Two stems with different characteristics and an internal loop in an RNA aptamer contribute to spermine-binding.
JOURNAL OF BIOCHEMISTRY(2017)
摘要
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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关键词
polyamines,aptamer,NMR spectroscopy,interaction analysis,RNA structure
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