title: Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.
abstract: RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin-streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin-SAv roadblocks. We then show that randomly distributed biotin-SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin-SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq.
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60201, USA.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Strobel
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Kyle E
authors.initials: KE
authors.lastname: Watters
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA.|The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Nedialkov
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA.|The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Artsimovitch
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60201, USA.
authors.forename: Julius B
authors.initials: JB
authors.lastname: Lucks
mesh_terms: D000215:Acylation; D020029:Base Pairing; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry* / Q000235:genetics; D002623:Chemistry Techniques, Analytical; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry / Q000235:genetics; D006878:Hydroxides / Q000737:chemistry; D009690:Nucleic Acid Conformation; D012313:RNA / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D059370:RNA Folding; D058928:Riboswitch; D017423:Sequence Analysis, RNA; D019809:Streptavidin / Q000737:chemistry* / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D006878:Hydroxides; D058928:Riboswitch; D012313:RNA; D001710:Biotin; D019809:Streptavidin; C031356:hydroxide ion; D015246:Deoxyribonuclease EcoRI