UC San Diego

Fluorescent nucleobase analogues (FBAs) are essential tools to study nucleic acid structures and interactions. Despite the great advancement in fluorescent nucleobase analogues design, they are not as bright as conventional fluorophores. We designed a new tricyclic pyrimidine analogue by resembling the structural features of Rhodamine B, while maintaining the Watson-Crick base pair motif. This C-linked 8-(diethylamino)benzo[b][1,8]naphthyridine-2(1H)-one, called ABN, exhibits ε442 = 20,000 M−1 cm−1 and Φem,540 = 0.39 in buffer, increasing to Φem = 0.50–0.53 when base paired with adenine in duplex DNA. Single-molecule fluorescence measurements of ABN using both one-photon and two-photon excitation demonstrate its excellent photostability and indicate that the nucleoside is present to > 95% in a bright state with count rates of at least 15 kHz/molecule. This new fluorescent nucleobase analogue, which, in duplex DNA, is the brightest and most red-shifted known, is the first to offer robust single-molecule fluorescence detection capabilities.While ABN is exceptionally bright, DEAtC shows a fluorescence turn-on effect when correctly base paired against guanosine in dsDNA. The eight-step synthesis of DEAtC deoxyribonucleoside phosphoramidite is optimized and improved into a reliable protocol. To synthesize the ribonucleoside phosphoramidite, an additional protection of the 2′-hydroxyl group with a (triisopropylsiloxy)methyl group is performed to prevent it from interfering with solid-phase RNA synthesis. In addition to using nucleoside analogues as study tools, they are widely utilized in medicinal chemistry as therapeutic agents. GCYH-I is the first enzyme in the folate biosynthesis pathway in bacteria. Some pathogenic bacteria, such as N. gonorrhoeae, use a special type of this enzyme, which is called GCYH-IB. This enzyme has the same catalytic activity as GCYH-IA, found in human cell, however they have limited structural similarities. By examining the crystal structures of both enzymes bound to 8-oxo-GTP, a potent inhibitor for both enzymes, we identified the main 3D structural differences between GCYH-IA and -IB. A small set of 8-oxoguanosine derivatives were designed according to these differences, to selectively inhibit the bacterial enzyme. Of this set of inhibitors, G3 is 3-fold more selective to GCYH-IB, which represents a 31-fold reversal of the selectivity of 8-oxo-GTP, the starting point of the study.