Isomorphic Fluorescent Nucleosides.

Area of Science:

• Photophysics and Biophysics of Nucleic Acids
• Chemical Biology and Synthetic Chemistry

Background:

• Tryptophan's fluorescence revolutionized protein studies, but canonical nucleobases in RNA and DNA have low emission, hindering fluorescence applications.
• Developing intrinsically emissive nucleoside surrogates is crucial for real-time, nucleobase-resolution monitoring of nucleic acid transformations.

Purpose of the Study:

• To design and synthesize isomorphic and isofunctional emissive nucleoside surrogates for nucleic acids.
• To enable real-time fluorescence-based monitoring of nucleic acid structures, functions, and enzymatic transformations.

Main Methods:

• Minimal atomic/structural perturbations were introduced to create synthetic nucleoside analogs with favorable photophysical properties.
• Developed thieno[3,4-d]pyrimidine and isothiazolo[4,3-d]pyrimidine cores to create fluorescent purine and pyrimidine analogs.
• Tested analogs as substrates for various enzymes, including polymerases, metabolic enzymes, and those involved in cofactor/coenzyme synthesis.

Main Results:

• Created fluorescent ribonucleoside alphabets (purine and pyrimidine analogs) that maintain native conformations and pairing.
• Demonstrated the utility of emissive analogs as substrates for enzymes, enabling real-time fluorescence assays for enzyme activity and inhibitor discovery.
• Synthesized emissive cofactors, coenzymes, and second messengers, allowing real-time monitoring of their biosynthesis and transformations.

Conclusions:

• Highly isomorphic and isofunctional emissive nucleoside surrogates can be fabricated and implemented for diverse biochemical studies.
• These probes offer optical windows into nucleic acid biochemistry, complementing established methods like 2-aminopurine.
• Future work will focus on overcoming limitations in emission wavelength and brightness through advanced spectroscopy and structural modifications.