Stringent Nucleotide Recognition by the Ribosome at the Middle Codon Position.

Accurate translation of the genetic code depends on mRNA:tRNA codon:anticodon base pairing. Here we exploit an emissive, isosteric adenosine surrogate that allows direct measurement of the kinetics of codon:anticodon University of California base formation during protein synthesis. Our results suggest that codon:anticodon base pairing is subject to tighter constraints at the middle position than at the 5'- and 3'-positions, and further suggest a sequential mechanism of formation of the three base pairs in the codon:anticodon helix

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

The thienoguanine nucleobase (thGb) is an isomorphic fluorescent analogue of guanine. In aqueous buffer at neutral pH, thGb exists as a mixture of two ground-state H1 and H3 keto-amino tautomers with distinct absorption and emission spectra and high quantum yield. In this work, we performed the first systematic photophysical characterization of thGb as a function of pH (2 to 12). Steady-state and time-resolved fluorescence spectroscopies, supplemented with theoretical calculations, enabled us to identify three additional thGb forms, resulting from pH-dependent ground-state and excited-state reactions. Moreover, a thorough analysis allowed us to retrieve their individual absorption and emission spectra as well as the equilibrium constants which govern their interconversion. From these data, the complete photoluminescence pathway of thGb in aqueous solution and its dependence as a function of pH was deduced. As the identified forms differ by their spectra and fluorescence lifetime, thGb could be used as a probe for sensing local pH changes under acidic conditions

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

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 (th G). To demonstrate the potency of 3HCnt and th 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

Dynamics of Methylated Cytosine Flipping by UHRF1

DNA methylation patterns, which are critical for gene expression, are replicated by DNA methyltransferase 1 (DNMT1) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) proteins. This replication is initiated by the recognition of hemimethylated CpG sites and further flipping of methylated cytosines (mC) by the Set and Ring Associated (SRA) domain of UHRF1. Although crystallography has shed light on the mechanism of mC flipping by SRA, tools are required to monitor in real time how SRA reads DNA and flips the modified nucleobase. To accomplish this aim, we have utilized two distinct fluorescent nucleobase surrogates, 2-thienyl-3-hydroxychromone nucleoside (3HCnt) and thienoguanosine ((th)G), incorporated at different positions into hemimethylated (HM) and nonmethylated (NM) DNA duplexes. Large fluorescence changes were associated with mC flipping in HM duplexes, showing the outstanding sensitivity of both nucleobase surrogates to the small structural changes accompanying base flipping. Importantly, the nucleobase surrogates marginally affected the structure of the duplex and its affinity for SRA at positions where they were responsive to base flipping, illustrating their promise as nonperturbing probes for monitoring such events. Stopped-flow studies using these two distinct tools revealed the fast kinetics of SRA binding and sliding to NM duplexes, consistent with its reader role. In contrast, the kinetics of mC flipping was found to be much slower in HM duplexes, substantially increasing the lifetime of CpG-bound UHRF1, and thus the probability of recruiting DNMT1 to faithfully duplicate the DNA methylation profile. The fluorescence-based approach using these two different fluorescent nucleoside surrogates advances the mechanistic understanding of the UHRF1/DNMT1 tandem and the development of assays for the identification of base flipping inhibitors

Oligodeoxynucleotides Containing Multiple Thiophene-Modified Isomorphic Fluorescent Nucleosides

[Image: see text] 5-(Thien-2-yl)-2’-deoxyuridine, an isomorphic fluorescent nucleoside analog, was incorporated into multiple positions within single stranded oligodeoxynucleotides. With minimal impact on duplex stability and overall structure, oligonucleotides containing three identical isomorphic fluorescent nucleosides in alternating or neighboring positions display enhanced, sequence-dependent on-signals for either duplex formation or dissociation