Variable Inhibition of DNA Unwinding Rates Catalyzed by the SARS-CoV-2 Helicase Nsp13 by Structurally Distinct Single DNA Lesions.

The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5' → 3' direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6-4) pyrimidone (6-4PP) photolesion. The experimentally observed unwinding rate constants (kobs) and processivities (P) were examined. Relative to undamaged DNA, the kobs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2-5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest kobs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions' inhibitory effects than unwinding rate constants.

The DNA damage-sensing NER repair factor XPC-RAD23B does not recognize bulky DNA lesions with a missing nucleotide opposite the lesion.

The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (KD) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants KD of unmodified and lesion-containing G*:Del complexes, showed that the KD values were -2.5-3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 ß-hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes.

Combining aminocyanine dyes with polyamide dendrons: a promising strategy for imaging in the near-infrared region.

Cyanine dyes are known for their fluorescence in the near-IR (NIR) region, which is desirable for biological applications. We report the synthesis of a series of aminocyanine dyes containing terminal functional groups such as acid, azide, and cyclooctyne groups for further functionalization through, for example, click chemistry. These aminocyanine dyes can be attached to polyfunctional dendrons by copper-catalyzed azide alkyne cycloaddition (CuAAC), strain-promoted azide alkyne cycloaddition (SPAAC), peptide coupling, or direct S(NR)1 reactions. The resulting dendron-dye conjugates were obtained in high yields and displayed high chemical stability and photostability. The optical properties of the new compounds were studied by UV/Vis and fluorescence spectroscopy. All compounds show large Stokes shifts and strong fluorescence in the NIR region with high quantum yields, which are optimal properties for in vivo optical imaging.

Absolute configurations of DNA lesions determined by comparisons of experimental ECD and ORD spectra with DFT calculations.

The usefulness of modern density functional theory (DFT) methods is considered for establishing the absolute configurations of DNA lesions by comparisons of computed and experimentally measured optical rotatory dispersion (ORD) and electronic circular dichroism (ECD) spectra. Two rigid, structurally different DNA lesions (two spiroiminodihydantoin stereoisomers and four equine estrogen 4-hydoxyequilenin-DNA stereoisomeric adducts) have been investigated. In all cases, the signs and shapes of the computed ORD spectra reproduced the experimentally measured ORD spectra, although the magnitudes of the computed and experimental ORD values do not coincide exactly. The computed ECD spectra also reproduced the shapes of the experimental ECD spectra rather well, but are blue-shifted by 10-20 nm. Since the assignments of the absolute configurations of the DNA lesions studied based on computed and experimental ORD and ECD spectra are fully consistent with one another, the computational DFT method shows significant promise for determining the absolute configurations of DNA lesions. Establishing the stereochemistry of DNA lesions is highly useful for understanding their biological impact, especially when sufficient amounts of material are not available for other methods of structural characterization.

Synthesis, conformational interconversion, and photophysics of tethered porphyrin-fullerene dyads with parachute topology.

The synthesis of a porphyrin-fullerene dyad with "parachute" topology is reported. To determine whether the dyad is "flexing" at room temperature, low-temperature NMR experiments were used. Computational modeling has shown the low-energy conformation of the dyad to be nonsymmetric. Although, (1)H NMR spectroscopy at room temperature is consistent with a molecule with C(2v) symmetry, the spectrum changes on lowering the temperature consistent with "windshield wiper"-like motion, in which the porphyrin moiety rotates from one side of the C(60) sphere to the other. Nanosecond and picosecond fluorescence lifetime experiments show two components contribute to the fluorescence decay, also consistent with the presence of more than one conformer.

Absolute configurations of spiroiminodihydantoin and allantoin stereoisomers: comparison of computed and measured electronic circular dichroism spectra.

The assignment of absolute configurations is of critical importance for understanding the biochemical processing of DNA lesions. The diastereomeric spiroiminodihydantoin (Sp) lesions are oxidation products of guanine and 8-oxo-7,8-dihydroguanine (8-oxoG), and the absolute configurations of the two diastereomers, Sp1 and Sp2, have been evaluated by experimental and computational optical rotatory dispersion (ORD) methods. In order to support our previous assignments by the ORD method, we calculated the electronic circular dichroism spectra (ECD) of the Sp stereoisomers. Comparison of the experimentally measured and computed ECD spectra indicates that Sp1 has (-)-S absolute configuration, while Sp2 has (+)-R absolute configuration. Thus, the S and R assignments, based on the ECD spectra of Sp1 and Sp2, are consistent with our previous assignments of absolute configurations. To further test the validity of this approach, we performed a proof-of-principle computation of the ECD and ORD of the R and S enantiomers of allantoin (similar in chemical composition to Sp) of known absolute configurations. The calculations provide the correct assignment of the absolute configurations of the allantoin enantiomers, indicating that the computational TDDFT approach is robust for identifying the absolute configurations of allantoins and probably the Sp stereoisomers, as has been shown previously for other organic molecules.

Determination of absolute configurations of 4-hydroxyequilenin-cytosine and -adenine adducts by optical rotatory dispersion, electronic circular dichroism, density functional theory calculations, and mass spectrometry.

Estrogen components of some hormone replacement formulations have been implicated in the initiation of breast cancer. Some of these formulations contain equine estrogens such as equilin and equilenin that are metabolized to the genotoxic catechol 4-hydroxyequilenin (4-OHEN). Auto-oxidation generates the o-quinone form that reacts with dC and dA in oligodeoxynucleotides to form unusual stable cyclic bulky adducts, with four different stereoisomers identified for each base adduct. The dC and dA adducts have the same unsaturated bicyclo[3.3.1]nonane type linkage site with identical stereochemical characteristics. Stereochemical effects may play an important part in the biological consequences of the formation of 4-OHEN-DNA adducts, and the assignment of the absolute configurations of the stereoisomeric 4-OHEN-dC and -dA adducts is therefore needed to understand structure-function relationships. We used density functional theory (DFT) to compute the specific optical rotations and electronic circular dichroism (ECD) spectra of the four 4-OHEN-C stereoisomers, and the results were compared with experimentally measured optical rotatory dispersion (ORD) and ECD spectra. The predicted ORD curves for the four stereoisomeric base adducts reproduced the shapes and signs of experimental spectra in the transparent spectral region. The stereochemistry of the C3' atom was determined by comparison of the calculated and experimental ORD and ECD spectra, and the stereochemistry of C2' was determined by mass spectrometric methods. Combining the ORD and mass spectrometry data, the absolute configurations of the four 4-OHEN-C and the stereochemically identical -dC adducts have been identified. The molecular architecture of the linkage site at the 4-OHEN-C/A and 4-OHEN-dC/dA is identical, and it is shown that the deoxyribose group does not substantially contribute to the optical activities. The absolute configurations of the 4-OHEN-dA adducts were thus deduced by comparing the experimental ORD with computed ORD values of 4-OHEN-A adducts.

Oxidation of guanine in G, GG, and GGG sequence contexts by aromatic pyrenyl radical cations and carbonate radical anions: relationship between kinetics and distribution of alkali-labile lesions.

Oxidatively generated DNA damage induced by the aromatic radical cation of the pyrene derivative 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT), and by carbonate radicals anions, was monitored from the initial one-electron transfer, or hole injection step, to the formation of hot alkali-labile chemical end-products monitored by gel electrophoresis. The fractions of BPT molecules bound to double-stranded 20-35-mer oligonucleotides with noncontiguous guanines G and grouped as contiguous GG and GGG sequences were determined by a fluorescence quenching method. Utilizing intense nanosecond 355 nm Nd:YAG laser pulses, the DNA-bound BPT molecules were photoionized to BPT*+ radicals by a consecutive two-photon ionization mechanism. The BPT*+ radicals thus generated within the duplexes selectively oxidize guanine by intraduplex electron-transfer reactions, and the rate constants of these reactions follow the trend 5'-..GGG.. > 5'-..GG.. > 5'-..G... In the case of CO3*- radicals, the oxidation of guanine occurs by intermolecular collision pathways, and the bimolecular rate constants are independent of base sequence context. However, the distributions of the end-products generated by CO3*- radicals, as well as by BPT*+, are base sequence context-dependent and are greater than those in isolated guanines at the 5'-G in 5'-...GG... sequences, and the first two 5'- guanines in the 5'-..GGG sequences. These results help to clarify the conditions that lead to a similar or different base sequence dependence of the initial hole injection step and the final distribution of oxidized, alkali-labile guanine products. In the case of the intermolecular one-electron oxidant CO3*-, the rate constant of hole injection is similar for contiguous and isolated guanines, but the subsequent equilibration of holes by hopping favors trapping and product formation at contiguous guanines, and the sequence dependence of these two phenomena are not correlated. In contrast, in the case of the DNA-bound oxidant BPT*+, the hole injection rate constants, as well as hole equilibration, exhibit a similar dependence on base sequence context, and are thus correlated to one another.

Assignment of absolute configurations of the enantiomeric spiroiminodihydantoin nucleobases by experimental and computational optical rotatory dispersion methods.

The diastereomeric spiroiminodihydantoin (Sp) lesions are oxidation products of guanine and 8-oxo-7,8-dihydroguanine (8-oxoG) and have generated considerable interest because of their stereochemistry-dependent mutagenic properties in vivo (Henderson, P. T., et al. (2003) Biochemistry 42, 9257-9262). However, the absolute configurations of the two diastereomers have not yet been elucidated, and such information may prove valuable for understanding relationships between biological function and structure at the DNA level (Jia, L., Shafirovich, V., Shapiro, R., Geacintov, N. E., and Broyde, S. (2005) Biochemistry 44, 13342-13353). We have synthesized the two chiral Sp nucleobases by hydrolysis of the nucleosides denoted by dSp1 and dSp2 according to their elution order in HPLC experiments using a Hypercarb column, and determined their absolute configurations using a combination of experimentally measured optical rotatory dispersion (ORD) spectra in aqueous solutions and computed ORD specific rotations using density functional theory (DFT). Recent developments have shown that DFT methods are now sufficiently robust for predicting ORD values of chiral molecules (Polavarapu, P. L. (2002) Chirality 14, 768-781). The nucleobases Sp1 and Sp2 exhibit experimentally measured CD and ORD spectra that are very close to those of the respective precursor nucleosides dSp1 and dSp2 in shape and sign. The first nucleoside stereoisomer (dSp1) to elute from a typical Hypercarb HPLC column has (-)-S, while the second (dSp2) has (+)-R absolute configuration. The R and S assignments are applicable to the amino tautomeric forms in each case.

A sensitive probe for the detection of Zn(II) by time-resolved fluorescence.

A new, highly sensitive fluorescent sensor for Zn(II) ion (a tris(2-pyridylmethyl)amine derivative) shows very strong binding and Zn(II) concentration-dependent biexponential time-resolved fluorescence (TRF) decay profiles that can be used for ratiometric estimates of Zn(II) concentrations. The ligand-metal complexes were characterized in solution by spectroscopic techniques and in the solid state by X-ray crystallography. The TRF studies revealed that the sensor aggregates in the absence of Zn(II) in a ligand concentration-dependent manner, a complication that is discerned by TRF but not by steady-state fluorescence ratiometric sensing techniques. It is shown that the same TRF methods are highly useful for monitoring Zn(II) concentrations in A549 epithelial lung cells in vitro and that the results were consistent with those in solution.