PHD Student

KEY WORDS: NMR spectroscopy, nucleic acids, paramagnetic effects

Research topic: “”

Principal Investigator: dr Witold Andrałojć

I. Project description

Biomolecular NMR spectroscopy is one of the most accomplished and widely used methods in high resolution structural biology and its methodological arsenal is still being actively expanded. One branch of methodologies finding increasing application in the biomolecular NMR community is a family of techniques known under the common name of “paramagnetic biomolecular NMR”. The great appeal of paramagnetic effects in NMR spectroscopy stems from their inherent long-range nature (> 30 Å), which allows to translate them into extremely long-range structural restraints. Over the last two decades these “paramagnetic restraints” have been applied to great effect in the field of protein NMR. In a stark contrast, the paramagnetic effects – with the exception of Paramagnetic Relaxation Enhancements (PRE) – have, thus far, found almost no application in the field of NMR studies of nucleic acids. This situation is caused by the lack of reliable techniques for introducing paramagnetic centers into this type of biomolecules. The objective of the current proposal is the development and testing of lanthanide binding oligonucleotides (LBOs) – a direct counterpart of lanthanide binding peptides used to great effect in NMR spectroscopy of proteins – as a general method of paramagnetic tagging of nucleic acid systems.

The development of LBOs will be based on three families of tightly lanthanide-binding DNA molecules previously reported in the literature. The original systems are several times too large to be practically useful as paramagnetic tags and concurrently none of them have been structurally characterized. The main objective of the current project is to thoroughly structurally characterize the three known families of lanthanide binding DNAs (preferentially by solving their 3D structures in solution), identify the exact position and geometry of each lanthanide binding site and then use this knowledge to extract what may be called the “minimal lanthanide binding DNA sequences” for each family. As the final step of the project these sequences will then be “implanted” at carefully selected positions into various “host” DNA and RNA molecules to test their capability to induce paramagnetic effects in these hosts and thus serve to as LBOs for paramagnetic NMR.

If successful, the proposed research will provide a new, much desired tool for NMR spectroscopists, hopefully meaningfully expanding capacities of nucleic acid NMR towards dealing with more complex systems and addressing more sophisticated structural and dynamic inquiries.

The main techniques to be used in the scope of the project are biomolecular NMR spectroscopy (including paramagnetic effects), molecular dynamics protocols for structure calculations and chemical synthesis of nucleic acids on solid support as the source of samples. Other biophysical techniques (like UV an CD spectroscopies and gel electrophoresis) will also be used as supporting experiments.

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