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System for single-molecule isolation for cell populations and single cells, and methods and uses thereof

A cell and molecular technology, applied in the field of protein and/or cell detection, can solve the problems that are not widely used, easy to be destroyed, and cannot be used in ordinary biological laboratories

Pending Publication Date: 2021-02-26
THE HONG KONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These weak PPIs are easily destroyed by the multiple washing steps used in conventional co-IP assays
However, SiMPull has not been widely used since its development, nor has it been commercialized on a large scale, because the method presents high technical barriers and is time-consuming
[0010] Existing single-cell pull-down methods can isolate single cells but involve too many difficulties in expanding their use for analysis of cellular components, as evidenced by the complete lack of any commercial systems or methods for their use (Wedeking, T. .et al., "SingleCell GFP-Trap Reveals Stoichiometry and Dynamics of Cytosolic Protein Complexes", Nano Lett., 15(5):3610-3615(2015 ); Wang, X. et al., "Toward Single-Cell Single-Molecule Pull-Down", Biophysical Journal, 115(2):283-288(2018); Ryu, J.Y. et al., " Profiling Protein-Protein Interactions of Single Cancer Cells with in situ Lysis and Co-Immunoprecipitation", Lab Chip.,19(11):1922-1928 (2019)
The applicability of these methods is questionable because (1) the methods can be used to analyze slowly diffusing cells only in bacterial or adherent cell cultures and not in primary or suspension culture cells such as blood cells and circulating tumor cells. molecule; 2) the method can only be used to analyze cell surface proteins using antibodies against the extracellular domain of cell surface proteins; and 3) the technical barriers to the method are extremely high and thus the method cannot be used in general biological laboratories middle
[0011] Thus, there remains a considerable unmet need for single-cell pull-downs for cellular component analysis

Method used

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  • System for single-molecule isolation for cell populations and single cells, and methods and uses thereof
  • System for single-molecule isolation for cell populations and single cells, and methods and uses thereof
  • System for single-molecule isolation for cell populations and single cells, and methods and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0109]General representative method for single cell separation and analysis based on nanobeads for cell populations

[0110]The streptavidin-coated magnetic nanobeads are modified by immobilizing a primary antibody on their surface and are used to capture the target protein (bait protein) (as described in the Examples herein). The cells are lysed and magnetic nanobeads are added to the lysate and incubated for 30 minutes, and after the prey protein is captured by the beads, the protein is detected using a specific primary antibody and a fluorescently labeled secondary antibody. The nanobeads were washed three times to remove non-specifically bound proteins, transferred to a glass slide and covered with a cover slip, and imaged using a TIRF microscope. In some embodiments, the bait protein or prey protein is fluorescently labeled for direct visualization. (figure 1 )

Embodiment 2

[0112]Nanobead-based SiMPull for green fluorescent protein (GFP) pull-down in cell populations

[0113]The SiMPull method based on nanobeads first verifies protein pulldown by pulling down GFP, because GFP can be directly visualized without immunostaining, which simplifies verification and, more importantly, because GFP can be used in photobleaching experiments to evaluate the fluorescence of pulldown The single-molecule state of the spot.

[0114]Use magnetic nanobeads to pull down the ectopic GFP in HEK293T cells (figure 2 a). In contrast, in the negative control experiment, very few GFP molecules were pulled down (figure 2 a), and the calculated measured signal-to-noise (SN) ratio is about 10-20 (figure 2 b). The SN ratio is equivalent to or better than the original SiMPull method of Jain et al. By applying the algorithm previously used by Jain et al.,figure 2 About 95% of the fluorescent spots in a show Gaussian distribution. A photobleaching experiment was performed on these fluoresc...

Embodiment 3

[0116]Pull down the cAMP-dependent protein kinase A (PKA) complex in the cell population

[0117]The holoenzyme or complex of PKA (one of the most widely studied protein kinases) exists as a heterotetramer comprising a regulatory (R) subunit dimer and two catalytic (C) subunits (image 3 a); cAMP can bind to the R subunit and release the C subunit from the PKA complex at physiological levels (image 3 a). We used the well-characterized interaction between the PKA-R subunit and the PKA-C subunit to validate the nanobead-based SiMPull assay used to study PPI.

[0118]The expression vectors used in this study are pEGFP-n1 (Addgene catalog number 6085-1) for GFP expression, pcDNA3-mouse PKA-C-α-mEGFP (Addgene catalog number 45521) and pcDNA3-mouse PKA-RII -α-mEGFP (Addgene catalog number 45527). By replacing the GFP sequence in pcDNA3-PKA-C-α-mEGFP with the mCherry sequence, pcDNA3-PKA-C-α-mCherry was constructed.

[0119]The interaction of PKA-C-eGFP and PKA-R-mCherry was detected in HEK293T cell...

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Abstract

The invention relates to the systems and methods for isolating single cells, capturing selected prey biomolecules within said cells, and detecting said prey biomolecules using a sensitive assay method. Also described herein are functionalized substrates and compositions for enabling the low limit of detection of selected prey biomolecules of selected cells.

Description

[0001]Related application[0002]This patent application claims the benefit of U.S. Provisional Application No. 62 / 922,657 filed on August 22, 2019, the entire disclosure of which is incorporated herein by reference.Technical field[0003]The invention described herein generally relates to the field of protein and / or cell detection. Specifically, the embodiments described herein relate to systems and methods for isolating single cells, capturing selected prey biomolecules within the cells, and detecting the prey biomolecules using sensitive assay methods.[0004]Incorporated by reference[0005]All U.S. patents, U.S. patent application publications, foreign patents, foreign and PCT published applications, articles and other documents, references and publications referred to herein, as well as acts cited in any one or more patents issued thereby All documents listed in the references are hereby incorporated by reference in their entirety. The incorporated information is part of this applicat...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/68C12M1/00C12N5/00C12N15/10C07K1/14
CPCG01N33/53G01N33/68C12M47/04C12M25/16C12N5/00C12N15/1013C07K1/14C12N2509/00G01N2021/6439G01N21/6458G01N21/648G01N21/6452G01N27/745G01N33/54333G01N33/553G01N33/582
Inventor 黄平波赵琦睿
Owner THE HONG KONG UNIV OF SCI & TECH
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