A lambda-exonuclease SELEX method for generating aptamers to bacterial targets

Robert Gowland, Darren M. Gowers

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

Abstract

Nucleic acid aptamers are short sequences of single-stranded (ss) DNA or RNA that fold into a three-dimensional shape with useful binding properties. Traditionally, these properties have included specific recognition and binding of ions, small-molecules, proteins, and enzyme targets. Increasingly though, aptamers are being raised against complex subcellular or cellular targets. These broader-affinity aptamers can be usefully employed for detection, labeling, or therapeutic targeting of intact/living cells, whether prokaryotic or eukaryotic. Aptamers are usually developed from a random-sequence oligonucleotide library by repeated rounds of selection and amplification, a process named “systematic evolution of ligands by exponential enrichment” (SELEX). We describe here a widely applicable cell-SELEX method for raising aptamers against bacteria, using Escherichia coli strain HB101 as an example. Our cell-SELEX method uses a cycle of four stages: (1) incubation of a fluorescently labeled random-sequence ssDNA library with bacterial cells; (2) separation of cell-associated ssDNA from free ssDNA; (3) amplification of bound ssDNA by PCR, and (4) use of lambda-exonuclease to selectively regenerate ssDNA for further rounds of selection.
Original languageEnglish
Title of host publicationDNA Manipulation and Analysis
EditorsGarry Scarlett
Place of PublicationNew York
PublisherHumana Press
Chapter12
Pages145-161
Number of pages17
Edition1st
ISBN (Electronic)9781071630044
ISBN (Print)9781071630037
DOIs
Publication statusPublished - 1 Mar 2023

Publication series

NameMethods in Molecular Biology
Volume2633
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • SELEX
  • aptamer
  • oligonucleotide library
  • lambda exonuclease
  • E. coli

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