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Biomedical Optics & Medical Imaging

New fluorescent nucleosides for real-time exploration of nucleic acids

Synthetic chemistry enables creation of emissive nucleoside surrogates for applications in sensor development, discovery assays, and biophysical measurements.
18 December 2009, SPIE Newsroom. DOI: 10.1117/2.1200912.002508

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 deflected. 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 fluorescence 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 biophysical studies aimed at understanding the dynamics and structure, and recognition events involving nucleic acids.

One approach to address this challenge is to develop fluorescent 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, sensitivity to changes in their microenvironment, and red-shifted absorption maxima. Our research group, along with many others, 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 antibiotics6 and anti-HIV (human immunodeficiency 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 potent antibiotics, and their target, the A site, which is responsible for the high fidelity in protein synthesis by appraising codon (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 conformation similar to that induced by the cognate acyl-tRNA-mRNA (transfer-messenger RNA) complex is stabilized that causes the ribosome to lose its ability to correctly distinguish among anticodon-codon hybrids, resulting in faulty protein synthesis.10

Thus, the A site is a key target for the discovery and development of new antibiotics. Previously, a single fluorophore was incorporated into A-site constructs to report on the binding of aminoglycosides.6 While useful, singly labeled RNA might not respond well to all varieties of microenvironmental differences resulting from antibiotic binding. A system involving a pair of FRET-donor and acceptor fluorophores that would faithfully report on binding and displacement events, regardless of the binding mode, represents a far more robust probing strategy (see Figure 1).

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 aminoglycoside (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 unmodified 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.).

We replaced a native nucleobase in the A site located near the binding site, but not part of it, with an emissive isomorphic nucleobase analog to act as a FRET donor—see Figure 2(a)—and labeled the aminoglycosides with an appropriate FRET acceptor in positions that are not essential for RNA binding: see Figure 2(b). As FRET is distance dependent, the donor and acceptor would be brought together when the antibiotic is bound to the A site, leading to increased emission of the acceptor and quenching of the donor. To test our system, we recorded the emission changes in the acceptor and donor fluorophores while titrating in labeled neomycin and tobramycin antibiotics, and we successfully obtained EC50 (half maximal effective concentration) values that were in agreement with previously reported trends.

Figure 2. (a) Fluorescent-nucleoside analog (dark green), based on a uridine nucleoside core (shown in bold), acting as the FRET donor. (b) Tobramycin antibiotic labeled with the FRET acceptor, 7-diethylaminocoumarin (red).

In addition to monitoring binding, the FRET pair can also report the displacement of the labeled aminoglycoside by a range of competitors. When a potential drug competes off the originally bound aminoglycoside, the donor and acceptor are parted, which decreases the sensitized emission of the acceptor and simultaneously increases the donor's fluorescence. We validated this behavior with a variety of other A-site binding antibiotics, as well as semi-synthetic aminoglycosides never before shown to bind to the A site.8,11

A FRET-based system, therefore, provides information on both association and competitive dissociation, independently of specific binding modes. Most importantly, while a singly labeled RNA construct could generate false-positive signals caused by remote binding in a nonfunctional state that could alter a probe's environment, our FRET-based technique requires specific binding to the A-site pocket. Nonspecific RNA binders would fail to generate an intense FRET signal in monitoring direct-binding experiments and cannot displace an antibiotic from its cognate recognition site.

In addition to being a useful tool for drug discovery, we recently demonstrated that fluorescent nucleosides can detect the enzymatic activity of ribosomal-inactivating proteins (RIPs), a family of plant toxins.5 RIPs interfere with protein biosynthesis by catalyzing the depurination (alteration) of a specific nucleotide in a ribosomal-RNA sequence called the α-sacrin/ricin loop: see Figure 3(a).12,13 Depurination leads to a loss of a nucleobase from a nucleotide, thus creating an abasic site, which rarely appears otherwise in RNA. Earlier methods for detecting RIPs, like antibodies employed in enzyme-linked immunosorbent assays, are complicated and difficult to use in practice.

Figure 3. (a) Ribosome-inactivating proteins (RIPs) depurinate an α-sarcin/ricin hairpin RNA substrate at position A15 (shown in blue, corresponding to A4324 of rat 28S ribosomal RNA) to yield an RNA product that contains an abasic site (in red). (b) Fluorescent nucleoside, Y (pink), used to detect depurination by RIPs. (c) Sequences of the emissive synthetic-oligonucleotide probes that contain the fluorescent nucleoside Y.

Our strategy involved synthesizing a fluorescent nucleoside that is highly emissive only when found opposite an abasic site in RNA: see Figure 3(b). We created an oligonucleotide probe by including the modified nucleoside into a strand of RNA that complements the α-sacrin/ricin loop. When the probe is in an RNA-RNA duplex with an abasic site, it displays the most intense emission, signaling the presence of RIPs, while otherwise the emission is relatively quenched. The probe is therefore a simple sensor of depurinations in the α-sacrin/ricin loop, making it a potentially useful tool for detection of RIPs.

These two recent advances provide a glimpse into the vast range of possibilities and opportunities in this evolving field and highlight its challenges. The difficulty in enhancing the emissive properties of nucleic acids with fluorescent nucleobase analogs lies in the strict size, shape, and pairing requirements that are imposed on the surrogates. Deviations from these criteria, while possibly synthetically easier to construct, are likely to perturb the native system. Nevertheless, it is a challenge that can be met (which represents our next steps in this field), especially as nature provides an abundant number of highly fluorescent small molecules, like coumarin, for inspiration.

This work has been supported by the National Institutes of Health (GM 069773).

Yitzhak Tor, Yun Xie
Department of Chemistry and Biochemistry
University of California at San Diego
La Jolla, CA

Yitzhak Tor is a professor of chemistry and Traylor Scholar in organic chemistry. His main research interests include nucleic acids/ligand interactions, new emissive nucleosides and oligonucleotides, and novel cellular-delivery agents. He is editor in chief of Perspectives in Medicinal Chemistry.