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

Abstract

The abundant Watson-Crick face methylations in biological RNAs such as N¹ -methyladenosine (m¹ A), N¹ -methylguanosine (m¹ G), N³ -methylcytosine (m³ C), and N² ,N² -dimethylguanosine (m²₂ 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 m¹ A, m¹ G, m³ C modifications in transfer RNA (tRNA), but they work poorly on m²₂ G. Here we report the design and evaluation of a series of AlkB mutants against m²₂ 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 m²₂ G modification to N² -methylguanosine (m² G). We also show that this new enzyme improves the efficiency of tRNA sequencing.

Keywords: AlkB mutant; demethylase; methylguanosine; reverse transcriptase reaction; tRNA sequencing.

Figure 1. AlkB mutant design for m²₂G demethylation

(A) Chemical structure of m²₂G. (B) Location of m²₂G modification in tRNA tertiary structure (PDB 4tna). The m²₂G residue is in orange, and the two methyl groups are in blue. (C) View of AlkB active site stereochemistry model with m²₂G coordination based on Protein data bank (PDB) ID 3BIE. m²₂G is labeled in cyan.

Figure 2

Evaluations of a series of AlkB mutants for efficient m²₂G 5′-GAGCXUUAG, X= m²₂G (ON- m²₂G), m²G (ON-m²G) or G (ON-G). (A) HPLC traces of the digestion products after ON-m²₂G was treated with demethylases showed that AlkB D135S/L118V mutant quantitatively converts m²₂G to m²G. (B) Comparison of MALDI-TOF MS of unmodified RNA oligo ON-G, m²G-containing RNA oligo ON-m²G, m₂²G-containing RNA oligo ON-m²₂G, and ON-m²₂G which was treated with AlkB D135S/L118V mutant confirmed that the demethylation product of ON-m²₂G is ON-m²G instead of ON-G.

Figure 3

Application of AlkB D135S/L118V to high throughput tRNA-seq. (A) tRNA-seq heatmap result showing the modification index (MI) change at position 26 in tRNAs with known m²₂G sites. 2DM = wild-type AlkB+D135S; 3DM = wild-type AlkB + D135S + D135S/L118V. The higher the MI value, the higher the fraction of modifications. Therefore, decreased modification fraction corresponds to lower MI value or more intense green color for the corresponding tRNA isoacceptor. (B) Full MI plot for the full-length mitochondrial tRNA^Ile. MI at the m²₂G nucleotide is 0.972, 0.343, 0.178 for untreated (black), +2DM (red), and +3DM (blue), respectively.

Scheme 1

Improved syntheses of m²G and m²₂G phosphoramidites and their incorporation into RNA to prepare demethylase substrates. i). (a) (t-Bu)₂Si(OTf)₂, DMF; (b) imidazole, TBDMS-Cl, 90 °C, 75%. ii). p-nitrophenylethanol, Ph₃P, DIAD, 1,4-dioxane 100 °C, 92%. iii). (HCHO)_x, NaBH₃CN, AcOH, 40 °C. iv) HF·Py/THF. v). DMTr-Cl/Py. vi). (i-Pr)₂NP(Cl)OCH₂CH₂CN, CH₂Cl₂, (i-Pr)₂NEt, 1-methyl-imidazole. vii). RNA synthesis and deprotection.