Environment-responsive fluorescent nucleoside analogue probe for studying oligonucleotide dynamics in a model cell-like compartment.

The majority of fluorescent nucleoside analogue probes that have been used in the in vitro study of nucleic acids are not suitable for cell-based biophysical assays because they exhibit excitation maxima in the UV region and low quantum yields within oligonucleotides. Therefore, we propose that the photophysical characterization of oligonucleotides labeled with a fluorescent nucleoside analogue in reverse micelles (RM), which are good biological membrane models and UV-transparent, could provide an alternative approach to studying the properties of nucleic acids in a cell-like confined environment. In this context, we describe the photophysical properties of an environment-sensitive fluorescent uridine analogue (1), based on the 5-(benzo[b]thiophen-2-yl)pyrimidine core, in micelles and RM. The emissive nucleoside, which is polarity- and viscosity-sensitive, reports the environment of the surfactant assemblies via changes in its fluorescence properties. The nucleoside analogue, incorporated into an RNA oligonucleotide and hybridized to its complementary DNA and RNA oligonucleotides, exhibits a significantly higher fluorescence intensity, lifetime, and anisotropy in RM than in aqueous buffer, which is consistent with the environment of RM. Collectively, our results demonstrate that nucleoside 1 could be utilized as a fluorescent label to study the function of nucleic acids in a model cellular milieu.

Heavy atom containing fluorescent ribonucleoside analog probe for the fluorescence detection of RNA-ligand binding.

Although numerous biophysical tools have provided effective systems to study nucleic acids, our current knowledge on how RNA structure complements its function is limited. Therefore, development of robust tools to study the structure­function relationship of RNA is highly desired. Toward this endeavor, we have developed a new ribonucleoside analog, based on a (selenophen-2-yl)pyrimidine core, which could serve as a fluorescence probe to study the function of RNA in real time and as an anomalous scattering label (selenium atom) for the phase determination in X-ray crystallography. The fluorescent selenophene-modified uridine analog is minimally perturbing and exhibits probe-like properties such as sensitivity to microenvironment and conformation changes. Utilizing these properties and amicability of the corresponding ribonucleotide analog to enzymatic incorporation, we have synthesized a fluorescent bacterial ribosomal decoding site (A-site) RNA construct and have developed a fluorescence binding assay to effectively monitor the binding of aminoglycoside antibiotics to the A-site. Our results demonstrate that this simple approach of building a dual probe could provide new avenues to study the structure­function relationship of not only nucleic acids, but also other biomacromolecules.

Synthesis, photophysical characterization, and enzymatic incorporation of a microenvironment-sensitive fluorescent uridine analog.

The synthesis of a microenvironment-sensitive base-modified fluorescent ribonucleoside analog based on a 5-(benzo[b]thiophen-2-yl)pyrimidine core, enzymatic incorporation of its corresponding triphosphate into RNA oligonucleotides, and photophysical characterization of fluorescently modified oligoribonucleotides are described.