Scientists at IISER-Pune have synthesized a new fluorescent ribonucleoside analog, which can slip in as a native nucleoside during transcription and report changes in RNA conformation via changes in fluorescence properties.
Results on synthesis and characterization of this uridine analog have been described in a recent issue of Organic Letters. “An ideal fluorescent analog causes minimum perturbation to the system and offers a strong enough signal for measurement. Importantly, for a fluorescent analog to be a useful conformation-sensitive probe, its fluorescence properties should be sensitive to changes in environment,” says Seergazhi Srivatsan, Assistant Professor at IISER-Pune and lead author on this publication.
Along with graduate student Maroti Pawar, Srivatsan began with the aim of finding an alternative to 2-aminopurine, which is widely used in fluorescence-based assays addressing nucleic acid dynamics. 2-aminopurine is limited to in vitro assays as its excitation and emission maxima are in the ultraviolet wavelength range. His team was able to synthesize benzothiophene-conjugated uridine analog (ribonucleoside 1), which emitted fluorescence in the visible wavelength range and could distinguish solvents of different polarity by displaying distinctive fluorescence characteristics. When modified with a triphosphate, ribonucleoside 1 could be incorporated into RNA transcripts through in vitro transcription reactions.
But the real test is to check the extent to which the photophysical properties of the analog may be affected when incorporated into oligonucleotides. “A fluorescent nucleoside analog that otherwise might seem like a promising candidate might disappoint you when incorporated in a target site—in an oligoribonucleotide in this case,” says Srivatsan referring to a bothersome caveat.
Ribonucleoside 1, however, fares exceptionally well in this aspect as well—its fluorescence does not get drastically quenched when placed in oligoribonucleotides, as is the case with many fluorescent nucleoside analogs. What’s even better, ribonucleoside 1 seems to be able to distinguish between a pyrimidine and a purine base pointing to its ability to sense and distinguish neighboring microenvironment. This puts forth a strong case for the use of ribonucleoside 1 as a fluorescent reporter to study nucleic acid dynamics.
This work titled “Synthesis, photophysical characterization and enzymatic incorporation of a microenvironment-sensitive fluorescent uridine analog” has appeared in Organic Letters (13:1114-1117) and is authored by Maroti G. Pawar and Seergazhi Srivatsan. Funding for the study was provided by Department of Science and Technology (DST), India, Council of Scientific and Industrial Research (CSIR), India and IISER-Pune.
–Reported by Shanti Kalipatnapu