Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.

Appending conformationally restraining ring systems to the cyanine chromophore creates exceptionally bright fluorophores in the visible range. Here, we report the application of this strategy in the near-infrared range through the preparation of the first restrained heptamethine indocyanine. Time-resolved absorption spectroscopy and fluorescence correlation spectroscopy verify that, unlike the corresponding parent unrestrained variant, the restrained molecule is

Protein-induced fluorescence enhancement (PIFE) is an increasingly used approach to investigate DNA-protein interactions at the single molecule level. The optimal probe for this type of application is highly photostable, has a high absorption extinction coefficient, and has a moderate fluorescence quantum yield that increases significantly when the dye is in close proximity to a large macromolecule such as a protein. So far, the green-absorbing symmetric cyanine known as Cy3 has been the probe of choice in this field because the magnitude of the increase observed upon protein binding (usually 2–4 –fold) is large enough to allow for the analysis of protein dynamics on the inherently noisy single-molecule signals. Here, we report the characterization of the photophysical properties of the red-absorbing hemicyanine dye Dy-630 in the context of its potential application as a single-molecule PIFE probe. The behavior of Dy-630 in solution is similar to that of Cy3; the fluorescence quantum yield and lifetime of Dy-630 increase with increasing viscosity, and decrease with increasing temperature indicating the existence of an activated nonradiative process that depopulates the singlet state of the dye. As in the case of Cy3, the results of transient spectroscopy experiments are consistent with the formation of a photoisomer that reverts to the ground state thermally in the microsecond timescale. Unfortunately, experiments with DNA samples paint a more complex scenario. As in the case of Cy3, the fluorescence quantum yield of Dy-630 increases significantly when the dye interacts with the DNA bases, but in the case of Dy-630 attachment to DNA results in an already long fluorescence lifetime that does not provide a significant window for the protein-induced enhancement observed with Cy3. Although we show that Dy-630 may not be well-suited for PIFE, our results shed light on the optimal design principles for probes for PIFE applications.

Uracil DNA-glycosylase (UNG) is a DNA repair enzyme that removes the highly mutagenic uracil lesion from DNA using a base flipping mechanism. Although this enzyme has evolved to remove uracil from diverse sequence contexts, UNG excision efficiency depends on DNA sequence. To provide the molecular basis for rationalizing UNG substrate preferences, we used time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations to measure UNG specificity constants (kcat/KM) and DNA flexibilities for DNA substrates containing central AUT, TUA, AUA, and TUT motifs. Our study shows that UNG efficiency is dictated by the intrinsic deformability around the lesion, establishes a direct relationship between substrate flexibility modes and UNG efficiency, and shows that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and UNG activity. The finding that substrate flexibility controls UNG efficiency is likely significant for other repair enzymes and has major implications for the understanding of mutation hotspot genesis, molecular evolution, and base editing.

Uracil DNA-glycosylase (UNG) is a base excision repair enzyme that removes the highly mutagenic uracil lesion from DNA by a base flipping mechanism. UNG excision efficiency depends on DNA sequence, yet the underlying principles that dictate UNG substrate specificity have remained elusive. Here, we show that UNG efficiency is dictated by the intrinsic local deformability of the substrate sequence around the uracil. UNG specificity constants (kcat/KM) and DNA flexibilities were measured for an engineered set of DNA substrates containing AUT, TUA, AUA, and TUA motifs. Time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations of the bare DNA indicated significant differences in substrate flexibilities. A strong correlation between UNG efficiency and substrate flexibility was observed, with higher kcat/KM values measured for more flexible strands. DNA bending and base flipping were observed in simulations, with more frequent uracil flipping observed for the more bendable sequences. Experiments show that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and resultant UNG activity. The finding that substrate flexibility controls UNG efficiency has implications in diverse fields, including the genesis of mutation hotspots, molecular evolution, and understanding sequence preferences of emerging base editors.