4-Cyanoindole-2'-deoxyribonucleoside as a Dual Fluorescence and Infrared Probe of DNA Structure and Dynamics.

Ismail A Ahmed,Feng Gai,Jeffrey M Rodgers,Christina M Eng,William F Degrado,Hyunil Jo,Arusha Acharyya

doi:10.3390/molecules24030602

Abstract

Unnatural nucleosides possessing unique spectroscopic properties that mimic natural nucleobases in both size and chemical structure are ideally suited for spectroscopic measurements of DNA/RNA structure and dynamics in a site-specific manner. However, such unnatural nucleosides are scarce, which prompts us to explore the utility of a recently found unnatural nucleoside, 4-cyanoindole-2′-deoxyribonucleoside (4CNI-NS), as a site-specific spectroscopic probe of DNA. A recent study revealed that 4CNI-NS is a universal nucleobase that maintains the high fluorescence quantum yield of 4-cyanoindole and that among the four natural nucleobases, only guanine can significantly quench its fluorescence. Herein, we further show that the C≡N stretching frequency of 4CNI-NS is sensitive to the local environment, making it a useful site-specific infrared probe of oligonucleotides. In addition, we demonstrate that the fluorescence-quencher pair formed by 4CNI-NS and guanine can be used to quantitatively assess the binding affinity of a single-stranded DNA to the protein system of interest via fluorescence spectroscopy, among other applications. We believe that this fluorescence binding assay is especially useful as its potentiality allows high-throughput screening of DNA–protein interactions.

Highlights

Bio-macromolecules, including proteins and polynucleotides such as DNA and RNA, can adopt various complex structures that confer specific functions
The fluorescence quantum yield (QY) of 4-cyanoindole-20 -deoxyribonucleoside (4CNI-NS), determined using 4CNI as a reference, is 0.85 ± 0.5 in water, which is in agreement with that measured by Passow and Harki [10]
Similar to that observed for the 4CN-Trp [12], in the hydrophobic solvent tetrahydrofuran (THF), the fluorescence QY of 4CNI-NS is decreased to 0.72, while the peak wavelength of its fluorescence spectrum is blue-shifted to 380 nm

Summary

Introduction

Bio-macromolecules, including proteins and polynucleotides such as DNA and RNA, can adopt various complex structures that confer specific functions. Many past studies have focused on identifying molecular moieties that afford the required spectroscopic attributes and can be incorporated into the bio-macromolecules of interest, and minimally perturb their native structure. In this regard, the ideal extrinsic spectroscopic probes for protein (DNA) would be simple analogues of naturally-occurring amino acids (nucleosides). Little has been done in the development of minimally-perturbing, unnatural nucleoside-based spectroscopic probes This is due, at least in part, to the fact that the fundamental building blocks of DNA involve only four nucleobases in comparison to the 20 canonical amino acids for protein, and specific base pairing in double stranded DNA is achieved via well-defined hydrogen-bonding interactions. We aim to show that an unnatural nucleoside (UNS), 4-cyanoindole-20 -deoxyribonucleoside (4CNI-NS) (Figure 1), can be used as a site-specific IR and fluorescence probe of DNA structure and dynamics

Objectives

Methods

Results

Conclusion