Rapid determination of RNA accessible sites

摘要

RNA secondary structure formation occurs in a simple fashion, yet is one of the more complicated biological processes to understand. The RNA structure gives rise to a diverse set of functions, including hydrolysis of other RNAs and translation of RNA into proteins. In order to improve the models that are currently used to determine RNA secondary structure, fast and informative experimental methods to evaluate structure are needed. In addition, knowing how RNA structures form and how they may be manipulated is important in targeting genes implicated in diseases. Two different methods were developed to evaluate RNA structure. The first uses reverse transcription of a random oligonucleotide library of short probes to determine locations of hybridization, called accessible sites, by analysing with mass spectrometry. While it worked, it was neither fast nor very informative. The second approach uses microarray technology in conjunction with surface plasmon resonance (SPR) detection, recently developed amorphous carbon thin film chemistry, and a custom Maskless Array Synthesizer (MAS) for the light-directed fabrication of DNA arrays. Several DNA arrays were designed containing tiling features complementary to short (∼75mer) RNA sequences known to have structure. Binding to specific sites is seen within 1 minute of the exposure to RNA solution. The hybridization regions are then used to map the accessible sites onto predicted structures. This method was extended to higher density arrays to make universal arrays that could be used with any RNA molecule. Two universal arrays were produced, a 5mer, 1050 feature array, and a 6mer, 4122 feature array. Both arrays provided hybridization information similar to that obtained from the custom tiling arrays. The universal arrays are especially useful due to their capability to be used with any RNA sequence.