US 12,339,291 B2
Lipid-DNA labeling of lipid bilayer particles for amplification quantitation
Thomas Robert Carey, Berkeley, CA (US); Molly Aliza Kozminsky, Berkeley, CA (US); and Lydia L. Sohn, Berkeley, CA (US)
Assigned to The Regents of the University of California, Oakland, CA (US)
Filed by The Regents of the University of California, Oakland, CA (US)
Filed on May 20, 2022, as Appl. No. 17/749,163.
Application 17/749,163 is a continuation of application No. PCT/US2020/062957, filed on Dec. 2, 2020.
Claims priority of provisional application 62/942,216, filed on Dec. 2, 2019.
Prior Publication US 2022/0276266 A1, Sep. 1, 2022
Int. Cl. G01N 33/92 (2006.01); C12Q 1/6851 (2018.01)
CPC G01N 33/92 (2013.01) [C12Q 1/6851 (2013.01); G01N 2458/10 (2013.01)] 20 Claims
 
1. A method to quantify lipid bilayer particles displaying specific surface antigens, comprising:
(a) labeling the particles with single-stranded DNAs (ssDNAs) each end-labeled with a membrane self-embedding lipid;
(b) specifically capturing different subpopulations of the particles by specific antigen binding or complementary oligonucleotide hybridization;
(c) employing a restriction enzyme to release the ssDNA label after the capture; and
(d) using quantitative polymerase chain reaction (qPCR) quantification of the ssDNA to yield a quantitative readout that is directly correlated with the number of particles captured;
wherein the labeling step comprises combining the particles with:
i. an anchor oligo comprising: lipid-anchor sequence-adhesion sequence;
ii. a co-anchor oligo comprising: lipid-anchor sequence′; and
iii. a detection oligo comprising: adhesion sequence′-detection sequence;
wherein the anchor oligo and at least one of the co-anchor oligo and detection oligo comprise complementary restriction sequences;
wherein the ′ indicates reverse complement, and the anchor is in opposite orientation from the co-anchor and detection oligos, such that:
the lipids self-embed into the lipid bilayer membrane of the particles and anchor sequences hybridize via complementary base pairing, preventing the anchor oligo from dissociating from the membrane; the adhesion sequences hybridize, capturing the detection oligo onto the particles; and the restriction sequences hybridize, forming a double-stranded restriction site.