Despite its relatively simple primary sequence, ribonucleic acids (RNA) fold into complex three-dimensional structures that, often together with tightly bound protein partners, play important roles within the cell. It is therefore of major importance to decipher the three dimensional structures of these fascinating molecules at an atomic scale. NMR has often proven the method of choice in structure determination of nucleic acids. 42.4% of all nucleic acid structures deposited in the PDB database by July 2009 were solved by NMR. This chapter will focus on in vitro transcription and purification of RNA samples for investigation by NMR. Crucial parameters for the success of a transcription reaction will be discussed and strategies to avoid and detect RNase contamination will be outlined. The problem of distinguishing between a hairpin and a duplex RNA will be considered briefly, as this is a recurring issue when pursuing structural studies with RNA. Often it is desirable to obtain RNAs with 100% homogeneous ends. This can be achieved by self-cleaving ribozymes at each end of the transcript, a strategy that will be discussed herein. This chapter aims to provide a toolkit for successful sample preparation in your own laboratory and provide you the opportunity to immerse yourself into the RNA world.
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