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US-20260125670-A1 - ANALYSIS SYSTEM FOR ORTHOGONAL ACCESS TO AND TAGGING OF BIOMOLECULES IN CELLULAR COMPARTMENTS

US20260125670A1US 20260125670 A1US20260125670 A1US 20260125670A1US-20260125670-A1

Abstract

The invention relates to a system and methods for enhancing access to nuclear informational molecules, such as DNA, RNA, and proteins, by analytical biomolecules, such as transposome complexes, by treating nuclei with a nuclear permeability enhancer, and to methods of using nuclear membrane, cell membrane, and external compartmentalization approaches as contiguity preserving elements.

Inventors

  • Ramesh Ramji
  • Frank J. Steemers
  • Lena Christiansen
  • Dmitry K. Pokholok
  • Fan Zhang

Assignees

  • ILLUMINA, INC.

Dates

Publication Date
20260507
Application Date
20250820

Claims (20)

  1. 1 - 50 . (canceled)
  2. 51 . A method of increasing yield of tagged double-stranded nucleic acid fragments from a population of nuclei comprising: (a) isolating nuclei each comprising cell nuclear target nucleic acid from a population of cells; (b) contacting the isolated nuclei with Pitstop-2 (N-[5-(4-bromobenzylidene)-4-oxo-4,5-dihydro-1,3-thiazol-2-yl]naphthalene-1-sulfonamide) and a transposome complex comprising a transposase and two transposon end compositions comprising transposon end sequences; (c) reacting the transposome complex with the target nucleic acid, wherein the reacting comprises fragmenting the target nucleic acid into double-stranded nucleic acid fragments and tagging with transferred strands from the transposon end compositions to form tagged double-stranded nucleic acid fragments; and (d) determining yield of tagged double-stranded nucleic acid fragments, wherein the yield of tagged double-stranded nucleic acid fragments is greater in comparison to the yield wherein nuclei are not contacted with Pitstop-2.
  3. 52 . The method of claim 51 , wherein the Pitstop-2 concentration is 5 μM to 300 μM, optionally wherein the Pitstop-2 concentration is 10 μM to 100 μM.
  4. 53 . The method of claim 52 , wherein the Pitstop-2 concentration is 30 μM to 60 μM.
  5. 54 . The method of claim 51 , wherein the target nucleic acid is genomic DNA.
  6. 55 . The method of claim 54 , wherein fragmenting occurs at genomic DNA regions free of nucleosomes.
  7. 56 . The method of claim 51 , wherein isolating nuclei comprises incubating cells with lysis buffer and pelleting nuclei with centrifugation.
  8. 57 . The method of claim 51 , wherein the yield of tagged double-stranded nucleic acid fragments is 50% or more, 100% or more, 200% or more, or 300% or more, in comparison to the yield wherein nuclei are not contacted with Pitstop-2.
  9. 58 . The method of claim 51 , further comprising after step (d): (e) sequencing the tagged double-stranded nucleic acid fragments; and (f) analyzing the sequencing results.
  10. 59 . The method of claim 58 , wherein the sequencing results exhibit improved sequencing coverage of intergenic regions of genomic DNA, in comparison to results wherein nuclei are not contacted with Pitstop-2.
  11. 60 . The method of claim 58 , wherein the sequencing results show increased normalized coverage over promoter regions in comparison to exons, in comparison to results wherein nuclei are not contacted with Pitstop-2.
  12. 61 . The method of claim 58 , wherein the sequencing results show increased diversity and uniqueness of sequencing results, in comparison to results wherein nuclei are not contacted with Pitstop-2.
  13. 62 . The method of claim 51 , further comprising compartmentalizing nuclei into a plurality of first vessels after step (a) and before step (b) to prepare isolated single nuclei, wherein contacting the isolated nuclei with Pitstop-2 and a transposome complex comprises tagmentation to attach indexes.
  14. 63 . The method of claim 58 , further comprising amplifying tagged double-stranded nucleic acid fragments after step (c) and before step (e).
  15. 64 . The method of claim 63 , wherein the amplifying is performed with a strand-displacing polymerase.
  16. 65 . The method of claim 64 , wherein the amplifying with a strand-displacing polymerase removes transposases from the tagged double-stranded nucleic acid fragments.
  17. 66 . The method of claim 63 , wherein the amplifying introduces an indexed oligonucleotide.
  18. 67 . The method of claim 63 , wherein the amplifying is performed on a bead with a PCR primer.
  19. 68 . The method of claim 67 , wherein the PCR primer is biotinylated.
  20. 69 . The method of claim 68 , further comprising enriching for biotinylated fragments after amplifying and before sequencing.

Description

RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 16/456,763 filed Jun. 28, 2019 which is a continuation of International Application No. PCT/US2017/068672, filed Dec. 28, 2017, which claims the benefit of, U.S. Provisional Application No. 62/440,089, filed on Dec. 29, 2016, the contents of which are each incorporated herein by reference in its entirety. REFERENCE TO ELECTRONIC SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on Nov. 17, 2025, is named “IP-1537-US-A.xml” and is 14,498 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND Detection of nucleic acids and proteins in biological samples is useful for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with disease onset or progression, studying genetic susceptibility to disease, and measuring response to disease treatments. Analysis of single cells or single nuclei, or allow for discrete analysis of cytoplasmic and nuclear compartments, offer broad insight into assessments of cell type, cell differentiation, cell status, protein synthesis and regulation, evolution, disease progression and diagnosis, and responses to disease treatment. In particular, analysis of DNA and RNA, as well as histones and other nuclear proteins, at the nucleus level can be used to do entire genome sequencing, to reveal information about quiescence states of a cell and open chromatin states, or to provide real-time information about protein regulation. Such information is useful for applications such as gene editing, cell type conversion, analysis of protein regulation, and disease therapy. Current techniques for accessing cellular nucleic acid and protein content for subsequent analysis generally employ cell lysis methods that disrupt the cellular compartments. In some methods, lysis media such as ionic detergents are used, yielding mixtures of cytoplasmic and nuclear content and preventing resolution of molecular information between the compartments (e.g., due to cross-contamination between cytoplasmic mitochondrial DNA and nuclear DNA). Alternative lysis methods employ mild, non-ionic detergents to disrupt the cellular membrane while leaving the nuclei intact. In another approach, isolated nuclei may be disrupted with digestion enzymes such as proteases. Next-generation sequencing (NGS) techniques routinely employ a sample or library preparation step in which genomic DNA or RNA is converted into a library of fragmented, sequenceable templates. Fragmentation of genomic DNA is a crucial step in DNA sample preparation for high-throughput sequencing. In one approach, transposome complexes are used to fragment and tag target nucleic acids. Transposases mediate the fragmentation of double-stranded DNA and ligate synthetic oligonucleotides at both ends. The appended oligonucleotides enable subsequent amplification and sequencing steps. The cellular and nuclear lysis methods discussed above are incompatible with such approaches because they degrade the enzymes (e.g., ionic detergents, digestion enzymes), or because the enzymes are unable to sufficiently penetrate intact nuclei. For example, transposome complexes, such as Nextera Tn5 (dimer ˜106 kDa), that are used in current tagmentation protocols are unable to access nuclear material efficiently due to the complexity of the nuclear envelope (see FIG. 1). The nuclear envelope is composed of an outer nuclear membrane and an inner nuclear membrane, which together form a lipid bilayer that restricts diffusion of biomolecules from the cytoplasm into the nucleus. Nuclear pore complexes (NPCs) span the nuclear membrane and tightly regulate transport of biomolecules in and out of the nucleus, typically allowing only molecules less than 40 kDa in size to pass through. Adjacent to the inner nuclear membrane is the nuclear lamina, which includes protein filaments, such as scaffold/matrix attachment elements, clathrin, and other proteins, that create a supportive framework to maintain nuclear rigidity and control size-selected entry of molecules into the nucleus. Because of these issues of enzymatic degradation and limited diffusion across the nuclear membrane, cell lysis methods would require additional purification and isolation steps to isolate target nucleic acids or proteins before further sample preparation could be effected. Analysis of single cell content may be accomplished by isolating single cells in discrete compartments. In one technique, cells are distributed into water droplets in an oil medium. However, the oil impedes transfer of materials and sample preparation enzymes in and out of the aqueous droplets, so all suc