Selective Enzymatic Demethylation of N2,N2-Dimethylguanosine in RNA and Its Application in High-Throughput tRNA Sequencing

The abundant Watson-Crick face methylations in biological RNAs such as N-1-methyladenosine (m(1)A), N-1-methylguanosine (m(1)G), N-3-methylcytosine (m(3)C), and N-2,N-2-dimethylguanosine (m(2)(2)G) cause significant obstacles for high-throughput RNA sequencing by impairing cDNA synthesis. One strategy to overcome this obstacle is to remove the methyl group on these modified bases prior to cDNA synthesis using enzymes. The wild-type E. coli AlkB and its D135S mutant can remove most of m(1)A, m(1)G, m(3)C modifications in transfer RNA (tRNA), but they work poorly on m(2)(2)G. Here we report the design and evaluation of a series of AlkB mutants against m(2)(2)G-containing model RNA substrates that we synthesize using an improved synthetic method. We show that the AlkB D135S/L118V mutant efficiently and selectively converts m(2)(2)G modification to N-2-methylguanosine (m(2)G). We also show that this new enzyme improves the efficiency of tRNA sequencing.