Methods for the selective analysis of cells or organelles

Patent metadata

Field	Value
Publication number	US-2024167082-A1
Application number	US-202218550598-A
Country	US
Kind code	A1
Filing date	Mar 18, 2022
Priority date	Mar 19, 2021
Publication date	May 23, 2024
Grant date	—

Abstract

The present invention relates to a method for the selective analysis of a subpopulation of cells or organelles based determining the level of at least one cell or organelle marker, via the implementation of an adapted DNA toolbox able to convert the presence of a cell or organelle marker into a signal. This method is particularly useful for selecting cells or organelles having a phenotype of interest, analyzing the proteins of said cells or organelles and/or sequencing the DNA and/or RNA of said cells. The present invention also relates to a kit for implementing the method of selective analysis of cells or organelles as defined above.

First claim

1 . A method for the selective analysis of a subpopulation of cells or organelles based on determining the level of at least one cell or organelle marker, said method comprising:
a) partitioning said cells or organelles into a plurality of compartments, so that the majority of the compartments do not comprise more than a single cell of one cellular type or a single organelle of one cellular type, together with at least one cell or organelle marker-specific ligand coupled to at least one activator oligonucleotide or repressor oligonucleotide and with a DNA toolbox mixture comprising at least one polymerase, at least one restriction or nicking enzyme and at least one amplification oligonucleotide able to bind and amplify a signal oligonucleotide, b) in each compartment, letting the activator oligonucleotide(s) and/or the repressor oligonucleotide(s) bound to the cell or organelle marker, interact together and with the components of the DNA toolbox mixture, to generate one or more signal oligonucleotides and/or one or more anti-signal oligonucleotides and to amplify said signal oligonucleotide(s), c) analyzing the cells or organelles of the compartments in which at least one signal oligonucleotide is present at a concentration over or below a threshold value.

2 . The method according to claim 1 , wherein the DNA toolbox mixture comprises one or more leak-absorption oligonucleotides able to bind the signal oligonucleotides.

3 . The method according to claim 1 , wherein the cell or organelle marker is selected from the group consisting of a surface marker, an internal marker, a secreted or released marker and a membrane marker.

4 . The method according to claim 1 , wherein the level of at least one cell or organelle marker is modified by the interaction between at least two co-compartmentalized cells or organelles and wherein step c) allows analysis of the interaction between the cells or organelles.

5 . The method according to claim 1 , wherein the marker is not a secreted or released marker and wherein said method comprises, before step a):
mixing said cells or organelles with at least one cell or organelle marker-specific ligand coupled to an activator or repressor oligonucleotide, and optionally, washing the cells or organelles to remove unbound cell or organelle marker-specific ligands coupled to an activator or repressor oligonucleotide.

6 . The method according to claim 1 , wherein
the activator oligonucleotide coupled to the cell or organelle marker-specific ligand generates one or more signal oligonucleotide by acting as template and/or as a primer for the polymerase, and/or the repressor oligonucleotide coupled to the cell or organelle marker-specific ligand generates one or more anti-signal oligonucleotide by acting as a template and/or as a primer for the polymerase.

7 . The method according to claim 1 , wherein the cell or organelle marker-specific ligands coupled to at least one activator oligonucleotide or repressor oligonucleotide comprise at least one pair of ligands, said pair of ligands comprising a first cell or organelle marker-specific ligand and a second cell or organelle marker-specific ligand, and wherein the binding of said first and second ligands to their marker induces proximity extension or proximity ligation between the activator or repressor oligonucleotide coupled to said first ligand and the activator or repressor oligonucleotide coupled to said second ligand, said proximity extension or proximity ligation leading to the generation of one or more signal oligonucleotides or one or more anti-signal oligonucleotide(s) and to amplification of said signal oligonucleotide(s).

8 . The method according to claim 1 , wherein the cell or organelle marker-specific ligand is a membrane anchor and in step b) the amount of activator or repressor oligonucleotide(s) present in the compartment is proportional to the surface of the membrane of the enclosed cell(s), so that the concentration of the signal oligonucleotide(s) in step c) is a function of the presence or absence of a compartmentalized cell and/or of the number of compartmentalized cell(s) and/or of the size of the compartmentalized cell(s).

9 . The method according to claim 1 , wherein the signal oligonucleotide is a master signal oligonucleotide produced from at least two different signal oligonucleotides.

10 . The method according to claim 1 , wherein in step a) the DNA toolbox mixture further comprises at least one enzyme selected from the group consisting an exonuclease, an endonuclease, a reverse transcriptase, a ligase, a recombinase, a glycosylase and a DNA-processing enzyme.

11 . The method according to claim 1 , wherein the compartment is a microdroplet, a microcompartment or a cage.

12 . The method according to claim 1 , wherein step b) is performed at a temperature comprised from 20° C. to 45° C.

13 . The method according to claim 1 , wherein step c) comprises determining the concentration of a signal oligonucleotide by adding a reporter probe, wherein said reporter probe is specifically activated by said signal oligonucleotide, and detecting a signal emitted by the reporter probe bound to said signal oligonucleotide.

14 . The method according to claim 1 , wherein step c) comprises recovering the contents of the compartments in which at least one signal oligonucleotide is present at a concentration over or below a threshold value.

15 . The method according to claim 14 , wherein the contents of said recovered contents are analyzed by sequencing cellular or organellar nucleic acids and/or labeling nucleic acid sequence(s).

16 . The method according to claim 1 , wherein step c) comprises recovering the cells of the compartments in which at least one signal oligonucleotide is present at a concentration over or below a threshold value and cultivating the cells prior to further analysis.

17 . The method according to claim 15 , wherein the cellular or organellar nucleic acids and/or labeling nucleic acid sequence(s) located in a same compartment are associated with a compartment-specific barcode sequence.

18 . The method according to claim 17 , wherein the cellular or organellar nucleic acids and/or labeling nucleic acid comprise a compartment-specific barcode sequence further to extension of a cDNA on a barcoded primer, extension of a cDNA on a barcoded template switching oligonucleotide, ligation of a barcoded adapter, or recombination with a barcoded recombination site.

19 . The method according to claim 18 , wherein the barcoded primer, the barcoded template-switching oligonucleotide, the barcoded adaptor or the barcoded recombination site comprises at least one photolabile protecting group and wherein in step c), for the compartments wherein the signal(s) from the reporter probe(s) is above or below a threshold value, photodeprotection of said barcoded primer, barcoded template-switching oligonucleotide, barcoded adaptor or barcoded recombination site, thereby allowing the selective association of the cellular or organellar nucleic acids and/or labeling nucleic acids with a compartment-specific barcode sequence.

20 . The method according to claim 17 , wherein the barcoded primer, the barcoded template-switching oligonucleotide, the barcoded adaptor or the barcoded recombination site comprises photocleavable linkage and, wherein in step c), for the compartments wherein the signal(s) from the reporter probe(s) is above or below a threshold value, photocleavage of said barcoded primer sequence, barcoded template-switching

Assignees

• Paris Sciences Et Lettres
• Centre Nat Rech Scient
• Ecole Superieure Physique & Chimie Ind Ville De Paris

Inventors

• Rondelez Yannick
• Griffiths Andrew
• Blivet-Bailly Gaël
• Ibanez Pablo
• Banerjee Satyam
• Gines Guillaume
• Masurier Antoine

Classifications

• C12Q1/682 Primary

  Signal amplification · CPC title

• C12N9/1241

  Nucleotidyltransferases (2.7.7) · CPC title

• C12N15/1075

  by coupling phenotype to genotype, not provided for in other groups of this subclass · CPC title

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title