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.

In Vivo Imaging of the Tumor‐Associated Enzyme NCEH1 with a Covalent PET Probe

“…Gel‐based measurements of active enzyme in microdissected tumor cells from the edge and core of xenografts were made using a specific fluorescent probe that labels active NCEH1, JW576, which confirmed significantly higher NCEH1 activity in cells isolated from the edge of both MDA‐MB231 and PC3 xenografts (Figure B and Figures S7 and S8 C). This difference in active NCEH1 was also confirmed using a more sensitive method, soluble activity‐dependent proximity ligation (Figure C) . Likewise, western blot revealed decreased NCEH1 abundance in the tumor core relative to the edge (Figure D).…”

Ultrasensitive, multiplexed chemoproteomic profiling with soluble activity-dependent proximity ligation

“…In parallel, we synthesized antibody-oligonucleotide (Ab-oligo) conjugates for specific proteins within each enzyme family, as well as probe-specific streptavidin-oligonucleotide (SA-oligo) conjugates (SI Appendix, Fig. S1) (20). Using monoacylglycerol lipase (MGLL) as a model enzyme, we optimized the absolute and relative concentrations of Ab-oligo and SA-oligo conjugates required for the sADPL signal and dynamic range in whole-cell proteome (SI Appendix, Fig.…”

“…While the distinct biological mechanisms underlying these differences are the subject of future dedicated investigations, these results establish the potential for sADPL profiling in clinical samples to provide novel biological hypotheses as well as potential diagnostic correlations based on activity and not solely on protein expression. We expect that optimized protocols, automation, and contemporary methods to generate oligonucleotide-protein conjugates (20) will further augment these capabilities, and that sADPL will find many applications in the molecular analysis of biological and clinical samples.…”

“…In the same samples, we simultaneously measured the abundance of a loading control protein, GAPDH, using proximity ligation assay (PLA)-qPCR as a way to normalize for sample, processing, and amplification differences across patients (SI Appendix, Fig. S8) (20). The resulting activity profile confirmed that NCEH1, MGLL, CTSB, and CTSL activities were generally correlated within the same patient spheroids, whereas active FAAH levels were more variable and DPP4 activity was undetectable (SI Appendix, Fig.…”

In Vivo Imaging of the Tumor‐Associated Enzyme NCEH1 with a Covalent PET Probe

“…This difference in active NCEH1 was also confirmed using a more sensitive method, soluble activity-dependent proximity ligation (Figure 3 C). [24,25] Likewise, western blot revealed decreased NCEH1 abundance in the tumor core relative to the edge (Figure 3 D). Finally, immunofluorescent imaging of whole xenograft tissue sections confirmed NCEH1 localization along the outer edges of the tumor, with comparatively little NCEH1 observed within the tumor, reinforcing the microenvironmental heterogeneity observed by PET imaging with [ 18 F]JW199 (Figure 3 E).…”