This grant proposal explores the development of a nascent and novel technology, effector-activated ribozymes, also known as aptazymes. In vitro selection techniques have previously yielded a wide variety of nucleic acid binding species, aptamers (B.1). Aptamers can be appended to ribozymes to yield ligand-dependent, allosteric catalysts (B.2). It may be possible to use the broad-ranging molecular recognition abilities of aptamers to generate a similar range of aptazymes. Since aptazymes in effect transduce molecular recognition into catalysis, they can potentially be used as biosensors. We propose to develop aptazyme arrays that can be used to detect and quantitate proteins in organismal proteomes. In particular, by developing aptazymes that can recognize peptide epitopes and protein targets it may prove possible to readily create both targets and biosensors for array construction (B.3.). As a starting point for these goals we have chosen to use a small ribozyme ligase (L1) that was selected in our laboratory (C.1.). The L1 ligase has already been engineered to be responsive to oligonucleotide and small molecule effectors, and the allosteric activation parameters of the engineered variants are far superior to their protein counterparts (C.2-C.4). However, the ability to engineer the L1 ligase to be responsive to peptide or protein effectors should greatly potentiate the development of novel proteome chips. To this end the development efforts for the L1 ligase fall into two major areas: (1)Adapting the L1 ligase to be activated by peptide ad protein effectors (D.1.-D.3), and (2)Adapting peptide and protein-activated aptazymes to function in chip arrays (D.4).
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