Allosteric nucleic acid catalysts can directly transduce ligand recognition into catalysis, and thus are particularly useful as biosensors. We have previously developed a variety of aptazymes, based on ribozyme and deoxyribozyme ligases and Group I self-splicing introns, and have also developed novel assay formats for these aptazymes. Now, we propose to adapt aptazymes to function in vivo, in order to build genetic circuits that can control gene regulation. In particular, we propose to replace or augment regulatory proteins with regulatory aptazymes in a particularly well-studied model system, catabolite repression in E. coli. In greater detail:
Specific Aim 1. In vitro selection of aptazymes dependent on cAMP and CRP. The small molecule cAMP and cAMP receptor protein (CRP) are instrumental in modulating the expression of operons involved in the utilization of sugars, such as the lac operon. Starting with three different ribozyme platforms (hammerhead, L1 ligase, and Group I self-splicing intron) we will select and characterize aptazymes that are activated by cAMP or CRP. It should be noted that a cAMP-dependent hammerhead aptazyme is already available, and thus that Specific Aims 2 and 3 do not completely rely upon the execution of Specific Aim 1.
Specific Aim 2. Aptazyme optimization for multiple turnover and in vivo function. The aptazymes selected as a result of Specific Aim 1 will be further optimized for multiple turnover reactions using a novel trans-selection method based on in vitro compartmentalization (IVC) in water-in-oil emulsions. The fastest aptazymes will then be further screened for function in E. coli cells. We believe that both of these technologies will ultimately be necessary to identify aptazymes that are highly efficient in vivo. The IVC method will allow us to screen larger pool sizes prior to assessing smaller, functional pools in vivo.
Specific Aim 3. Constructing genetic circuits with aptazymes. The aptazymes from Specific Aims 1 and 2 will be activated by either cAMP or CRP, and will be able to either cleave, ligate, or splice either Lacl or LacZ mRNAs. They thus represent modular tools for the construction of a variety of genetic circuits for catabolite repression. We will generate several such circuits in which the addition of glucose leads, for example, to the cleavage of Lacl mRNA or to the ligation of LacZ mRNA. These circuits will be further evolved for in vivo function by iterative growth in the presence of glucose and lactose. The development of technologies for the production of aptazymes and genetic circuits that can function in vivo should potentiate the construction of organismal biosensors, regulatable gene therapies, and new tools for the burgeoning field of synthetic biology. ? ?
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