EP-4737589-A2 - OLIGO-MODIFIED NUCLEOTIDE ANALOGUES FOR NUCLEIC ACID PREPARATION

Abstract

Nucleic acid techniques are disclosed. Embodiments include modified nucleotides with oligonucleotide adapters that are coupled via cleavable linkers. Incorporation of the modified nucleotide at a 3' end of a nucleic acid permits end-adapterization via ligation of a free 5' end of the oligonucleotide adapter to a 3' reactive group of the modified nucleotide and cleavage at the cleavable linker to liberate a free 3' end.

Inventors

• GORMLEY, NIALL ANTHONY
• RANDISE-HINCHLIFF, Carlo
• BRODIN, Jeffrey
• MUSGRAVE-BROWN, ESTHER
• Shultzaberger, Sarah E
• SLATTER, ANDREW
• FISHER, JEFFREY S

Assignees

• Illumina, Inc.

Dates

Publication Date

20260506

Application Date

20220526

Claims (15)

1. An oligo-modified nucleic acid analogue composition, comprising: a modified nucleotide comprising: a ribose; a 5' phosphate group coupled to the ribose; a 3' reactive group coupled to the ribose; and an oligonucleotide adapter coupled to the ribose by a linker and terminating in a 3' oligonucleotide end.
2. The composition of claim 1, wherein the oligonucleotide adapter is coupled to 3' position of the ribose via the linker.
3. The composition of claim 1, wherein the oligonucleotide adapter is single-stranded.
4. The composition of claim 1, wherein the oligonucleotide adapter is coupled to a 1' position of the ribose via the linker.
5. The composition of claim 1, wherein the 3' reactive group comprises a hydroxyl group or an azide.
6. The composition of claim 1, wherein the modified nucleotide comprises a nucleobase coupled to a 1' position of the ribose.
7. The composition of claim 1, wherein the oligonucleotide adapter is coupled to an affinity binder.
8. A nucleic acid fragment comprising: a single or double-stranded nucleic acid fragment; and a modified nucleotide coupled to a 3' end of the nucleic acid fragment, the modified nucleotide comprising: an oligonucleotide adapter coupled to a ribose by a linker at a first end and terminating in a 5' or 3' oligonucleotide end at a second end.
9. The nucleic acid fragment of claim 8, wherein the oligonucleotide adapter is coupled to a 1' position of the ribose via the linker.
10. The nucleic acid fragment of claim 8, wherein the modified nucleotide comprises a nucleobase coupled to a 1' position of the ribose, wherein the linker extends from the nucleobase.
11. The nucleic acid fragment of claim 10, wherein the nucleobase is uracil, thymine, cytosine, adenine, or guanine.
12. The nucleic acid fragment of claim 8, wherein the oligonucleotide adapter is hybridized to a tail of a forked adapter.
13. The nucleic acid fragment of claim 8, wherein the nucleic acid fragment is a double-stranded DNA.
14. The nucleic acid fragment of claim 13, wherein the modified nucleotide is incorporated at a 3' recessed end of the double-stranded DNA.
15. The nucleic acid fragment of claim 13, wherein the modified nucleotide is incorporated at a 3' blunt end of the double-stranded DNA.

Description

BACKGROUND The disclosed technology relates generally to techniques for preparing nucleic acids, e.g., sequencing library preparations, using oligo-modified nucleotide analogues. The oligo-modified nucleotide analogues including oligonucleotide adapters can be used to directly incorporate adapters onto nucleic acids as part of sample preparation for downstream processing steps. The subject matter discussed in this section should not be assumed to be prior art merely as a result of its mention in this section. Similarly, a problem mentioned in this section or associated with the subject matter provided as background should not be assumed to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which in and of themselves can also correspond to implementations of the claimed technology. Molecular biology now makes intensive use of nucleic acid analysis. Various nucleic acid analysis techniques involve a sample preparation stage in which the sample is manipulated to generate an end product that is compatible with the desired analysis platform. For example, certain sequencing platforms are compatible with sequencing libraries that include specific adapter sequences to permit strand capture and synthesis. These adapter sequences may include universal adapters for high throughput parallel processing of large numbers of nucleic acids. The addition of universal adapters for sequencing can be achieved by a variety of methods. In one example, adapters that contain universal priming sequences can be ligated onto the ends of template nucleic acids. A single adapter or two different adapters may be used in a ligation reaction. If a template nucleic acid has been manipulated such that its ends are the same, i.e., both are blunt or both have the same overhang, then ligation of a single compatible adapter will generate a template with that adapter on both ends. However, if two compatible and different adapters, e.g., adapter A and adapter B, are used, then three permutations of ligated products are formed: template with adapter A on both ends, template with adapter B on both ends, and template with adapter A on one end and adapter B on the other end. This last product is, under some circumstances, the only desired product from the ligation reaction and, consequently, additional purification steps are necessary following the ligation reaction to purify it from the undesired ligation products that have the same adapter at both ends. Thus, certain techniques for adding universal adapters to a sample involve inherent loss of the sample as well as additional purification steps when creating and subsequently selecting suitably modified fragments. Accordingly, more efficient techniques for adding adapters to nucleic acids are of interest. BRIEF DESCRIPTION In one embodiment, the present disclosure provides an oligo-modified nucleic acid analogue composition. The composition includes a modified nucleotide comprising: a ribose (e.g., a deoxyribose, a ribonucleic acid, a dideoxyribose); a 5' phosphate coupled to the deoxyribose; a 3' reactive group coupled to the ribose; and an oligonucleotide adapter coupled to the ribose by a linker, e.g., a cleavable linker, and terminating in a reactive 5' oligonucleotide end. In an embodiment, the oligonucleotide can be coupled to the linker with a terminating 3' end or a reactive 3' end, depending on desired subsequent reactions after reactions with the modified nucleotide. In one embodiment, the present disclosure provides a method of modifying a nucleic acid. The method includes providing a nucleic acid and contacting the nucleic acid with a modified nucleotide. The modified nucleotide includes a deoxyribose; a 5' phosphate group coupled to the deoxyribose; a 3' reactive group coupled to the deoxyribose; and an oligonucleotide adapter coupled to the deoxyribose by a cleavable linker and terminating in a 5' oligonucleotide end. The method includes incorporating the modified nucleotide onto a 3' end of the nucleic acid to generate an extended nucleic acid; reacting the 5' oligonucleotide end of the oligonucleotide adapter on the extended nucleic acid with the 3' reactive group to couple the 5' oligonucleotide end to the deoxyribose and such that the oligonucleotide adapter forms a loop. In an embodiment, the method includes cleaving the linker to liberate a 3' end of the oligonucleotide adapter. In one embodiment, the present disclosure provides a method of modifying a nucleic acid. The method includes contacting a single-stranded nucleic acid with a modified nucleotide comprising: a deoxyribose; a 5' phosphate group coupled to the deoxyribose; and a single-stranded oligonucleotide adapter coupled to the deoxyribose and terminating in a 5' oligonucleotide end. The method also includes using a polymerase to incorporate the modified nucleotide onto a 3' end of the single-stranded nucleic acid to generate an extended single-str