Richard C. Conrad

IN VITRO SELECTION OF NUCLEIC ACID APTAMERS THAT BIND PROTEINS

Publisher Summary Nucleic acid sequences can fold into a huge variety of shapes. Some of these shapes can form complexes, with chemically complementary compounds. The development of more sophisticated in vitro selection methods has made possible the isolation of nucleic acid-binding species (aptamers) that can tightly and specifically interact with a variety of proteins and small molecules. Specifically, in vitro selection has been used to probe interactions between nucleic acids and proteins that normally bind nucleic acids. Subsequently, in vitro selection has been used both to define natural nucleic acid-binding sites for proteins and to identify novel high-affinity sites for these proteins. In vitro selection experiments have not only increased the understanding of the biology of nucleic acid-binding proteins, but have also identified aptamers that can potentially act as therapeutically useful “decoys.” Surprisingly, in vitro selection can also be used to isolate aptamers that can specifically interact, with proteins that are not normally thought to bind nucleic acids.

Conversion of a trans-spliced C. elegans gene into a conventional gene by introduction of a splice donor site.

In Caenorhabditis elegans, pre‐mRNAs that are trans‐spliced are distinguished by the presence of an ‘outron’, intron‐like RNA at the 5′ end followed by a splice acceptor. We report that trans‐splicing of the rol‐6 gene can be completely suppressed simply by introducing a donor site into its 173 nt outron, at a site 50 nt upstream of the trans‐splice site, thereby converting rol‐6 into a conventional gene with a spliced intron near its 5′ end. When the consensus donor site was inserted at sites further upstream it was less effective in replacing transplicing with cis‐splicing. Surprisingly, the length of the intron was not the important variable, since lengthening of the 50 nt intron to 250 nt did not restore trans‐splicing. Apparently the context into which the splice site was introduced determined the efficiency of its use. These results support the conclusion that the sole signal for trans‐splicing is the presence of an outron. Clearly, cis‐ and trans‐splice acceptor sites are interchangeable, allowing the possibility of competition between the two types of splicing.

In vitro selection methodologies to probe RNA function and structure

SummaryIn vitro selection, or SELEX, has been used both to characterize the interaction of natural nucleic acids with proteins and to generate novel nucleic acid-binding species, or aptamers. Although numerous reports have demonstrated the power of the technique, they have not expanded on the methodologies that can be used for selection. This review focuses on the considerations and problems involved in selecting protein-binding aptamers from a random-sequence RNA pool. As an illustration, we describe two approaches to selecting aptamers to a particular target, the HTLV-I Rex protein. In the first, complete randomization is used to find an artificial, high-affinity RNA binding site. In the second, the contributions of individual nucleotides and/or base pairs to the natural Rex-binding element are determined by mutating the wild-type sequence and selecting active binding variants.