期刊
JOURNAL OF BIOCHEMISTRY
卷 161, 期 2, 页码 197-206出版社
OXFORD UNIV PRESS
DOI: 10.1093/jb/mvw062
关键词
polyamines; aptamer; NMR spectroscopy; interaction analysis; RNA structure
资金
- Japan Society for the Promotion of Science (JSPS) [22501036]
- Jikei University
- MEXT
- Grants-in-Aid for Scientific Research [22501036] Funding Source: KAKEN
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 mu M 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.
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