A Lambda-Exonuclease SELEX Method for Generating Aptamers to Bacterial Targets

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.

Keywords: Aptamer; E. coli; Lambda exonuclease; Oligonucleotide library; SELEX.