A methylation-switchable conformational probe for the sensitive and selective detection of RNA demethylase activity

Abstract

We describe a novel methylation-sensitive nucleic acid (RNA) probe which switches conformation according to its methylation status. When combined with a differential scanning fluorimetry technique, it enables highly sensitive and selective detection of demethylase activity at a single methylated-base level. The approach is highly versatile and may be adapted to a broad range of RNA demethylases.

Multiprotein Dynamic Combinatorial Chemistry: A Strategy for the Simultaneous Discovery of Subfamily‐Selective Inhibitors for Nucleic Acid Demethylases FTO and ALKBH3

“…We were also able to demonstrate that the thermal tags had little impact on the catalytic activities of AlkB demethylases. Notably, ALKBH5 and FTO preferentially demethylate N 6 ‐methyladenosine (m 6 A) substrates, whereas ALKBH3 favors N 1 ‐methyladenosine (m 1 A) substrates . Consistent with their inherent substrate specificity, detailed kinetic analyses revealed that ALKBH5 and FTO modified with either KE or RD tags displayed similar affinities ( K m ) and catalytic efficiencies ( k cat / K m ) for the m 6 A substrate as that of their unmodified proteins (Figure G and H and Figure S2 in the Supporting Information), whereas ALKBH3 conjugated with either KE or RD tags retained >90 % of its canonical demethylase activity against the m 1 A substrate (Figure I and Figure S2 I in the Supporting Information).…”

“…Notably,A LKBH5 and FTO preferentially demethylate N 6 -methyladenosine (m 6 A) substrates, whereas ALKBH3 favors N 1 -methyladenosine (m 1 A) substrates. [55,56] Consistent with their inher- ent substrate specificity,d etailed kinetic analyses revealed that ALKBH5 and FTO modified with either KE or RD tags displayed similar affinities( K m )a nd catalytic efficiencies (k cat /K m )f or the m 6 As ubstrate as that of their unmodified proteins ( Figure 2G and Ha nd Figure S2 in the Supporting Information),w hereas ALKBH3 conjugated with either KE or RD tags retained > 90 % of its canonical demethylase activity against the m 1 As ubstrate ( Figure 2I and Figure S2 Ii nt he Supporting Information). This finding is interesting because, contrary to our results, ap revious study showed that the attachment of larger zwitterionic peptides, such as poly(Glu-Lys) 10 and 30 KDa in length, led to as ignificant increase in the substrate affinities of b-lactamases.…”

“…[53,55] DNA for full-length human ALKBH3 was cloned into pET28a to generate an N-terminal His 6 -tagged ALKBH3 1-286 construct, as previously described. [56] The above constructs were then subjected to mutagenesis experiments by using the QuikChange mutagenesis kit (Agilent Te chnologies), whereby DNA encoding various KE and RD tag sequences (commercially synthesized by GenScript USA) was fused to the Ct erminus of the respective genes. The sequences of all fusion protein constructs were confirmed by DNA sequencing.…”

Active Site Breathing of Human Alkbh5 Revealed by Solution NMR and Accelerated Molecular Dynamics

“…Alkbh5 belongs to the nonheme Fe 2þ -and a-ketoglutarate (aKG)-dependent AlkB dioxygenases and catalyzes oxidative demethylation of single-stranded RNAs (4). Although several different methyl modifications have been described and cataloged in nucleic acids (5), Alkbh5 specifically demethylates N 6 -methyladenosine (m 6 A), with little to no activity for other methylated nucleotides (6,7). m 6 A is the most abundant internal modification of eukaryotic mRNA and long-noncoding RNA (8).…”

Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses

“…We validated the ability of probe 13 to assay ALKBH2 inhibitors by measuring the IC 50 of pyridine 2,4-dicarboxylic acid (PDCA), a broad-spectrum inhibitor of 2-OG-dependent enzymes [16] (Figure 3). The assay was conducted in a 60 µL reaction volume on a 384-well plate, yielding an IC 50 of 2.43 ± 0.12 µ m , fully consistent with reported values.…”