Fluorescent Pyrimidine Ribonucleotide:  Synthesis, Enzymatic Incorporation, and Utilization

Abstract

Fluorescent nucleobase analogs that respond to changes in their microenvironment are valuable for studying RNA structure, dynamics and recognition. The most commonly used fluorescent ribonucleoside is 2-aminopurine, a highly responsive purine analog. Responsive isosteric fluorescent pyrimidine analogs are, however, rare. Appending 5-membered aromatic heterocycles at the 5-position on a pyrimidine core has recently been found to provide a family of responsive fluorescent nucleoside analogs with emission in the visible range. To explore the potential utility of this chromophore for studying RNA-ligand interactions, an efficient incorporation method is necessary. Here we describe the synthesis of the furan-containing ribonucleoside and its triphosphate, as well as their basic photophysical characteristics. We demonstrate that T7 RNA polymerase accepts this fluorescent ribonucleoside triphosphate as a substrate in in vitro transcription reactions and very efficiently incorporates it into RNA oligonucleotides, generating fluorescent constructs. Furthermore, we utilize this triphosphate for the enzymatic preparation of a fluorescent bacterial Asite, an RNA construct of potential therapeutic utility. We show that the binding of this RNA target to aminoglycoside antibiotics, its cognate ligands, can be effectively monitored by fluorescence spectroscopy. These observations are significant since isosteric emissive U derivatives are scarce and the trivial synthesis and effective enzymatic incorporation of the furan-containing U triphosphate make it accessible to the biophysical community.

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

“…In most cases the dNTP modifications were introduced to the nucleobase. In cases where pyrimidines are employed the modifications are almost exclusively at the C5 position to avoid disturbing of Watson-Crick base pairing ability (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In addition, the C5 modification is well accommodated into the major groove of DNA without perturbing the DNA structure.…”

“…Mainly C5 alkyne modified dNTPs are used because they are accessible via metal mediated cross coupling reactions (21-23) in a straightforward manner. While many C5 modified dNTP analogs are already applied, because they are processed by DNA polymerases at least to some extent, it is still unpredictable which modification will be accepted (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) because the detailed molecular mechanism by which C5 modifications are accepted by a DNA polymerase is not at all understood. This is largely due to the absence of suitable structural data.…”

Using an emissive uridine analogue for assembling fluorescent HIV-1 TAR constructs

“…28 We have also demonstrated that T7 RNA polymerase recognizes this modified ribonucleoside triphosphate as a substrate in run-off in vitro transcription reactions and very efficiently incorporates it into RNA oligonucleotides. We have further illustrated that fluorescent RNA constructs, generated in this fashion, can be employed for the development of biophysical assays.…”

“…Specifically, we have applied this method for the examination of a fluorescent bacterial A-site and its interactions with aminoglycoside antibiotics. 28 Here we employ similar experimental procedures for the preparation of fluorescently tagged TAR constructs as described below.…”

“…We have also demonstrated the effective incorporation of this fluorescent nucleoside analogue into RNA constructs using in vitro transcription reactions, and its use in exploring RNA recognition events such as RNA-antibiotics interactions. 28 To illustrate the potential of this fluorescent probe for the study of HIV-1 RNA targets, we describe here the synthesis of fluorescent TAR constructs by transcription reactions and subsequent development of an effective fluorescence-based Tat-TAR binding assay.…”