10.1117/2.1200912.002508 New fluorescent nucleosides for real-time exploration of nucleic acids Yitzhak Tor and Yun Xie Synthetic chemistry enables creation of emissive nucleoside surrogates for applications in sensor development, discovery assays, and biophys- ical measurements. Along the natural-selection pathways leading to the building blocks of our genetic material, components called nucleobases that can effectively dissipate their excitation energy have proved advantageous because photochemical damage could be de- flected. The fittest surviving nucleobases—adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U)—have excited-state lifetimes on the picoseconds scale, which are associated with flu- orescence quantum yields of 0.03% or less. 1 Such features are beneficial for the photochemical stability and maintenance of our hereditary information, but present a challenge for biophys- ical studies aimed at understanding the dynamics and structure, and recognition events involving nucleic acids. One approach to address this challenge is to develop fluo- rescent analogs that are chemically and physically similar to native A, G, C, T, and U but are endowed with favorable photophysical properties, such as sufficient quantum yields, sen- sitivity to changes in their microenvironment, and red-shifted absorption maxima. Our research group, along with many oth- ers, has shown that fluorescent-nucleoside analogs have great potential for detection of single-nucleotide polymorphisms, 2 DNA lesions, 3, 4 and protein toxins, 5 and can also assist in the discovery of new antibiotics 6 and anti-HIV (human immunode- ficiency virus) agents. 7 We recently developed a method to unambiguously monitor interactions of small-molecule drugs with their RNA target by labeling the components with a fluorescence-resonance energy- transfer (FRET) donor and acceptor. 8 As a proof of concept we examined the interactions of aminoglycosides, a family of po- tent antibiotics, and their target, the A site, which is responsible for the high fidelity in protein synthesis by appraising codon Figure 1. When a fluorescence-resonance energy-transfer (FRET) donor within an A-site model is excited, emission from the incorporated fluorescent nucleoside (green) is observed. When bound to an amino- glycoside (a molecule composed of a sugar group and an amino group) labeled with a FRET acceptor (orange), emission from the acceptor is observed and emission from the donor is reduced (see inset emission spectra). Upon displacement of the labeled aminoglycoside by an un- modified small molecule, the emission of the donor is regained, while the sensitized emission of the acceptor is lost. PL: Photoluminescence (in arbitrary units, a.u.). (a sequence of three nucleotides)-anticodon (corresponding sequence that binds to the codon) matching. 9 When such antibiotics bind to the A site (the decoding site), an RNA confor- mation similar to that induced by the cognate acyl-tRNA-mRNA (transfer-messenger RNA) complex is stabilized that causes Continued on next page