oligomer comprising said adapter sequence of said capture oligomer, optionally said blocker oligomer binding said complement of said adapter sequence with higher affinity than said adapter sequence of said capture oligomer; The combination is configured to form a complex with the complement of the adapter sequence that has a higher melting temperature than the complex with the complement of the adapter sequence.

A method of capturing a target polynucleotide from a composition, the method comprising:
contacting the target polynucleotide with a capture oligomer according to any one of claims 1 to 12 , wherein the target hybridization sequence of the capture oligomer comprises a 3' end of the target polynucleotide. annealing or contacting the target polynucleotide with;
elongating the 3' end of the target polynucleotide using a DNA polymerase with strand displacement activity, thereby annealing to the capture oligomer such that the capture sequence of the capture oligomer is available for binding. , forming a complement of said complement of said capture sequence;
The capture sequence of the capture oligomer is contacted with the complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby binding the target polynucleotide, the capture oligomer, and forming a complex comprising the secondary capture reagent; and isolating the complex from the composition, thereby capturing the target polynucleotide.