With the discoveries of the last 5-10 years of many different types of RNA molecules, the need to quantitatively image and track live RNA molecules has become crucial. A testament to this rapid pace of discovery is the recent report by the ENCODE collaboration that approximately 1/3 of the human genome is transcribed.
The question is into what?
The RNA world or the transcriptome is considered to be biology’s “dark-matter”. We would like to turn on the light – per say on the transcriptome – and using synthetic biology in parallel to developing complex imaging solutions, we hope to develop probes that will allow us to quantitatively analyze the many processes and interactions that the different RNA molecules take part in.
Some specific projects:
- Exploring RBP-RNA binding and the role of repeats in RNA scaffolds.
In order to fulfill our vision of developing RNA scaffolds for localized complex reactions, we need to first develop a quantitative understanding of RNA binding protein (RBP) – RNA interactions. To that end, we developed an in-vivo high-throughput quantitative binding assay, which will not only allow us to explore how RBP binding is affected by RNA secondary structure, but also to explore the space of specific binding sites. Or in other words, how many orthogonal binding sites with a minimal sequence homology to the consensus sequence can be identified? In addition, we would like to explore when and under what conditions RBP-RNA interactions lead to down-regulation or up-regulation of translation and so on. Together, this quantitative molecular level understanding will allow us to distill crude design rules, which will later be used to design our scaffolds.
- Reduced noise for poised promoters
- In order to apply the concept of synthetic enhancers, and build synthetic enhancer circuits in Eukaryotic organisms, we must first find the analog eukaryotic parts to the ones used in E. coli. While Eukaryotic transcription factors that function as activator, repressors, and drivers are common-place, for the most part whether a particular promoter is poised or not remains unknown.
- We hypothesized that poised promoters may be characterized by a reduced thermal noise profile in transcription. If this turns out to be correct, then it is possible to develop tools that will use this property as a biological marker to characterize whether a given promoter is poised or not.
- We will engineer novel DNA sequences that contain a binding site cassette for the RNA binding PP7 protein. When bound by a fusion of PP7-FP, the RNA will form a localized puncta of light, which can be imaged dynamically and quantified via single cell microscopy methods. We will use different cassette with varying numbers and arrangement of PP7 binding sites to analyze our promoters.
- Developing Genetically Encoded Nanoprobes for Tracking and Detection of RNA interactions
In this project we plan to simultaneously develop special genetically encoded probes that upon interaction with an RNA molecule of some type, structural changes will occur in the probe that will lead to a signal detectible on a specialized imaging set-up. The final goal is to develop a tool that can probe a single RNA-RNA interaction inside a single cell.
- Developing an optical bar-code for live-tracking of multiple promoters in single cell.
Another application is to develop individual scaffolds which will bind varying number of RBPs, each fused to a different fluorescent protein. Thus, each scaffold can be programmed to be an optical bar-code based on the colors, which it will emit and the intensity of each color. Using such an approach, multiple promoters can be tagged and tracked simultaneously in single cells. One of our larger aims is to develop this technology, and track in single cells multiple promoters using simple epi-fluorescent microscopy apparatus.