Structure-property Relationships and the Optimization of Fluorescent Nucleoside Analogues

San Diego State University Foundation, San Diego CA

Investigators

Abstract

With the support of the Chemical Mechanism, Function, and Properties Program of the Division of Chemistry, Professor Byron Purse of the Department of Chemistry and Biochemistry at San Diego State University is studying the design and properties of fluorescent nucleobase analogues (FBAs). FBAs are small synthetic molecules that mimic the natural genetic bases — adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) — in DNA and RNA. Ideal FBAs preserve base-pairing properties while allowing sensitive detection of structural changes, molecular interactions, and other features relevant to genetic regulation. However, designing effective FBAs is challenging due to the difficulty of predicting how fluorescence responds in complex and dynamic biological environments like nucleic acids. This research aims to determine how molecular structure and local environment influence key FBA properties: brightness, photostability, and the ability to report on significant changes in biomolecular environment. The intellectual merit lies in advancing the understanding of how fluorophore chemical structure affects fluorescence and how fluorescence changes in response to the local chemical environment, and specifically when incorporated within DNA and RNA. Broader impacts include enabling new tools for genetic research and biotechnology, and training graduate students and undergraduates in synthetic and physical organic chemistry with interdisciplinary applications. Two fluorescent nucleobase analogues (FBAs) have been created that (1) mimics cytosine but becomes fluorescent upon base pairing with guanine (G) and (2) is sufficiently bright for single molecule detection, respectively. However, the bulkiness of the cytosine mimic (DEAtC) limits its biological compatibility, and the relatively rapid photobleaching of these and other FBAs limits applications in confocal fluorescence microscopy and single-molecule total internal reflection fluorescence (smTIRF) microscopy. The mechanisms of fluorescence turn-on and photobleaching are not known for these compounds, limiting the ability to improve on their properties. Accordingly, this research seeks to address these knowledge gaps and create improved FBAs in the following specific aims. (1) Investigate mechanisms of fluorescence turn-on in response to base pairing and stacking. (2) Improve the photostability of pyrimidine analogue FBAs. (3) Investigate two alternative FBA scaffolds designed to attain (i) high brightness from a small size and (ii) excitation at > 450 nm. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.