Environmentally Sensitive Fluorescent Nucleoside Analogues for Surveying Dynamic Interconversions of Nucleic Acid Structures

Abstract

Nucleic acids are characterized by a variety of dynamically interconverting structures that play a major role in transcriptional and translational regulation as well as recombination and repair. To monitor these interconversions, Förster resonance energy transfer (FRET)-based techniques can be used, but require two fluorophores that are typically large and can alter the DNA/RNA structure and protein binding. Additionally, events that do not alter the donor/acceptor distance and/or angular relationship are frequently left undetected. A more benign approach relies on fluorescent nucleobases that can substitute their native counterparts with minimal perturbation, such as the recently developed 2-thienyl-3-hydroxychromone (3HCnt) and thienoguanosine ( G). To demonstrate the potency of 3HCnt and G in deciphering interconversion mechanisms, we used the conversion of the (-)DNA copy of the HIV-1 primer binding site (-)PBS stem-loop into (+)/(-)PBS duplex, as a model system. When incorporated into the (-)PBS loop, the two probes were found to be highly sensitive to the individual steps both in the absence and the presence of a nucleic acid chaperone, providing the first complete mechanistic description of this critical process in HIV-1 replication. The combination of the two distinct probes appears to be instrumental for characterizing structural transitions of nucleic acids under various stimuli.

Deciphering the pH-dependence of ground- and excited-state equilibria of thienoguanine

“…We then characterized the fluorescence quantum yields of th Gb as a function of pH. For each pH value, we determined the QYs at multiple excitation wavelengths (310-340 nm at pH 2 and 3, and 310-370 nm at pH [4][5][6][7][8][9][10][11][12]. At all pH values, the QY values were found to be constant over the explored excitation wavelength range.…”

“…Numerous cellular mechanisms and pathways rely on dynamic interactions of nucleic acids with proteins that induce local and transient changes in their secondary and tertiary structures as well as in their properties and functions (1)(2)(3)(4)(5)(6). Though X-ray diffraction, NMR spectroscopy or electron microscopy provide invaluable information on the structure of protein/nucleic acid complexes, they are less suited for monitoring the dynamics of these interactions and their associated changes, especially in dilute solutions.…”

“…In addition, this fluorescent G analogue remains strongly emissive when incorporated into ODNs, and exhibits environmental sensitivity. This fluorescent nucleoside has already been extensively used to monitor protein/ODN interactions (6,14,15), protein-induced base flipping (16), conformational changes in ODNs (6,17), single nucleotide polymorphism (14); ribozyme-mediated cleavage processes (18), cellular activity of siRNAs (19) and distance measurements in DNA (15,20).…”

What Makes Thienoguanosine an Outstanding Fluorescent DNA Probe?

“…5,6 The equilibrium between these tautomers informs on the local environment of th G. Due to its unique properties, th G was successfully used to monitor the local conformational changes associated with the binding of the HIV-1 nucleocapsid protein NCp7 to the (−)Primer Binding Site (PBS) and to decipher the mechanism of the NCp7-promoted (+)/(−)PBS annealing reaction. 7 The fluorescent nucleoside analogue in its deoxyribo or in its ribo form has also been successfully used to monitor B to Z transitions of DNA, 2 single nucleotide polymorphism, 7 distance measurements in DNA, 8,9 base flipping reactions, 10 ribozyme-mediated cleavage, 11 and cellular activity of siRNAs. 12 Most of the applications of th G reported so far rely on empirical changes in fluorescence intensity and thus, do not exploit the full potential of this information-rich probe.…”

Probing of Nucleic Acid Structures, Dynamics, and Interactions With Environment-Sensitive Fluorescent Labels

“…The 3HC label was employed to study the conversion of the (-)DNA copy of the HIV-1 primer binding site (-)PBS stem-loop into the (+/-)PBS duplex in the absence and presence of the NC chaperone protein. In contrast to 2AP, the 3HC probe provided the first complete mechanistic description of this critical process in HIV-1 replication (Sholokh et al, 2018). In a comparison study with commercially available FNAs, the fluorescent label was exploited to investigate the mechanism of the DNA repair enzyme, endonuclease VIII, in interactions with damaged DNA.…”