A Concise, Modular Antibody-Oligonucleotide Conjugation Strategy Based on Disuccinimidyl Ester Activation Chemistry.

ABSTRACT: The synthesis of antibody-oligonucleotide conjugates has enabled the development of highly sensitive bioassays for specific epitopes in the laboratory and clinic. Most synthetic schemes to generate these hybrid molecules require expensive reagents, significant quantities of input antibody, and multistep purification routes; thus limiting widespread application. Herein a facile and robust conjugation strategy is reported that involves "plug-and-play" antibody conjugation with succinimidyl-functionalized oligonucleotides, which are high yielding and compatible for use directly after buffer exchange. The succinimidyl-linked oligonucleotides are synthesized with 5'-amine-modified oligonucleotides and disuccinimidyl suberate (DSS), both of which are inexpensive and commercially available. Direct incubation of the resulting stable succinimidyl- oligonucleotide conjugates with commercial antibodies yields conjugates ready for use after benchtop buffer exchange. It is demonstrated that the resulting oligonucleotide-antibody and oligonucleotide-streptavidin conjugates retain potent and specific binding in activity-dependent proximity ligation imaging, and proximity ligation-mediated qPCR detection of endogenous proteins in native cellular contexts down to picogram levels of whole proteome. This DSS conjugation strategy should be widely applicable in the synthesis of protein-oligonucleotide conjugates.

SUBMITTER: Li G 

PROVIDER: S-EPMC6773266 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature