UC San Diego

As our understanding of complex cellular processes advances, nucleic acids, the biomolecules of life, consistently appear as key players. The ability to explore the structure, dynamics, and recognition of nucleic acids has been accessible due to many techniques. Specifically, the sensitivity of fluorescent spectroscopy has made it one of the most useful tools for the detailed study of macromolecules. The development of synthetically modified nucleosides has been driven by the lack of naturally occurring nucleosides with favorable photophysical features that allow for probing of nucleic acid-containing systems. The design of the ideal probe should ensure high structural resemblance to the native nucleosides, minimizing the perturbation of the biomolecular architecture, a feature commonly referred to as isomorphicity. This is particularly challenging when one aims at shifting the emission bands further into the red spectral domains.

Three related structural designs of synthetic 6-aza-uridines were implemented to facilitate a bathochromic shift of emission bands. First, the electronics of 5-substituted uridines were altered by replacing the pyrimidine core with the more electron withdrawing 6-aza-uridine, yielding a red shift as well as hyperchromic effect and higher brightness. In the second design, to further shift the emission into the red region, the polarization of this conjugated electron-poor/electron-rich biaryl system was enhanced by directly conjugating donor groups through an extended aromatic system. The last design resulted in a synthetic uridine surrogate, which can undergo a proton transfer in the excited state (a phenomenon known as ESIPT), thus facilitating a relatively large bathochromic shift while fulfilling the requirements of isomorphic nucleosides.

Furthermore, due to their structural similarity, these isomorphic mimics should fabricate functions and maintain bio-compatibility similar to native building blocks. It has been shown that T7 RNA polymerase accepts the triphosphate-functionalized thiophene 6-aza-uridines, which can determine the affinity of aminoglycoside antibiotics for the singly-modified fluorescent A-site. This finding is important to the biochemical community as it introduces a new attractive pyrimidine analogue, which can be used for probing RNA targets with therapeutic potentials.

To investigate more than uridine analogues, the affinity of T7 RNA polymerase for synthetic pyrimidine and purine mimics was compared and closely evaluated. Subsequently, the partially and fully modified transcripts were successfully synthesized and utilized in a ligation reaction, thereby showing that they can moderately mimic functions and bio-compatibility of the native strands. These results point to intriguing future applications of all four mimics, either site specific modification can be envisioned or partially and fully modified constructs can be utilized for biochemical assays.