N2,N2-Dimethylguanosine

N2,N2-Dimethylguanosine is a methylated modified nucleoside present in RNA and serves as a structural modification component of tRNA. N2,N2-Dimethylguanosine inhibits reverse transcriptase-mediated cDNA synthesis and is one of the key modifications affecting sequencing efficiency in high-throughput RNA sequencing. N2,N2-Dimethylguanosine can be selectively demethylated at one methyl group by AlkB mutant enzymes (such as D135S/L118V) and converted to N2-methylguanosine, thereby reducing the inhibition of reverse transcription^[1][2]. In Vitro:N²,N²-dimethylguanosine (5-95 μM for 12m₂²GA; 2.3-84 μM for 13m₂²GA; 2.9-105 μM for 11m₂²GA) increases hairpin stability by 3.5°C in the 12m₂²GA oligoribonucleotide, slightly destabilizes hairpin stability by 1°C and significantly destabilizes duplex stability in the 13m₂²GA oligoribonucleotide, and shifts the secondary structure equilibrium toward a mixture of hairpin and duplex forms in the 11m₂²GA oligoribonucleotide^[1].
N²,N²-dimethylguanosine (0.5 mM; ~1 week at 291 K) forms imino-hydrogen bonded pairs with A in the 13m₂²GA RNA duplex, inducing local conformational changes including helical bending and altered hydrogen bond geometry, and reducing minor groove hydration, which contributes to reduced duplex thermodynamic stability^[1].
N2,N2-dimethylguanosine (m₂²G) (1 nmol; 2 h) in a synthetic 9mer RNA oligo substrate is fully converted to N²-methylguanosine (m²G) via selective removal of one methyl group by the AlkB^D135S/L118V mutant, with no further demethylation to unmodified guanosine^[2].
N2,N2-dimethylguanosine at position 26 (m₂²G26) in HEK293T cell tRNAs has its modification index reduced via selective demethylation to N²-methylguanosine (m²G) by the AlkB D135S/L118V mutant (as part of a three-demethylase mixture), with the greatest effect observed in mitochondrial tRNA^Ile and cytosolic tRNA^Thr, tRNA^Trp, and tRNA^Tyr[2].