Development, characterization, and application of next generation fluorescent RNA aptamers

Abstract

As deciphering the functions of various types of RNAs has been instrumental to better understand developmental biology and other disease progressions, tracking and imaging intracellular RNA has been an exciting, yet challenging, study field. The most common in vivo imaging approaches utilize fluorescent protein as a reporter whose maturation process and high background signal characteristic limit the possibility of real-time monitoring with clear visualization. Thus, in the past decade, researchers have begun to explore fluorogenic RNA aptamer system to tackle this challenge. Hitherto, there are at least a dozen variants of fluorescent aptamers with different properties, however their applications have been limited by relatively poor molecular brightness and thermostability in vivo. Therefore, there is an urgent need to evolve next generations of RNA fluorescent aptamers with higher brightness and thermostability. In this thesis, microfluidic-assisted in vitro compartmentalization and fluorescence-activated sorting technologies were applied to improve both the Broccoli and Corn fluorescent aptamers. Random mutagenesis was initially introduced to Broccoli aptamer before five rounds of reselection. With the high temperature acted as selection pressure and rigorous sorting criteria, it was observed that new Broccoli mutant A36U fluoresces approximately 1.5 times brighter than the original Broccoli when measured in vitro at 37 and 45oC. Similarly, the rationally designed Corn library then went through the six rounds of reselection to isolate brighter and thermostable aptamers. After sequencing and preliminary screening, four new unique sequences ranging from 108 - 116 nucleotides appear to fluoresce 6.4 - 9.3 and 3.9 - 20.6 times brighter than the original Corn when measured in vitro at 37 and 45oC, respectively. The short new Broccoli mutant A36U was then applied to RNA biosensor system. Using theophylline as a small molecule target, Broccoli A36U fluoresced 1.5 times brighter than the original Broccoli when the target encountered RNA biosensor system. Other than Broccoli, this scheme was also applied to another next generation of short Mango fluorescent aptamer. Using the similar approach, after the initial optimization of inhibitory sequence length, this Mango-based RNA biosensor was able to gain 1.74 times signal turn-on ratio upon binding to the specific target. Taken together these results, this study lays the foundation of development and potential applications of next generation RNA fluorescent aptamers for a wide range of RNA imaging and biosensing experiments.