IMPATIENT-qPCR: Monitoring SELEX Success During in Vitro Aptamer Evolution

Overview

Journal Appl Microbiol Biotechnol

Specialties Biomedical Engineering
Biotechnology
Microbiology

Date 2024 Apr 4

PMID 38573322

Authors

Ann-Kathrin Kissmann

Grigory Bolotnikov

Runliu Li

Franziska Muller

Hu Xing

Markus Kramer

Kay-E Gottschalk

Jakob Andersson

Tanja Weil

Frank Rosenau

Affiliations

Soon will be listed here.

Abstract

SELEX (Systematic Evolution of Ligands by Exponential enrichment) processes aim on the evolution of high-affinity aptamers as binding entities in diagnostics and biosensing. Aptamers can represent game-changers as constituents of diagnostic assays for the management of instantly occurring infectious diseases or other health threats. Without in-process quality control measures SELEX suffers from low overall success rates. We present a quantitative PCR method for fast and easy quantification of aptamers bound to their targets. Simultaneous determination of melting temperatures (T) of each SELEX round delivers information on the evolutionary success via the correlation of increasing GC content and T alone with a round-wise increase of aptamer affinity to the respective target. Based on nine successful and published previous SELEX processes, in which the evolution/selection of aptamer affinity/specificity was demonstrated, we here show the functionality of the IMPATIENT-qPCR for polyclonal aptamer libraries and resulting individual aptamers. Based on the ease of this new evolution quality control, we hope to introduce it as a valuable tool to accelerate SELEX processes in general. IMPATIENT-qPCR SELEX success monitoring. Selection and evolution of high-affinity aptamers using SELEX technology with direct aptamer evolution monitoring using melting curve shifting analyses to higher T by quantitative PCR with fluorescence dye SYBR Green I. KEY POINTS: • Fast and easy analysis. • Universal applicability shown for a series of real successful projects.

Citing Articles

Specific gFET-Based Aptasensors for Monitoring of Microbiome Quality: Quantification of the Enteric Health-Relevant Bacterium Roseburia Intestinalis.

Zhang Y, Xing H, Li R, Andersson J, Bozdogan A, Strassl R Adv Healthc Mater. 2024; 14(4):e2403827.

PMID: 39663689 PMC: 11804837. DOI: 10.1002/adhm.202403827.

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