A. Oguro, A. Yanagida, Y. Fujieda, Ryotaro Amano, M. Otsu, T. Sakamoto, G. Kawai, S. Matsufuji
{"title":"Two stems with different characteristics and an internal loop in an RNA aptamer contribute to spermine-binding","authors":"A. Oguro, A. Yanagida, Y. Fujieda, Ryotaro Amano, M. Otsu, T. Sakamoto, G. Kawai, S. Matsufuji","doi":"10.1093/jb/mvw062","DOIUrl":null,"url":null,"abstract":"Though polyamines (putrescine, spermidine, and spermine) bind to the specific position in RNA molecules, interaction mechanisms are poorly understood. SELEX procedure has been used to isolate high-affinity oligoribonucleotides (aptamers) from randomized RNA libraries. Selected aptamers are useful in exploring sequences and/or structures in RNAs for binding molecules. In this study, to analyze the interaction mechanism of polyamine to RNA, we selected RNA aptamers targeted for spermine. Two spermine-binding aptamers (#5 and #24) were obtained and both of them had two stem-loop structures. The 3′ stem-loop of #5 (SL_2) bound to spermine more effectively than the 5′ stem-loop of #5 did. A thermodynamic analysis by an isothermal titration calorimetry revealed that the dissociation constant of SL_2 for spermine was 27.2 μM and binding ratio was nearly 1:1. Binding assay with base-pair replaced variants showed that two stem regions and an internal loop in SL_2 were important for their spermine-binding activities. NMR analyses proposed that a terminal-side and a loop-side stem in SL_2 take a loose and a stable structure, respectively and a conformational change of SL_2 is induced by spermine. It is conclusive that two stems with different characteristics and an internal loop in SL_2 contribute to the specific spermine-binding.","PeriodicalId":22605,"journal":{"name":"The Journal of Biochemistry","volume":"78 1","pages":"197–206"},"PeriodicalIF":0.0000,"publicationDate":"2016-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"11","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"The Journal of Biochemistry","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1093/jb/mvw062","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 11
Abstract
Though polyamines (putrescine, spermidine, and spermine) bind to the specific position in RNA molecules, interaction mechanisms are poorly understood. SELEX procedure has been used to isolate high-affinity oligoribonucleotides (aptamers) from randomized RNA libraries. Selected aptamers are useful in exploring sequences and/or structures in RNAs for binding molecules. In this study, to analyze the interaction mechanism of polyamine to RNA, we selected RNA aptamers targeted for spermine. Two spermine-binding aptamers (#5 and #24) were obtained and both of them had two stem-loop structures. The 3′ stem-loop of #5 (SL_2) bound to spermine more effectively than the 5′ stem-loop of #5 did. A thermodynamic analysis by an isothermal titration calorimetry revealed that the dissociation constant of SL_2 for spermine was 27.2 μM and binding ratio was nearly 1:1. Binding assay with base-pair replaced variants showed that two stem regions and an internal loop in SL_2 were important for their spermine-binding activities. NMR analyses proposed that a terminal-side and a loop-side stem in SL_2 take a loose and a stable structure, respectively and a conformational change of SL_2 is induced by spermine. It is conclusive that two stems with different characteristics and an internal loop in SL_2 contribute to the specific spermine-binding.