NMR monitoring of the SELEX process to confirm enrichment of structured RNA.
RNA aptamers are RNA molecules that bind to a target molecule with high affinity and specificity using uniquely-folded tertiary structures. RNA aptamers are selected from an RNA pool typically comprising up to 1015different sequences generated by iterative steps of selection and amplification known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Over several rounds of SELEX, the diversity of the RNA pool decreases and the aptamers are enriched. Hence, monitoring of the enrichment of these RNA pools is critical for the successful selection of aptamers, and several methods for monitoring them have been developed. In this study, we measured one-dimensional imino proton NMR spectra of RNA pools during SELEX. The spectrum of the initial RNA pool indicates that the RNAs adopt tertiary structures. The structural diversity of the RNA pools was shown to depend highly on the design of the primer-binding sequence. Furthermore, we demonstrate that enrichment of RNA aptamers can be monitored using NMR. The RNA pools can be recovered from the NMR tube after measurement of NMR spectra. We also can monitor target binding in the NMR tubes. Thus, we propose using NMR to monitor the enrichment of structured aptamers during the SELEX process.
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
Ribomic Inc., 3-16-13 Shirokanedai, Minato-ku, Tokyo, 108-0071, Japan.
Kazuteru
K
Aoki
Ribomic Inc., 3-16-13 Shirokanedai, Minato-ku, Tokyo, 108-0071, Japan.
Shin
S
Miyakawa
Ribomic Inc., 3-16-13 Shirokanedai, Minato-ku, Tokyo, 108-0071, Japan.|Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
Yoshikazu
0000-0001-8763-0424
Y
Nakamura
Research Institute for Clinical Oncology, Saitama Cancer Center, 818 Komuro, Ina, Kitaadachi-gun, Saitama, 362-0806, Japan.
Tomoko
T
Kozu
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 Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba, 275-0016, Japan. taiichi.sakamoto@p.chibakoudai.jp.
Taiichi
0000-0002-1450-9372
T
Sakamoto
D052157:Aptamers, Nucleotide / Q000032:analysis* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D052156:SELEX Aptamer Technique
D052157:Aptamers, Nucleotide