טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentFriedman Aya
SubjectDesign and Characterization of a Fluorescent Protein-based
imaging System for RNA in Live Cells
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Roee Amit
Full Thesis textFull thesis text - English Version


Abstract

Recently, the single-cell and single-molecule approaches have become increasingly popular. These approaches provide large amounts of data at the highest possible resolution in biology. However, they require expertise in advanced experimental techniques in order to obtain the desired data. One area that still requires development of such techniques is the study of RNA.  The fusion of a fluorescent protein to an RNA binding protein is a means of visualizing specific RNA molecules inside living cells. In order for the fusion protein to bind, the target RNA must include a sequence which the binding protein can recognize and attach to.  Gathering large amounts of data requires the use of multiple combinations of fusion proteins, which can recognize different binding sites and thus follow multiple RNA species simultaneously. For this purpose, we created a library of phage-derived RNA binding proteins fused to fluorescent proteins, and set out to characterize it.

The fluorescent function of the fusions was analyzed and compared to cells lacking them. This assay was examined on combinations including four different RNA binding proteins, but only three showed significant fluorescence levels. These three were the coat proteins of MS2, Qβ and PP7. The fluorescent genes used were mCherry, mDendra2 and cerulean.

A key characteristic of these proteins is to function as translational repressors of the phage's replicase gene, via the gene's translation initiation site. By placing a reporter gene after this site, scientists have studied the specificity of binding of these proteins. In this study, we placed these sequences upstream to genes encoding for fluorescent proteins, and observed the fluorescence levels in cells containing different combinations of fusion proteins and binding sites. In this manner, we were able to assess the binding affinities of each fusion protein to each RNA operator in vivo. We discovered that some of the coat proteins are not necessarily orthogonal in their binding site recognition as previously thought. Furthermore, we found out that one of our fusions, MS2-dendra, amplifies rather than represses expression of the reporter, regardless of the structure of the translation initiation site. This finding raises questions regarding the nature of the interaction between MS2-dendra and the reporter's RNA, and concerning the ability of synthetic biology to be a reliable platform for the study of natural processes.