Development of an RNA array to Protein array (RAPA) platform and its application to gene expression analysis of synthetic riboregulators
Student thesis: Doctoral Thesis
Microarray technology has revolutionised the high throughput molecular biology research.DNA oligonucleotide probe arrays have been extensively used for transcriptome analysis, functional RNA arrays have provided insight into RNA’s biosensing capabilities, and protein arrays have routinely enabled high throughput protein interaction studies. Nonetheless, there are still potential areas where the microarray technology can be employed to increase the throughput for new research possibilities. One area is the study of post-transcriptional gene regulation and riboregulatory devices which act post-transcriptionally.So far, no array-based methods have been described for in vitro post-transcriptional gene regulation analysis or riboswitch performance screening. The work presented here bridges this gap in the field by developing a microarray technology whichis amenable to post-transcriptional gene regulation studies as well as riboregulatoryperformance screening applications. Building on a recently published RNA array fabrication platform (from the Callaghan group), this thesis shows: (I) an optimised methodfor making a DNA array, (II) an optimised method for transcribing the DNA array into anRNA array, (III) an efficient novel strategy for translating the RNA array into a protein array,(IV) proof of concept for the new platform’s application in high throughput riboregulation research. In summary, the presented results describe a highly efficient protocol, in terms of time and reagent usage, for the fabrication of protein arrays from DNA array-programmed RNA arrays. In addition, a group of synthetic post-transcriptional riboregulatory devices were successfully tested on the new platform, demonstrating its utility in riboregulator development and application studies.
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