High-throughput sequencing allows the identification of binding molecules isolated from DNA-encoded chemical libraries

Abstract

DNA encoding facilitates the construction and screening of large chemical libraries. Here, we describe general strategies for the stepwise coupling of coding DNA fragments to nascent organic molecules throughout individual reaction steps as well as the first implementation of high-throughput sequencing for the identification and relative quantification of the library members. The methodology was exemplified in the construction of a DNA-encoded chemical library containing 4,000 compounds and in the discovery of binders to streptavidin, matrix metalloproteinase 3, and polyclonal human IgG. (5-7), the identification and relative quantification of library members before and after selection can often be achieved by using DNA-microarrays (5-10). By contrast, selections of binding molecules from larger DNA-encoded chemical libraries (comprising several thousand to millions of compounds) may require the use of highthroughput sequencing technologies to assess the relative abundance of library members before and after selection against a target protein of interest.Herein, we describe the construction of a DNA-encoded chemical library consisting of 4,000 compounds covalently attached to unique DNA fragments serving as amplifiable identification bar codes. Similar to our previous experiments with DNA-encoded libraries consisting of several hundreds of members (7), we have initially assessed the relative composition of the new library and its functionality by performing selection experiments on Sepharose resin coated with streptavidin. Because a variety of ligands were known with dissociation constants ranking from the millimolar to the femtomolar range (7) the challenge was to investigate whether binders with various affinities could be easily and rapidly isolated from a library containing 4,000 members. We have found that selections can conveniently be decoded by using a recently described high-throughput DNA sequencing technology (termed ''454 technology'') developed for genome sequencing (11), revealing chemical structures with submicromolar dissociation constants toward streptavidin. In addition, we have performed selections to against the target polyclonal human IgG and the catalytic domain of matrix metalloproteinase 3. To our knowledge a high-throughput sequencing application for decoding of DNA-encoded chemical libraries has not been reported previously. Furthermore, we have devised strategies for the construction and decoding of DNAencoded chemical libraries containing up to 10 6 compounds built on the basis of multiple independent sets of building blocks.

Barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics

“…We recovered the barcodes from tissues and cells and used deep sequencing to obtain counts for those barcodes in each sample of interest (18). Deep sequencing is a high throughput, cost-effective method to precisely quantitate nucleic acid species; it has led to the identification of molecules or peptides with specific biological activities and enabled pooled screening with shRNAs, cDNAs, and labeled pools of RNA (19)(20)(21). By associating specific nanoparticles with unique DNA barcode sequences, we can now reliably measure the biodistribution of many nanoparticles in a single animal (Fig.…”

A Handbook for DNA‐Encoded Chemistry

“…Thanks to the striking sensitivity and specificity of small-molecule DNA-encoding/decoding, today DNA-Encoded Chemical Library (DECL) technology holds a concrete promise to elegantly tackle this formidable task. The appeal of DECL technology mainly relies on the unrivalled opportunity to rapidly synthesize and probe by means of simple affinitycapture selection procedures chemical libraries of unprecedented size in a single test tube [1][2][3][4][5][6].…”

“…Today, cutting-edge, deep-sequencing platforms are capable of collecting millions to billions of DNA-sequence reads per run in just a week [22][23][24], thus allowing the simultaneous deconvolution of multiple panning experiments using libraries containing millions of compounds in a single shot. Employing DNA-encoded strategies, researchers are quickly provided with instant (structure-activity relationship) databases after each selection experiment: an invaluable set of information for medicinal chemistry optimization of the selected structures and/or the design of successive DNA-encoded affinity maturation libraries [1,2,25].…”

Methods for the Creation of Cyclic Peptide Libraries for Use in Lead Discovery

“…Single-pharmacophore libraries are made up of a small organic molecule bound to an oligonucleotide and can be further defined into two subdivisions: libraries that are created combinatorially using a split-and-pool method as first imagined by Brenner and Lerner or libraries that are created using DNA-templated synthesis (DTS). The combinatorial split-and-pool synthesis approach is a robust method for creating DNA-encoded libraries of large magnitude (between 10 5 and 10 8 ) 68 and has identified inhibitors for tumor necrosis factor, 69 carbonic anhydrase IX, 70 polyclonal human IgG, 71 and trypsin. 72 DTS involves the DNA fragments not just as encoding tags but also as a means of directing the generation of the library.…”