We developed a novel method for analyzing RNA sequences, deemed triplet analysis, and applied the method in an in vitro RNA selection experiment in which HIV-1 Tat was the target. Aptamers are nucleic acids that bind a desired target (bait), and to date, many aptamers have been identified by in vitro selection from enough concentrated libraries in which many RNAs had an obvious consensus primary sequence after sufficient cycles of the selection. Therefore, the higher-order structural features of the aptamers that are indispensable for interaction with the bait must be determined by additional investigation of the aptamers. In contrast, our triplet analysis enabled us to extract important information on functional primary and secondary structure from minimally concentrated RNA libraries. As a result, by using our method, an important unpaired region that is similar to the bulge of TAR was readily predicted from a partially concentrated library in which no consensus sequence was revealed by a conventional sequence analysis. Moreover, our analysis method may be used to assess a variety of structural motifs with desired function. 1. Introduction In vitro selection of nucleic acids is a powerful method used to isolate novel functional molecules [1–6]. Generally, in vitro selection involves many rounds of selection followed by sequencing and analysis of several recovered clones and determination of a consensus sequence. These consensus sequences are often highly functional molecules that have promise as pharmaceutical and chemical agents [7–9]. However, there are several inherent biases that often affect in vitro selection procedures, for example, the abundance of particular motifs in the library, differential efficiency of reversetranscription or amplification by PCR due to stable secondary structures. Therefore, confirmation that a particular consensus sequence is optimal is difficult and usually requires identification of a naturally occurring, functional counterpart. For the selection of aptamers, which interact with desired targets, a consensus motif may be a sequence in a local minimum rather than an absolute minimum in an energy diagram that represents interactions between all possible aptamers and the bait or target. Furthermore, it should be hard to identify general rules of intermolecular interactions from only such “highly evolved” motifs. To overcome this problem, the sequence changes in the library during the early stage of the selection should be analyzed. Evaluation of the sequences that are gradually concentrated from the starting library
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