The goals of this project were (1) to develop new fluorescent nucleoside analogues as molecular probes for studying how structural and dynamic features of DNA and RNA control gene expression, and (2) to determine how the structural features of these fluorescent probes and the chemical environment in DNA and RNA determine fluorescent properties. This research is significant because it advances the science of fluorescent probe molecular probe design and it provides novel tools for studying the biochemical mechanisms that control when and how the genetic code is expressed.

Under the category of intellectual merit, the following outcomes were attained. (1) We designed and synthesized a new fluorescent nucleoside analogue that we named ABN. ABN is one of the brightest known fluorescent nucleoside analogues and the first to be readily detectable in single-molecule fluorescence measurements. This capability is significant because it enables molecules of DNA and RNA to be studied in isolation, avoiding the signal averaging effect of examining multiple molecules concurrently in an ensemble. Accordingly, minor states important for understanding biological mechanisms can be studied. (2) We demonstrated that our fluorescent nucleoside analogue DEAtC is capable of a strong fluorescence turn-on response to matched base pairing with RNA. This capability will be useful for studying RNA folding and three-dimensional structure, changes of which underpin biological function. (3) Knowledge of the relationships between fluorescent nucleoside analogue structure and fluorescent properties was advanced through (i) computational studies showing the role of explicit solvation in accurate spectral prediction, (ii) NMR structure determination showing that DEAtC only minimally perturbs the native B conformation of duplex DNA, and (iii) time-resolved fluorescence studies showing that changes in excited state lifetime are responsible for DEAtC’s ability to distinguish between matched and mismatched base pairs.

Under the category of broader impacts, the following outcomes were attained. (1) The field of nucleic acid biology benefitted from sharing the products of this research. Fluorescent nucleoside analogues resulting from these studies were used to study nucleic acid replication by the reverse transcriptases of HIV, AMV, and M-MLV retroviruses. In another application, fluorescent nucleosides resulting in part from this work were used to develop a new method for metabolic labeling and live-cell imaging of RNA metabolism. (2) This project included the participation and/or training of 8 graduate students, 19 undergraduates, and 7 collaborating faculty members. Most of the student participants who have graduated have gone on to graduate school or professional school or taken jobs in industry. Five of the undergrads won competitive NIH IMSD (Initiative for Maximizing Student Development) awards and one was awarded an ACS Division of Organic Chemistry Summer Undergraduate Research Fellowship, one of only 16 nationally. (3) In a Course-based Undergraduate Research Experience, more than additional undergraduates were trained in fluorescent measurements aimed at understanding how fluorescent probes for biomolecules respond to changes in their local chemical environment.

Last Modified: 12/23/2022
Modified by: Byron Purse