Two stems with different characteristics and an internal loop in an RNA aptamer contribute to spermine-binding.
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.
Department of Molecular Biology, The Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan.
Akihiro
A
Oguro
Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
Asumi
A
Yanagida
Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
Yuta
Y
Fujieda
Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
Ryo
R
Amano
Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
Maina
M
Otsu
Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
Taiichi
T
Sakamoto
Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
Gota
G
Kawai
Department of Molecular Biology, The Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan.
Senya
S
Matsufuji
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D011073:Polyamines / Q000737:chemistry; D013096:Spermine / Q000737:chemistry*; D013816:Thermodynamics
D052157:Aptamers, Nucleotide; D011073:Polyamines; D013096:Spermine
polyamines; aptamer; NIMR spectroscopy; interaction analysis; RNA structure