Abstract
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 1015 different 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.
Highlights
Systematic Evolution of Ligands by EXponential enrichment (SELEX) for obtaining RNA aptamers comprises selection steps and amplification steps
The 0R RNA pool spectrum showed multiple imino proton signals, secondary structure prediction showed that the primer-binding sequences do not adopt stable structure by themselves (Supplementary Fig. S6)
The imino proton spectra were quite similar between the 0R RNA pool and primer-binding sequence RNA, indicating that the Nuclear magnetic resonance (NMR) spectrum of the 0R RNA pool was derived from the primer-binding sequence
Summary
SELEX for obtaining RNA aptamers comprises selection steps (target binding, separation of target-bound RNAs from unbound RNAs) and amplification steps (reverse transcription, PCR amplification and transcription, and purification of selected RNAs). HTS is expensive, and sample preparation for HTS is time-consuming Another effective way of monitoring is to assess the average affinity of the RNA pools to the target molecule[16, 23,24,25,26,27,28,29,30,31,32,33]. To conduct EMSA, the filter-binding assay and FACS, the RNA pools or target molecules must be labeled with tags such as fluorophores or radioisotopes Such immobilization and labeling are time consuming and sometimes change the structure and binding properties of the RNA or target molecules. To evaluate NMR monitoring for the enrichment of RNA aptamers during the SELEX process, we measured 1D imino proton spectra of the RNA pools obtained in our previous study.
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