A Mega-High-Throughput Screening Platform for the Discovery of Biologically Relevant Sequence-Defined Non-Natural Polymers

Abstract

Combinatorial methods enable the synthesis of chemical libraries on scales of millions to billions of compounds, but the ability to efficiently screen and sequence such large libraries has remained a major bottleneck for molecular discovery. We developed a novel technology for screening and sequencing libraries of synthetic molecules of up to a billion compounds in size. This method utilizes Fiber-optic Array Scanning Technology (FAST) to screen bead-based libraries of synthetic compounds at a rate of 5 million compounds per minute (~83,000 Hz). This ultra-high-throughput screening platform has been used to screen libraries of synthetic “self-readable” non-natural polymers that can be sequenced at femtomole scale by chemical fragmentation and highresolution mass spectrometry. The versatility and throughput of the platform was demonstrated by screening two libraries of non-natural polyamide polymers with sizes of 1.77M and 1B compounds against the protein targets K-Ras, asialoglycoprotein receptor (ASGPR), IL-6, IL-6 receptor and TNFα. Hits with nanomolar binding affinities were found against all targets, including competitive inhibitors of K-Ras binding to Raf and functionally active uptake ligands for ASGPR facilitating intracellular delivery.

Advances in Ultrahigh Throughput Hit Discovery with Tandem Mass Spectrometry Encoded Libraries

“…The sensitivity of modern mass spectrometry, however, now enables the identification of peptide quantities of ∼1 fmol, allowing for the use of ultrahigh diversity libraries, prepared on smaller resin beads. 1,55,56 For a quality control of the library synthesis a number of random beads can be picked and analyzed. 1,54 This validation represents an important advantage over DNA-encoded libraries, in which there is no direct way to assay the quality of library synthesis.…”

“…A similar approach was leveraged to identify sequences with unique cysteine reactivity toward perfluoro aryl moieties. 75,76 56 They used used fiber-optic array scanning technology (FAST), a technology initially developed for cancer diagnostics, 77 as a bead sorting methodology. The technology can screen up to 5 million beads per minute.…”