Probing Aldehyde Modification Propensity on tRNA

A single-step method was used for probing relative modification propensity of the aldehyde with aa-tRNA where 0.2 µM of Ala-tRNA^Ala was incubated with different concentrations of aldehydes (2 mM and 10 mM) along with 20 mM NaCNBH₃ (in 100 mM potassium acetate [pH 5.4]) as a reducing agent at 37°C for 30 min. The reaction mixture was digested with S1 nuclease and analysed on TLC. Except for decanal, all the aldehydes modified Ala-tRNA^Ala. The method for processing and quantification of modification on aa-tRNA utilised is discussed earlier (Mazeed et al., 2021 (link)). However, a two-step method was used for generating substrates for biochemical assays as discussed earlier (Mazeed et al., 2021 (link)). It was used to generate maximum homogenous modification on the aa-tRNAs for deacylation assays. Briefly, 2 µM aa-tRNAs were incubated with 20 mM of formaldehyde, and methylglyoxal or 1 M of propionaldehyde, butyraldehyde, valeraldehyde, and isolvaleraldehyde at 37°C for 30 min. Samples were dried to evaporate excess aldehydes using Eppendorf 5305 Vacufuge plus Concentrator. The dried mixture was then reduced with 20 mM NaCNBH₃ at 37°C for 30 min. All reactions were ethanol-precipitated at –30°C overnight or –80°C for 2 hr. Ethanol precipitated pellets were resuspended in 5 mM sodium acetate (pH 5.4) and used for biochemical assays.