Monitoring Genomic Sequences during SELEX Using High-Throughput Sequencing Neutral SELEX 英文参考文献.docVIP

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Monitoring Genomic Sequences during SELEX Using High-Throughput Sequencing Neutral SELEX 英文参考文献.doc

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Monitoring Genomic Sequences during SELEX Using High-Throughput Sequencing Neutral SELEX 英文参考文献

MonitoringGenomicSequencesduringSELEXUsing High-ThroughputSequencing:NeutralSELEX BobZimmermann1.,TanjaGesell1,2.,DorisChen1,ChristinaLorenz1¤,Rene′eSchroeder1* 1Max F. Perutz Laboratories, Department of Biochemistry, University of Vienna, Vienna, Austria, 2Center for Integrative Bioinformatics in Vienna, Max F. Perutz Laboratories,UniversityofVienna,MedicalUniversityofViennaandUniversityofVeterinaryMedicine,Vienna,Austria Abstract Background: SELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequencescalledaptamers.GenomicSELEXtransformsSELEXintoatooltodiscovernovel,genomicallyencodedRNAor DNAsequencesbindingaligandofinterest,calledgenomicaptamers.Concernshavebeenraisedregardingrequirements imposedonRNAsequencesundergoingSELEXselection. Methodology/PrincipalFindings:ToevaluateSELEXandassesstheextentoftheseeffects,wedesignedandperformeda Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of SELEX’samplificationsteps.Usinghigh-throughputsequencing,weobtainedthousandsoffull-lengthsequencesfromthe initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtainedfromaGenomicSELEXexperimentderivingfromthesameinitiallibrary,butscreeningforRNAsbindingwithhigh affinitytotheE.coliregulatorproteinHfq.WitheachroundofNeutralSELEX,sequencesbecamelessstableandchangedin nucleotidecontent,butnosequenceswereenriched.Incontrast,wedetectedsubstantialenrichmentintheHfq-selected set with enriched sequences having structural stability similar to the neutral sequences but with significantly different nucleotideselection. Conclusions/Significance: Our data indicate that positive selection in SELEX acts independently of the neutral selective requirements imposed on the sequences. We conclude that Genomic SELEX, when combined with high-throughput sequencing of positively and neutrally selected pools, as well as