UC San Diego

Nucleosides are the essential building blocks of DNA and RNA. Being that the chromophoric region of the canonical nucleosides is nonemissive, their biophysical examination is difficult. To circumvent this, illumination of these molecules has been accomplished using fluorescent nucleosides. One challenge in the design of emissive nucleoside analogs is maintaining isomorphicity while retaining isofunctionality. The Tor lab has designed and implemented the use of a series of RNA alphabets that resemble the native nucleosides but have useful photophysical features. These alphabets are based on thieno[3,4-d]pyrimidine (thN), isothiazole[4,3-d]pyrimidine (tzN), and methylthieno[3,4-d]pyrimidine (mthN) cores. One area that remained unexplored using the nucleoside analog alphabets developed by the Tor lab, was that of use of the nucleobase cores without their sugar moiety. These would naturally not contain the large contact forming sugar moiety, relying on their structural analogy to their native counterparts for biochemical recognition. Herein, the use of guanine based emissive analogs based on a thN and tzN cores are used for the evaluation of cellular modifications.

Guanine deaminase converts guanine to xanthine. The study of human guanine deaminase remains limited compared to other metabolic deaminases and as a result its substrate and inhibitor repertoire are limited. Additionally, recent observations have shown it is implicated in neuronal morphology, traumatic brain injury, memory dysfunction, and psychiatric diseases. In this work, we explore two emissive heterocyclic cores, based on thN and tzN, as surrogate guanine deaminase substrates. We demonstrate that the isothiazolo guanine surrogate, tzGN, does undergo effective enzymatic deamination by guanine deaminase to the isothiazolo xanthine analog, tzXN, while the thiophene guanine analog, thGN, does not. Further, we showcase the potential of this fluorescent nucleobase surrogate to provide a visible spectral window for a real-time study of guanine deaminase and its inhibition.

O6-Alklylguanine-DNA-transferase is a repair protein that provides protection from mutagenic events caused by O6-alkylguanine lesions. The activity of human O6-alklylguanine-DNA-transferase is tissue specific, indicative of tumor status, and correlated to chemotherapeutic success. For this reason, tracking its activity could prove informative for disease diagnosis and therapy. We explore two families of emissive O6-methyl- and O6-benzylguanine analogs based on thGN and tzGN as potential surrogates. We establish that the O6-benzyl thGN and tzGN derivatives provide a spectral window to optically monitor O6-alklylguanine-DNA-transferase activity, can be used as substrates for the widely used SNAP-Tag delivery system, and are sufficiently bright for visualization in cells using fluorescence microscopy.