US-20260125754-A2 - HIGH INTENSITY LABELED REACTANT COMPOSITIONS AND METHODS FOR SEQUENCING

Abstract

Compositions useful for the detection of single molecules in a sample are provided. In some aspects, the disclosure provides a nucleic acid connected to a nucleotide and two or more luminescent labels. In some embodiments, the nucleic acids described herein comprise one or more structural features that provide enhanced fluorescence intensity. In some aspects, methods of sequencing using the labeled nucleotides of the disclosure are provided.

Inventors

• Jonathan M. Rothberg
• Jeremy Lackey
• Brian Reed
• Xinghua Shi
• Haidong Huang
• David Dodd

Assignees

• Quantum-Si Incorporated

Dates

Publication Date

20260507

Application Date

20230322

Claims (20)

1. 1 . A labeled molecule comprising a reactant connected to two or more luminescent labels via a linker, wherein each luminescent label is at least 5 angstroms separated from any other luminescent label.
2. 2 . The labeled molecule of claim 1 , wherein each luminescent label comprises a center of mass that is at least 5 angstroms separated from the center of mass of any other luminescent label.
3. 3 . The labeled molecule of claim 1 , wherein one or more luminescent labels are attached to the linker via a spacer molecule that comprises at least 8 contiguous atoms between the luminescent label and an attachment site on the linker.
4. 4 . The labeled molecule of claim 3 , wherein the spacer molecule comprises fewer than 50, fewer than 40, fewer than 30, or fewer than 20 contiguous atoms between the luminescent label and the attachment site on the linker.
5. 5 . (canceled)
6. 6 . The labeled molecule of claim 1 , wherein the linker is an oligomer that comprises at least 10 monomeric units.
7. 7 . The labeled molecule of claim 6 , wherein the oligomer comprises fewer than 150, fewer than 100, or fewer than 50 monomeric units.
8. 8 . The labeled molecule of claim 6 , wherein one or more luminescent labels are attached to the linker at an attachment site that is at least 5 monomeric units separated from any other attachment site.
9. 9 . The labeled molecule of claim 1 , wherein the linker is a nucleic acid.
10. 10 - 13 . (canceled)
11. 14 . The labeled molecule of claim 1 , wherein the linker is a nucleic acid comprising a first oligonucleotide strand attached to the two or more luminescent labels at two or more attachment sites on the first oligonucleotide strand, and wherein each luminescent label comprises a steric volume having a center point that is at least 5 angstroms separated from that of any other luminescent label.
12. 15 . The labeled molecule of claim 14 , further comprising a second oligonucleotide strand hybridized with the first oligonucleotide strand, wherein the first oligonucleotide strand is attached to the reactant.
13. 16 . The labeled molecule of claim 14 , further comprising a second oligonucleotide strand hybridized with the first oligonucleotide strand, wherein the second oligonucleotide strand is attached to the reactant.
14. 17 . (canceled)
15. 18 . The labeled molecule of claim 14 , wherein the two or more attachment sites are separated from one another by at least 5 and fewer than 50 bases on the first oligonucleotide strand.
16. 19 . The labeled molecule of claim 14 , wherein each attachment site is at least 2 bases separated from a guanine or a cytosine on the first oligonucleotide strand.
17. 20 . The labeled molecule of claim 14 , wherein at least one attachment site occurs at an abasic site on the first oligonucleotide strand.
18. 21 - 22 . (canceled)
19. 23 . The labeled molecule of claim 14 , wherein the first oligonucleotide strand forms one or more stem-loops.
20. 24 . The labeled molecule of claim 23 , wherein a loop region of each stem-loop comprises an attachment site of the two or more attachment sites.

Description

RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 16/043,547, filed Jul. 24, 2018, which claims priority under 35 U.S.C. § 119(e) to U.S. Application Ser. No. 62/536,426, filed Jul. 24, 2017, each of which is hereby incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (R070870026US02-SEQ-GIC.xml; Size: 27,365 bytes; and Date of Creation: Mar. 21, 2023) is herein incorporated by reference in its entirety. FIELD OF THE APPLICATION The present application is directed generally to brightly labeled reactant compositions and methods of using the same for the detection of single molecules. BACKGROUND Advancements in next-generation sequencing technologies have made it possible to conduct massively parallel analysis of single molecules, which has fundamentally altered the landscape of life science research. Some of these techniques involve monitoring a biological reaction in real-time using luminescently labeled reaction components. The labels are illuminated with a light source to cause luminescence, and the luminescent light is detected with a photodetector. These events can be recorded and analyzed to identify individual reaction components based on corresponding luminescent properties. In identifying a specific type of labeled molecule among a plurality of types, it is critical that each type possess unique and readily identifiable luminescent properties. Furthermore, these parameters can be determinative of instrumental requirements such as excitation source power and overall instrument size. SUMMARY Aspects of the technology disclosed herein relate to labeled reaction components comprising two or more luminescent labels separated by a linker (e.g., a constrained linker). In some embodiments, the application relates to the separation of luminescent labels to prevent attenuation of detectable signals due to label-label interaction. In some aspects, the application provides labeled nucleotides comprising a nucleotide (e.g., a nucleoside polyphosphate) connected to two or more luminescent labels via a linker. In some aspects, the application provides compositions, methods, and kits for sequencing a template nucleic acid. In some aspects, the application provides labeled nucleotides comprising a nucleotide (e.g., a nucleoside polyphosphate) connected to two or more luminescent labels via a linker. In some embodiments, the nucleotide is configured for use as a substrate in a polymerization reaction. In some embodiments, labeled nucleotides of the application comprise two or more luminescent labels separated from one another by a minimum distance. In some embodiments, each luminescent label is at least 5 angstroms separated from any other luminescent label. For example, in some embodiments, each luminescent label is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, or at least 50 angstroms separated from any other luminescent label. In some embodiments, each luminescent label comprises a center of mass that is at least 5 angstroms separated from the center of mass of any other luminescent label. In some embodiments, labeled nucleotides of the application comprise one or more luminescent labels attached to the linker via a spacer molecule. In some embodiments, the spacer molecule connects a luminescent label to an attachment site on the linker. In some embodiments, a luminescent label is attached to the linker via a spacer molecule that comprises at least 8 contiguous atoms between the luminescent label and the attachment site on the linker. In some embodiments, the spacer molecule comprises fewer than 50, fewer than 40, fewer than 30, or fewer than 20 contiguous atoms between the luminescent label and the attachment site on the linker. In some embodiments, a luminescent label is integrated into the linker. In some embodiments, the linker is an oligomer (e.g., an oligomeric linker, or a polymeric linker). In some embodiments, the oligomer comprises monomeric units. In some embodiments, the oligomer comprises two or more different types of monomeric units. In some embodiments, the oligomer comprises a plurality of the same type of monomeric unit (e.g., the oligomer is a polymer of one type of monomeric units). In some embodiments, the oligomer comprises a first region with a plurality of a first type of monomeric units and a second region with a plurality of a second type of monomeric units. In some embodiments, the oligomer comprises a plurality of different regions (e.g., 2, 3, 4, 5, or more) each comprising a plurality of a different type of monomeric units. In some embodiments, the oligomer comprises at least 5 monomeric units. In some embodiments, the oligomer comprises at least 10 monomeric units. In some embodiments, the oligomer comprises fewer than 150, fewer than 100, or fewer than 50 monomeric units (e.g., at least 5 monomeric units and fewer than 200,