DNA-Encoded Library Hit Confirmation: Bridging the Gap Between On-DNA and Off-DNA Chemistry
Bing Xia, G. Joseph Franklin, Xiaojie Lu, Katie L. Bedard, LaShadric C. Grady, Jennifer D. Summerfield, Eric Shi, Bryan W. King, Kenneth Lind, Cynthia H. Chiu, Eleanor Watts, Vera Q. Bodmer, Xiaopeng Bai, Lisa A. Marcaurelle
Abstract
DNA-encoded library (DEL) technology is a powerful platform for hit identification in academia and the pharmaceutical industry. When conducting off-DNA resynthesis hit confirmation after affinity selection, PCR/sequencing, and data analysis, one typically assumes a "one-to-one" relationship between the DNA tag and the chemical structure of the attached small-molecule it encodes. Because library synthesis often yields a mixture, this approximation increases the risk of overlooking positive discoveries and valuable information. To address this issue, we apply a library synthesis "recipe" strategy for on-DNA resynthesis using a cleavable linker, followed by direct affinity selection mass spectrometry (AS-MS) evaluation and identification of binder(s) from the released small-molecule mixture. We validate and showcase this approach employing the receptor-interacting-protein kinase 2 (RIP2) DEL campaign. We also designed and developed two cleavable linkers to enable this method, a photocleavable linker (nitrophenyl-based) and acid-labile linker (tetrahydropyranyl ether). The strategy provides an effective means of hit identification and rapid determination of key active component(s) of the mixture.