Techniques to isolate the small RNA fraction (<200nt) by column-based methods are commercially available. However, their use is limited because of the relatively high cost. We found that large RNA molecules, including mRNAs and rRNAs, are aggregated together in the presence of salts when RNA pellets are over-dried. Moreover, once RNA pellets are over-dried, large RNA molecules are barely soluble again during the elution process, whereas small RNA molecules (<100nt) can be eluted. We therefore modified the acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA extraction protocol by skipping the 70% ethanol washing step and over-drying the RNA pellet for 1 hour at room temperature. We named this novel small RNA isolation method “mirRICH.” The quality of the small RNA sequences was validated by electrophoresis, next-generation sequencing, and quantitative PCR, and the findings support that our newly developed column-free method can successfully and efficiently isolate small RNAs from over-dried RNA pellets.
Aptamers are single-stranded nucleic acids that bind to molecular targets, including proteins, with high affinity and specificity. RNA aptamer can be discovered in vitro selection process on the basis on their high affinity for the targets. Recently, various applications of RNA aptamers have been introduced, not only to determine a drug target involved in disease pathology, but also to develop therapeutic agents. We have demonstrated the ability of RNA aptamer to bind to human immunoglobulin G (IgG) with high affinity and specificity. The aptamer was shown to bind to the Fc domain of human IgG, but not to other IgG's, with high affinity. The aptamer was observed to compete with protein A, but not with the Fcr receptor, for IgG binding. Here, we have determined the crystal structure of the aptamer in complex with human IgG. The electron density has clearly proved two aptamers bound to dimeric IgG. The tertiary structures of the recognition sites on the Fc domain differ significantly between human IgG and other species of IgGs. The structure also explains high specificity and affinity of the selected aptamer to human IgG
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.