A Pyrimido‐Quinoxaline Fused Heterocycle Lights Up Transfer RNA upon Binding at the Mg2+ Binding Site

Transfer RNAs (tRNAs) are fundamental molecules in cellular translation. In this study we have highlighted a fluorescence‐based perceptive approach for tRNAs by using a quinoxaline small molecule. We have synthesised a water‐soluble fluorescent pyrimido‐quinoxaline‐fused heterocycle containing a mandatory piperazine tail (DS1) with a large Stokes shift (∼160 nm). The interaction between DS1 and tRNA results in significant fluorescence enhancement of the molecule with Kd∼5 μM and multiple binding sites. Our work reveals that the DS1 binding site overlaps with the specific Mg2+ ion binding site in the D loop of tRNA. As a proof‐of‐concept, the molecule inhibited Pb2+‐induced cleavage of yeast tRNAPhe in the D loop. In competitive binding assays, the fluorescence of DS1‐tRNA complex is quenched by a known tRNA‐binder, tobramycin. This indicates the displacement of DS1 and, indeed, a substantiation of specific binding at the site of tertiary interaction in the central region of tRNA. The ability of compound DS1 to bind tRNA with a higher affinity compared to DNA and single‐stranded RNA offers a promising approach to developing tRNA‐based biomarker diagnostics in the future.