In Vitro Selection
The Szostak Laboratory has pioneered the development of in vitro techniques for selecting novel RNA and protein molecules with high-affinity binding or enzymatic activity from highly diverse, random polymer pools. RNA or protein “aptamers” exhibiting high affinity and specific binding to a variety of small molecule targets have been isolated, including RNA molecules that selectively bind ATP, GTP, biotin, riboflavin, nicotinamide, and vitamin B12, as well as protein molecules that bind ATP and streptavidin. A number of in vitro-selected catalytic RNA molecules, or ribozymes, have been isolated and characterized, including ribozymes with ligase, kinase and acyl transferase activities, and efforts are underway to isolate novel proteins with catalytic functions.
We are also interested in exploring the capabilities of polymers with nucleic-acid like properties that have more plausible routes to prebiotic synthesis than either RNA or DNA. Threose Nucleic Acid (TNA) has been suggested as a candidate because of its ability to adopt A-form geometry and base-pair with RNA and DNA. To explore the functionality of TNA we have developed a system for performing in vitro selections for TNA sequences with desirable structural and functional properties. In this approach, TNA is enzymatically transcribed from a single-stranded portion of DNA using a haipin primer. A round of DNA synthesis is then used to displace a single-stranded region of TNA which may be subjected to in vitro selection. This design maintains a physical link between the TNA sequence and the complementary DNA sequence (see figure above), which greatly facilitates amplification for subsequent rounds of selection.
The work of our laboratory and by others has clearly demonstrated that in vitro selection techniques can be used to isolate RNA and protein sequences that bind to essentially any molecular target or catalyze any chemical reaction, providing powerful tools for drug discovery.