Comparative Analysis Reveals Furoyl in Vivo Selective Hydroxyl Acylation Analyzed by Primer Extension Reagents Form Stable Ribosyl Ester Adducts.
RNA molecules depend on structural elements that are critical for cellular function. Chemical methods for probing RNA structure have emerged as a necessary component of characterizing RNA function. As such, understanding the limitations and idiosyncrasies of these methods is essential for their utility. Selective hydroxyl acylation has emerged as a common method for analyzing RNA structure. Ester products as a result of 2'-hydroxyl acylation can then be identified through reverse transcription or mutational enzyme profiling. The central aspect of selective hydroxyl acylation analyzed by primer extension (SHAPE) experiments is the fact that stable ester adducts are formed on the 2'-hydroxyl. Despite its importance, there has not been a direct comparison of SHAPE electrophiles for their ability to make stable RNA adducts. Herein, we conduct a systematic analysis of hydrolysis stability experiments to demonstrate that furoyl imidazole SHAPE reagents form stable ester adducts even at elevated temperatures. We also demonstrate that the acylation reaction with the furoyl acylimidaole SHAPE reagent can be controlled with dithiothreitol quenching, even in live cells. These results are important for our understanding of the biochemical details of the SHAPE experiment.
Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
Dalen
D
Chan
Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
Chao
C
Feng
Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
Yuran
Y
Zhen
Department of Chemistry, Stanford University , Stanford, California 94305, United States.
Ryan A
RA
Flynn
Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.|Department of Chemistry, University of California, Irvine , Irvine, California 92697, United States.
Robert C
http://orcid.org/0000-0002-3511-8098
RC
Spitale
D000215:Acylation; D000255:Adenosine Triphosphate / Q000737:chemistry*; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D017665:Hydroxyl Radical / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry* / Q000494:pharmacology; D007202:Indicators and Reagents / Q000737:chemistry; D012313:RNA / Q000737:chemistry*
D017931:DNA Primers; D007093:Imidazoles; D007202:Indicators and Reagents; D017665:Hydroxyl Radical; D012313:RNA; D000255:Adenosine Triphosphate; D004229:Dithiothreitol