IN VITRO AND IN VIVO DETECTION OF NUCLEIC ACIDS AND PROTEINS USING SPLIT-PROTEIN REASSEMBLY

Abstract

The ability to directly monitor the presence of specific proteins or nucleic acids in a variety of in vitro and in vivo contexts has great utility for understanding biology as well as for the development of diagnostic agents. Herein I describe several methodologies, utilizing split-protein reassembly, which provides potentially general strategies for sequence-specific detection of DNA and RNA sequences as well as poly(ADP-ribose). I also provide a new split-protein approach for the direct detection of native proteins, such as the cancer marker HER2.The green fluorescent protein (GFP) provides a convenient sensor for reporting on a variety of cellular events. A series of spectroscopically distinct GFP variants was developed for sequence-specifically reporting on DNA. Each of these variants was demonstrated to provide a sensitive readout for the presence of a particular DNA sequence. Furthermore, utilizing a method of mixed split-protein complementation, I was able to simultaneously report on the presence of two distinct DNA sequences in the same solution.To provide a general solution for reporting on the presence of particular RNA sequences, a method was developed that utilized elements from a hybridization-based detection strategy coupled with split-protein reassembly. Specifically, DNA guide sequences complementary to an intended target were attached to hairpin sites that served as binding sites for high-affinity zinc fingers. Localization of the zinc fingers allowed for reassembly of the attached split enzyme, providing a sensitive readout for the presence of potentially any RNA sequence of interest. This methodology was applied to the detection of mRNA encoding VEGF, hDM2, and HER2, each of which may be overexpressed in cancer.A method was established for reporting on the presence of modifications to DNA and proteins that are elicited in response to DNA damage. Specifically, sensors were designed, which incorporated endogenous damage-recognition domains, to report on the global presence of particular DNA modifications, including the formation of 8-oxoguanine and pyrimidine dimers. Furthermore, to provide a technique for monitoring the general accrual of DNA damage and to interrogate the DNA damage response in cells, a sensor was developed which reported on the accrual of a posttranslational protein modification, poly(ADP-ribosyl)ation.Finally, I describe advances toward the adaptation of our protein-based biosensors for use in living cells, utilizing both GFP-based approaches for live cell imaging as well as luminescent-based strategies for reporting on proteins and nucleic acids following cell lysis.