Enabling precision distance measurements in long RNAs

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Technologies that explore the shape and motion of complex biomolecules such as ribonucleic acids (RNA) are fundamentally important for understanding the role of such biomolecules in human biology, or disease. The development of new analytical methods is of high importance, as this increases the range of tools available to the biologist for the precise investigation of biomolecule structure and behaviour.This project seeks to develop a new technology for the characterisation of RNA - the use of biosynthesis to introduce unnatural functional groups into RNA that can be reacted with probes that contain 'stable free radicals', which are termed 'spin labels'. This work will offer a new and totally unexplored method for the analysis of 'long' RNA (that is, RNA which is longer than can reliably be prepared through chemical synthesis).The key biosynthesis methodology is called 'Stepwise transcription', which uses the ability of certain RNA polymerase enzymes to accept unnatural nucleoside triphosphates (NTPs, the building blocks used by Nature to construct RNA) as an RNA chain is biosynthesised. These unnatural nucleosides contain a chemical functionality which is tolerated by the polymerase, but which enables post-biosynthesis 'labelling' of the RNA. In the case of our spin labels, this enables the use of electron paramagnetic resonance spectroscopy (EPR) to analyse the biomolecule. This technique has been applied with success for the analysis of small RNA and DNA motifs which can be prepared through chemical synthesis, but only very rarely to spin labelled long RNAs (>50 nucleotides) due to the current difficulties with their preparation. The EPR technique itself is highly informative, not only measuring distances, but also motion and orientation. The project itself will involve the synthesis of NTPs that contain appropriate functionality to install the spin label post-biosynthesis. While many modified NTPs already exist (which we will use in this project as a starting point), to obtain the best information in the EPR experiments, we will need to design new, more rigidified labelling handles in the NTP. This is an exciting challenge, and one that has consequences beyond EPR (other probes could be installed on these same handles). We will also synthesize a range of spin labels to attach to the biosynthesized RNA. To test the methodology, we have identified a 71 nucleotide RNA motif that is known to undergo structural change under defined conditions. We will label this motif, and explore by EPR spectroscopy the change in its shape, thus establishing this new technology as a viable and easily applied method for RNA spin labelling.