SELEX Screening and Characterization of Small RNA Molecules That Specifically Bind the Reactive Blue Dye

A 73-base single-stranded DNA library with a T7 promoter was chemically synthesized, in which 20 consecutive bases were completely randomized. After PCR amplification and T7 RNA polymerase transcription of the DNA random library, the in vitro transcribed RNA random library was subjected to 8 successive rounds of reactive blue dye column selection by SELEX method, resulting in a sharp increase of the percentage of the transcribed RNA pool that could bind reactive blue dye, from less than 0.03% in the first round pool to 22.4% in the eighth-round pool. Cloning and sequencing of the enriched RNAs pool led to three groups of RNA molecules in terms of molecular lengths. The correlation between the binding capacities and their corresponding secondary structures of the screened RNAs suggests that the RNA helix is the main motif in the interaction between RNAs and reactive blue dye. These results demonstrate that it is possible to find RNA candidates with a given function in a shorter RNA random library so that it is easier to follow the structure and function relationship. In addition, longer RNA sequences could theoretically be derived from short RNA sequences through a process of motif combination in the course of molecular evolution.