monitoring genomic sequences during selex using high-throughput sequencing neutral selex监控在使用高通量测序中性selex selex基因组序列.pdfVIP

Monitoring Genomic Sequences during SELEX Using High-Throughput Sequencing: Neutral SELEX 1. 1,2. 1 1¤ ´ 1 Bob Zimmermann , Tanja Gesell , Doris Chen , Christina Lorenz , Renee Schroeder * 1 Max F. Perutz Laboratories, Department of Biochemistry, University of Vienna, Vienna, Austria, 2 Center for Integrative Bioinformatics in Vienna, Max F. Perutz Laboratories, University of Vienna, Medical University of Vienna and University of Veterinary Medicine, Vienna, Austria Abstract Background: SELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA sequences binding a ligand of interest, called genomic aptamers. Concerns have been raised regarding requirements imposed on RNA sequences undergoing SELEX selection. Methodology/Principal Findings: To evaluate SELEX and assess the extent of these effects, we designed and performed a Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of SELEX’s amplification steps. Using high-throughput sequencing, we obtained thousands of full-length sequences from the initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtained from a Genomic SELEX experiment deriving from the same initial library, but screening for RNAs binding with high affinity to the E. coli regulator protein Hfq. With each round of Neutral SELEX, sequences became less stable and changed in nucleotide content, but no sequences were enriched