Figure 3.

Identifying a recognition residue for m⁶A. (A) The sheared G–A base pair found in many RNA structures (left). Within this purine–purine base pair N⁶H(A) and 2′OH(G) reside in close proximity to each other. The methyl group of m⁶A (right) might sterically clash with the phosphate backbone of G. (B) Synthetic scheme for N⁶-methyl-rA phosphoramidite. (C) Efficiency of A and m⁶A-directed ligation using different recognition residues. The 30-mer RNA templates contained at position 15 either unmodified adenosine (A) or N⁶-methyladenosine (m⁶A). Substrates contained one of the following recognition residues: dG (2′-deoxyguanosine), Gm (2′-methoxyguanosine), dT (thymidine) or Um (2′-methoxyuridine). (D) Ligation yield correlates linearly with the fraction of adenosine methylation f(m⁶A). Ligation reactions contained adenosine and N⁶-methyladenosine 30-mer RNA templates mixed together in defined ratios as indicated. Linear fit has a r-value of 0.964 and P-value of 0.002.