UC San Diego

The orchestrations that underlie the existence of even the simplest organisms are quite complex and extremely dynamic. In order to gain a greater understanding of the biochemistry underlying many unsolved biological problems, new tools are required to first visualize a phenomenon, and then perturb it to study its significance. Visualizing the dynamics of intracellular biochemistry has been enhanced greatly with widespread adoption of genetically encoded fluorescent proteins. Due to the large size of fluorescent proteins and their lack of chemical flexibility, the tetracysteine-biarsenical system was developed. This technology uses the combination of a short genetically encoded tag and a specific class of exogenous, membrane-permeant dyes. Since its introduction, the biarsenical-tetracysteine system has suffered from spontaneous background staining, preventing the detection of dilute proteins. To remedy this problem, a library of tetracysteine sequences was screened for improved dithiol resistance and brightness. Several new sequences were discovered and background staining was reduced significantly, resulting in a 20-fold increase in labeling contrast. One unique sequence isolated from the library found an unexpected mechanism to ensure its selection. Upon ReAsH labeling, YRECCPGCCMWR-GFP rapidly aggregates into tiny, highly fluorescent speckles. Upon bleaching ReAsH, the aggregates dissociate, dispersing the tagged protein throughout the cell. Fusions of this tag on several cellular proteins led to ReAsH dependent aggregation of the tagged protein as wells as endogenous binding partners. By sequestering protein complexes in the aggregates, activity is inhibited. Finally, the detection of specific RNAs in living cells remains a major challenge in biology with numerous potential applications. Trans- splicing repair of clinically relevant transcripts has been reported as an efficient and specific method for delivering exogenous message for translation. Therefore, a crippled reporter gene lacking translation initiation sites gene was targeted using existing trans-splicing techniques to an expressed RNAs. Trans-splicing then leads to the conversion of the targeted mRNA into a chimeric mRNA capable of translating an active protein. After significant effort and several novel approaches to enhance specificity, it became clear that new methods of RNA detection will be required to prevent non-specific splicing of cargo RNAs in cells