Redox Chemistry of B12-binding RNA Aptamers


Jason Willis

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Vitamin B12 has many biologically important roles in living systems today. It has been found to serve as a cofactor for methyl transferases, and as a reduction/oxidation cofactor for class II ribonucleotide reductases (RNRs). All organisms use RNRs to generate deoxyribonucleic acids from the ribose counterparts. Within the context of the 'RNA world' hypothesis, it is possible that ancient RNA sequences existed which may have catalyzed the same reaction using B12 as a reduction/oxidation (redox) cofactor. Our hope is to study the affect that recently isolated B12 RNA aptamers have on the reduction potential of vitamin B12 (cyanocobalomin). We have tethered 49nt RNA aptamers to a gold electrode surface through hybridization to a thiolated ssDNA monolayer; however, due to the nature of the buffer system the results are inconclusive. A slightly modified approach has been chosen which will use a dsDNA monolayer with a 5? overhang (sticky end). We have developed a random ssDNA library for use in the selection of new B12 aptamers that bind to B12 in a buffer system convenient for electrochemical analysis, and which do so while hybridized to the dsDNA monolayer. The synthesized library has been PCR amplified and preliminary analysis reveals that only 18% (1.1 x 1015 unique molecules) of the total synthetic yield is available for PCR amplification. Thus, our pool represents only a small fraction of the total number of random sequences possible. Further, through the cloning and sequencing of representatives from the pool, we find that a slight bias exists in the 72nt random sequence towards C and against G. Approximately 11.3mg of RNA was transcribed from this dsDNA library and we are using a portion of this final pool for selection on a B12-agarose column.


W. R. Hardy & M. G. Hill




Howard Hughes Medical Institute Fellowship

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