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G-quadruplex aptamer selection using capillary electrophoresis-LED-induced fluorescence and Illumina sequencing

Authors
  • Ric, Audrey1, 2, 3
  • Ecochard, Vincent2
  • Iacovoni, Jason S.4
  • Boutonnet, Audrey3
  • Ginot, Frédéric3
  • Ong-Meang, Varravaddheay1
  • Poinsot, Véréna1
  • Paquereau, Laurent2
  • Couderc, François1
  • 1 Université de Toulouse, Université Paul Sabatier, Laboratoire des IMRCP, UMR 5623, 118 Route de Narbonne, Toulouse, 31062, France , Toulouse (France)
  • 2 Université de Toulouse, CNRS, UPS, Institut de Pharmacologie et de Biologie Structurale, IPBS, 205 Route de Narbonne, Toulouse, 31077, France , Toulouse (France)
  • 3 Picometrics Technologies, 478 Rue de la Découverte, Labège, 31670, France , Labège (France)
  • 4 I2MC, UMR1048, 1 Avenue du Professeur Jean Poulhès, Toulouse Cedex 4, 31432, France , Toulouse Cedex 4 (France)
Type
Published Article
Journal
Analytical and Bioanalytical Chemistry
Publisher
Springer-Verlag
Publication Date
Jan 29, 2018
Volume
410
Issue
7
Pages
1991–2000
Identifiers
DOI: 10.1007/s00216-018-0865-5
Source
Springer Nature
Keywords
License
Yellow

Abstract

One of the major difficulties that arises when selecting aptamers containing a G-quadruplex is the correct amplification of the ssDNA sequence. Can aptamers containing a G-quadruplex be selected from a degenerate library using non-equilibrium capillary electrophoresis (CE) of equilibrium mixtures (NECEEM) along with high-throughput Illumina sequencing? In this article, we present some mismatches of the G-quadruplex T29 aptamer specific to thrombin, which was PCR amplified and sequenced by Illumina sequencing. Then, we show the proportionality between the number of sequenced molecules of T29 added to the library and the number of sequences obtained in Illumina sequencing, and we find that T29 sequences from this aptamer can be detected in a random library of ssDNA after the sample is fractionated by NECEEM, amplified by PCR, and sequenced. Treatment of the data by the counting of double-stranded DNA T29 sequences containing a maximum of two mismatches reveals a good correlation with the enrichment factor (fE). This factor is the ratio of the number of aptamer sequences found in the collected complex sample divided by the total number of sequencing reads (aptamer and non-aptamer) plus the quantity of T29 molecules (spiked into a DNA library) injected into CE.

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