Research Projects

In the past three decades, RNA has emerged as a major player in cellular biology14. However, tools that can be used to image and track RNAs in live cells are not abundant. To overcome this challenge, fluorogenic RNA aptamers (or “RNA mimics of GFP”) were developed as an RNA imaging tool4,15. These RNA aptamers activate the fluorescence of small molecule mimics of the GFP chromophores or other dyes as well (Fig. 1a). Since the discovery of the first fluorogenic RNA aptamer Spinach by the Jaffrey lab, several fluorogenic RNA aptamers have been developed by several other groups. Some of those fluorogenic RNA aptamers such as RhoBAST and Okra have high photostability as well. However, it is not well understood why RhoBAST and Okra have high photostability while other aptamers such as Broccoli or Pepper do not. On the other hand, the fluorogenic RNA aptamer Squash can bind to several GFP chromophore mimics spanning from blue to far red emssion7,9. We have observed that the photostability of the Squash-dye complex increases as the fluorescence emission shifts from blue to red (Fig. 2c & 2d). Similar trends have also been observed for the Pepper aptamer as well16. However, the reason behind this trend in photostability is not well understood. A general mechanism of photobleaching is proposed for the GFP chromophore-based fluorogenic RNA aptamers where the fluorogenic dye (GFP chromophore mimic) undergoes a cis-to-trans isomerization upon absorbing a photon even when bound to the RNA aptamer bringing it to the “Off” state (Fig. 1c)5. Then, the photoconverted trans dye is ejected from the binding pocket of the RNA aptamer which allows a fresh dye from the solution to bind the RNA aptamer bringing it back to the “On” state (Fig. 1c). Faster the rate of the cis-to-trans isomerization, higher will be the photostability of the RNA-dye pair. However, any of photophysical processes that happens between the dye going from “On” to “Off” state is not characterized. In this project, we would like to bridge this gap by studying the photophysics of the RNA-fluorogenic dye interaction using ultrafast fluorescence spectroscopy. The rate of going from the cis or “On” to trans or “Off” state can be dependent on two factors: the intrinsic ability of the dye to prevent cis-to-trans isomerization and the ability of the RNA aptamer to prevent the cis-to-trans isomerization. To understand this, we will use a series of fluorogenic dyes with increasing conjugations and study their photophysics in the RNA aptamer bound state. This will help us to characterize the different photophysical processes and their rates that occurs when the dye goes from “On” to “Off” state in the RNA bound form. From this knowledge, we will be able to design highly photostable fluorogenic dyes which will allow us to image and track RNAs both in live cells and in live animals. We will also develop a suite of new RNA aptamers that can bind and activate these new fluorogenic dyes using SELEX. Then, we will systematically study the photophysics of the RNA-dye complex using a ultrafast fluorescence techniques which will help us understand why some fluorogenic RNA aptamers are photostable and others are not. Finally, we will use the brightest and most photostable fluorogenic RNA aptamers for imaging and tracking of single mRNAs in live cells. Additionally, the new fluorogenic dyes with extended π-conjugation that will be synthesized in this project are expected to show high extinction coefficient and near-IR emission. Dyes with high extinction coefficient will give us bright fluorogenic RNA aptamers. Fluorescent probes with excitation and emission in the far-red region of the spectrum are beneficial for in vivo applications due to low phototoxicity and higher penetration depth of far-red light. Therefore, the new fluorogenic RNA aptamers will be highly useful for imaging and tracking of RNA in live animals.