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

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.

Visualization of Platelet Integrins via Two-Photon Microscopy Using Anti-transmembrane Domain Peptides Containing a Blue Fluorescent Amino Acid

“…Confirming the utility of 4CNI in 2PE fluorescence microscopy will open up new biological applications for this amino acid fluorophore and its nucleic acid analogue, 4cyanoindole 2′-deoxyribonucleoside. 25 We used 4CNI-3AA to label the N-termini of membranesoluble CHAMP peptides designed to specifically bind to integrin transmembrane domains in situ. Because these peptides bind to one component of the transmembrane heterodimer that maintains integrins in their inactive conformation, they cause heterodimer separation and subsequent integrin activation.…”

“…In addition, the absorption spectrum of 4CNI can be readily accessed by the femtosecond Ti:sapphire laser commonly equipped in commercial 2PE microscopes. Confirming the utility of 4CNI in 2PE fluorescence microscopy will open up new biological applications for this amino acid fluorophore and its nucleic acid analogue, 4-cyanoindole 2′-deoxyribonucleoside …”

PET and FRET utility of an amino acid pair: tryptophan and 4-cyanotryptophan

“…Therefore, development of amino acid-based FRET and PET pairs that are intrinsically less-perturbative and hence can minimize such pitfalls is needed; (3) the absorption spectrum of 4CN-Trp is significantly red-shifted from that of Trp, allowing selective excitation of its fluorescence (e.g., using λ ex = 330−360 nm) or that of Trp (e.g., using λ ex = 270 nm) in PET or FRET applications; (4) 4CN-Trp is only one atom larger than Trp, making it less perturbative to proteins than fluorescent dyes; (5) 4CN-Trp can now be conveniently synthesized via chemical 28 and bilogical 29 means; and (6) it is possible to incorporate 4CN-Trp into proteins genetically via amber codon suppression or chemically using a post-translational modification method. 30,31 Given the unique photophysical property 13,16,32 of 4CN-Trp and the less-perturbating nature of this unnatural amino acid and Trp, we believe that this pair will find valuable applications in biochemistry and biophysics, especially in cases where using dye-based PET or FRET reporters is deemed inappropriate. We are particularly excited about the possibility of using this pair of amino acids in single-molecule fluorescence studies, such as PET-FCS, 6,33,34 as well as using it to study the functional and/or conformational dynamics of Trp-rich proteins.…”

Synthesis of 4‐Cyanoindole Nucleosides, 4‐Cyanoindole‐2ʹ‐Deoxyribonucleoside‐5ʹ‐Triphosphate (4CIN‐TP), and Enzymatic Incorporation of 4CIN‐TP into DNA

“…In a recent publication, we described the synthesis and extensive characterization of 4CIN, which exhibits strong fluorescence not only as a monomer, but also in a DNA context (Passow & Harki, ). This molecule was subsequently used by Gai and coworkers in a proof of concept study showing that 4CIN could be used to report DNA‐protein binding including the calculation of a binding affinity (Ahmed et al., ). This, in turn, exemplifies the utilities of 4CI‐based nucleosides and nucleotides as biological reporters.…”