Quantitative PCR is a Valuable Tool to Monitor the Performance of DNA‐Encoded Chemical Library Selections

Abstract

Phage-display libraries and DNA-encoded chemical libraries (DECLs) represent useful tools for the isolation of specific binding molecules from large combinatorial sets of compounds. With both methods, specific binders are recovered at the end of affinity capture procedures by using target proteins of interest immobilized on a solid support. However, although the efficiency of phage-display selections is routinely quantified by counting the phage titer before and after the affinity capture step, no similar quantification procedures have been reported for the characterization of DECL selections. In this article, we describe the potential and limitations of quantitative PCR (qPCR) methods for the evaluation of selection efficiency by using a combinatorial chemical library with more than 35 million compounds. In the experimental conditions chosen for the selections, a quantification of DNA input/recovery over five orders of magnitude could be performed, revealing a successful enrichment of abundant binders, which could be confirmed by DNA sequencing. qPCR provided rapid information about the performance of selections, thus facilitating the optimization of experimental conditions.

DNA‐encoded chemical libraries – achievements and remaining challenges

“…The use of quantitative PCR in model screening experiments (e.g., by spiking binders into large libraries) has recently been described [42].…”

“…The problem can be tackled from different perspectives. On one hand, quantitative PCR methodologies provide direct experimental evidence regarding the efficiency and selectivity of ligand recovery [42,50]. Alternatively, certain estimates can be performed, using a computational analysis of selection results [51,52].…”

“…However, conventional technologies based on affinity capture methods are quite reliable for the isolation of nanomolar binders from encoded libraries, if and when they are present in the repertoire [42,61].…”

Quantitative Assessment of Affinity Selection Performance by Using DNA‐Encoded Chemical Libraries

“…The efficiency of DECL selection procedures has been scarcely investigated, so far. A direct quantification of individual DNA barcodes is possible by means of quantitative PCR (qPCR) techniques, but this technology has a limited sensitivity, due to the unspecific amplification of oligomeric primer structures . Alternatively, investigators have studied the performance of selection procedures by inspecting the features of library fingerprints from DNA sequencing results, comparing DECLs before and after selections .…”

“…A direct quantification of individual DNA barcodes is possible by means of quantitative PCR (qPCR) techniques, but this technology has a limited sensitivity, due to the unspecific amplification of oligomeric primer structures . Alternatively, investigators have studied the performance of selection procedures by inspecting the features of library fingerprints from DNA sequencing results, comparing DECLs before and after selections . In this work, we used ligands specific to carbonic anhydrase IX (CAIX; a tumor‐associated antigen) as tools for the quantitative evaluation of selection procedures, which are commonly used in academia and in industry .…”