Daniel Matthew Held
dmheld@indiana.edu

Indiana University
Department of Biology
1001 E. Third Street
Bloomington, IN 47405
USA

Advisors:
Dr. Donald Burke, Biochemistry, IU
Dr. John G. Foley, Medical Sciences, IU


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Research Interests


A hypothetical landscape for FAD-binding and GMP-binding phenotypes adjacent to surveyed sequence space. Sequence space is mapped in two dimensions, where the axes represent mutational distance. Individual boxes in the grid represent unique sequences within a landscape of all possible 35mers. Boxes bordering one another on a side or diagonally represent sequences differing by a single mutation. FAD binding activity is represented in gray, GMP binding activity in black, and inactive sequences in white. Assayed sequences are represented as overlapping regions of sequence space (A). A larger view of FAD aptamer-rich adjacent sequence space is represented where GMP aptamers are rare (B), or wher FAD- and GMP-binding sequences are abundant and lie in close mutational proximity (C).

We are interested in studying the evolutionary landscapes for ligand binding at the molecular level using RNA aptamers.  The use of in vitro selection techniques allows us to generate populations of RNA aptamers with high binding specificity for small molecule targets.  Our system has been devised to examine the sequence and structural requirements for binding to the nucleotide GMP and the nucleotide cofactor FAD (flavin adenine dinucleotide).

Mutagenized pools based the three published flavin-binding aptamers were combined for in vitro selections for GMP- or FAD-binding activity.  The FAD selection yielded three distinct populations displaying sequence conservation and covariation patterns consistent with known constraints for the parental molecules.  The GMP selection produced a less well-defined collection of sequences, derived predominantly from only one of the three flavin-binding parents.  Despite the absence of an FAD counter-selection, the acquisition of GMP recognition coincided with a loss of FAD recognition in all assayed selection isolates.  Overall, GMP aptamers were differentiated from FAD aptamers by an average of 20 mutations.  In order to more precisely examine the neutrality of sequence space around more closely related aptamer sequences, the complete sequence space between two FAD aptamers (F6-26 and F26m4) and between an FAD aptamer (F26m4) and a GMP aptamer were examined for FAD- and GMP-binding.  Within this well-defined sequence space, we find a neutral network for FAD-binding function in close proximity to (three mutations away from) GMP-binding function.  Our results illustrate the degree of phenotypic buffering available to a set of closely-related RNA sequences—defined as the set's tolerance for point mutations—and support neutral evolutionary theory by demonstrating the facility with which a new phenotype (ligand-binding) becomes accessible once that buffering capacity is exceeded.

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Publications

... none as yet ...

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