May DNA analyses be biased by hidden oxidative damage? Voltammetric study of temperature and oxidation stress effect.

The analysis of nucleic acids is one of the fundamental parts of modern molecular biology and molecular diagnostics. The information collected predominantly depends on the condition of the genetic material. All potential damage induced by oxidative stress may affect the final results of the analysis of genetic material obtained using commonly used techniques such as polymerase chain reaction or sequencing. The aim of this work was to evaluate the effects of high temperature and pH on DNA structure in the context of the occurrence of oxidative damage, using square-wave voltammetry and two independent research protocols. We resulted in visible oxidation damage registered in acidic conditions after the thermal denaturation process (pH 4.7) with changes in the intensity of guanine and adenine signals. However, using phosphate buffer (pH 7.0) for DNA denaturation negatively affected the DNA structure, but without any oxidized derivatives present. This leads to the conclusion that oxidation occurring in the DNA melting process results in the formation of various derivatives of nucleobases, both electrochemically active and inactive. These derivatives may distort the results of molecular tests due to the possibility of forming complementary bonds with various nucleobases. For example, 8-oxoguanine can form pairs with both cytosine and adenine.

Asymmetric Total Synthesis of Anti-HBV Drug Entecavir: Catalytic Strategies for the Stereospecific Construction of Densely Substituted Cyclopentene Cores.

We have successfully accomplished a catalytic asymmetric total synthesis of entecavir, a first-line antihepatitis B virus medication. The pivotal aspect of our strategy lies in the utilization of a Pd-catalyzed enyne borylative cyclization reaction, enabling the construction of a highly substituted cyclopentene scaffold with exceptional stereoselectivity. Additionally, we efficiently accessed the crucial 1,3-diol enyne system early in our synthetic route through a diarylprolinol organocatalyzed enantioselective cross-aldol reaction and Re-catalyzed allylic alcohol relocation. By strategically integrating these three catalytic protocols, we established a practical pathway for acquiring valuable densely heteroatom-substituted cyclopentene cores.

Ionization Patterns and Chemical Reactivity of Cytosine-Guanine Watson-Crick Pairs.

The front cover artwork is provided by Prof. Papadantonakis' group. The image shows a Watson-Crick Guanine-Cytosine pair, and the difference between vertical and adiabatic ionization potentials. Read the full text of the Research Article at 10.1002/cphc.202300946.

A DFTB study on the electronic response of encapsulated DNA nucleobases onto chiral CNTs as a sequencer.

Sequencing the DNA nucleobases is essential in the diagnosis and treatment of many diseases related to human genes. In this article, the encapsulation of DNA nucleobases with some of the important synthesized chiral (7, 6), (8, 6), and (10, 8) carbon nanotubes were investigated. The structures were modeled by applying density functional theory based on tight binding method (DFTB) by considering semi-empirical basis sets. Encapsulating DNA nucleobases on the inside of CNTs caused changes in the electronic properties of the selected chiral CNTs. The results confirmed that van der Waals (vdW) interactions, π-orbitals interactions, non-bonded electron pairs, and the presence of high electronegative atoms are the key factors for these changes. The result of electronic parameters showed that among the CNTs, CNT (8, 6) is a suitable choice in sequencing guanine (G) and cytosine (C) DNA nucleobases. However, they are not able to sequence adenine (A) and thymine (T). According to the band gap energy engineering approach and absorption energy, the presence of G and C DNA nucleobases decreased the band gap energy of CNTs. Hence selected CNTs suggested as biosensor substrates for sequencing G and C DNA nucleobases.

Enhanced recognition of G-quadruplex DNA oxidative damage based on DNA-mediated charge transfer.

G-quadruplex (G4) DNA is present in human telomere oligonucleotide sequences. Oxidative damage to telomeric DNA accelerates telomere shortening, which is strongly associated with aging and cancer. Most of the current analyses on oxidative DNA damage are based on ds-DNA. Here, we developed a electrochemiluminescence (ECL) probe for enhanced recognition of oxidative damage in G4-DNA based on DNA-mediated charge transfer (CT), which could specifically recognize damaged sites depending on the position of 8-oxoguanine (8-oxoG). First, a uniform G4-DNA monolayer interface was fabricated; the G4-DNA mediated CT properties were examined using an iridium(III) complex [Ir(ppy)2(pip)]PF6 stacked with G4-DNA as an indicator. The results showed that G4-DNA with 8-oxoG attenuated DNA CT. The topological effects of oxidative damage at different sites of G4-DNA and their effects on DNA CT were revealed. The sensing platform was also used for the sensitive and quantitative detection of 8-oxoG in G4-DNA, with a detection limit of 28.9 fmol. Overall, these findings present a sensitive platform to study G4-DNA structural and stability changes caused by oxidative damage as well as the specific and quantitative detection of oxidation sites. The different damage sites in the G-quadruplex could provide detailed clues for understanding the function of G4-associated telomere functional enzymes.

Direct Measurement of 8OG Syn-Anti Flips in Mutagenic 8OG·A and Long-Range Damage-Dependent Hoogsteen Breathing Dynamics Using 1H CEST NMR.

Elucidating how damage impacts DNA dynamics is essential for understanding the mechanisms of damage recognition and repair. Many DNA lesions alter their propensities to form low-populated and short-lived conformational states. However, NMR methods to measure these dynamics require isotopic enrichment, which is difficult for damaged nucleotides. Here, we demonstrate the utility of the 1H chemical exchange saturation transfer (CEST) NMR experiment in measuring the dynamics of oxidatively damaged 8-oxoguanine (8OG) in the mutagenic 8OGsyn·Aanti mismatch. Using 8OG-H7 as an NMR probe of the damaged base, we directly measured 8OG syn-anti flips to form a lowly populated (pop. ∼ 5%) and short-lived (lifetime ∼50 ms) nonmutagenic 8OGanti·Aanti. These exchange parameters were in quantitative agreement with values from 13C off-resonance R1ρ and CEST on the labeled partner adenine. The Watson-Crick-like 8OGsyn·Aanti mismatch also rescued the kinetics of Hoogsteen motions at distant A-T base pairs, which the G·A mismatch had slowed down. The results lend further support for 8OGanti·Aanti as a minor conformational state of 8OG·A, reveal that 8OG damage can impact Hoogsteen dynamics at a distance, and demonstrate the utility of 1H CEST for measuring damage-dependent dynamics in unlabeled DNA.

A pH-Responsive Topological Switch Based on a DNA Quadruplex-Duplex Hybrid.

A guanine-rich oligonucleotide based on a human telomeric sequence but with the first three-nucleotide intervening stretch replaced by a putative 15-nucleotide hairpin-forming sequence shows a pH-dependent folding into different quadruplex-duplex hybrids in a potassium containing buffer. At slightly acidic pH, the quadruplex domain adopts a chair-type conformation. Upon increasing the pH, a transition with a midpoint close to neutral pH to a major and minor (3+1) hybrid topology with either a coaxially stacked or orthogonally oriented duplex stem-loop occurs. NMR-derived high-resolution structures reveal that an adenine protonation is prerequisite for the formation of a non-canonical base quartet, capping the outer G-tetrad at the quadruplex-duplex interface and stabilizing the antiparallel chair conformation in an acidic environment. Being directly associated with interactions at the quadruplex-duplex interface, this unique pH-dependent topological transition is fully reversible. Coupled with a conformation-sensitive optical readout demonstrated as a proof of concept using the fluorescent dye thiazole orange, the present quadruplex-duplex hybrid architecture represents a potentially valuable pH-sensing system responsive in a physiological pH range of 7±1.

Enhancement of intrinsic guanine fluorescence by protonation in DNA of various structures.

Understanding the diversity of DNA structure and functions in biology requires tools to study this biomolecule selectively and thoroughly. Fluorescence methods are powerful technique for non-invasive research. Due to the low quantum yield, the intrinsic fluorescence of nucleotides has not been considered for use in the detection and differentiation of nucleic acid bases. Here, we have studied the influence of protonation of nucleotides on their fluorescence properties. We show that protonation of ATP and GTP leads to enhanced intrinsic fluorescence. Fluorescence enhancement at acidic pH has been observed for double-stranded DNA and single-stranded oligonucleotides. The formation of G4 secondary structures apparently protected certain nucleotides from protonation, resulting in less pronounced fluorescence enhancement. Furthermore, acid-induced depurination under protonation was less noticeable in G4 structures than in double-stranded and single-stranded DNA. We show that changes in the intrinsic fluorescence of guanine can be used as a sensitive sensor for changes in the structure of the DNA and for the protonation of specific nucleotides.

Identification of bona fide RNA G-quadruplex binding proteins.

RNAs often accomplish their diverse functions through direct interactions with RNA-binding proteins (RBPs) in a sequence- and/or structure-dependent manner. RNA G-quadruplexes (rG4s) are unique secondary structures formed by guanine-rich RNA sequences which impact RNA function independently and in combination with RBPs. Efforts from several labs have identified dozens of rG4 specific RBPs (rG4BPs), although the research is still in the growing phase. Here we present methods for the systematic identification of rG4BPs using a pull-down approach that takes advantage of the chemical modification of guanine bases. This allows abolishing the rG4 structures while still maintaining the base composition intact, and hence helps in recognizing true rG4BPS (in contrast to G-rich motif binders). In combination with other biochemical assays, such an approach can be efficiently used for the identification and characterization of bona fide rG4BPs.

Comprehensive analysis of intramolecular G-quadruplex structures: furthering the understanding of their formalism.

G-quadruplexes (G4) are helical structures found in guanine-rich DNA or RNA sequences. Generally, their formalism is based on a few dozen structures, which can produce some inconsistencies or incompleteness. Using the website ASC-G4, we analyzed the structures of 333 intramolecular G4s, of all types, which allowed us to clarify some key concepts and present new information. To each of the eight distinguishable topologies corresponds a groove-width signature and a predominant glycosidic configuration (gc) pattern governed by the directions of the strands. The relative orientations of the stacking guanines within the strands, which we quantified and related to their vertical gc successions, determine the twist and tilt of the helices. The latter impact the minimum groove widths, which represent the space available for lateral ligand binding. The G4 four helices have similar twists, even when these twists are irregular, meaning that they have various angles along the strands. Despite its importance, the vertical gc succession has no strict one-to-one relationship with the topology, which explains the discrepancy between some topologies and their corresponding circular dichroism spectra. This study allowed us to introduce the new concept of platypus G4s, which are structures with properties corresponding to several topologies.

Ionization Patterns and Chemical Reactivity of Cytosine-Guanine Watson-Crick Pairs.

Gas-phase and aqueous vertical ionization potentials, vIPgas and vIPaq respectively and measurements of the molecular electrostatic and local ionization maps calculated at the DFT/B3LYP-D3/ 6-311+G** level of theory and the C-PCM reaction field model for single- and double-stranded CpG and 5MeCpG pairs show that the vIPaq for single- and double-stranded pairs of C-G and 5MeC-G are practically the same, in the range of 5.79 to 5.81 eV. The aqueous adiabatic ionization potentials for single-stranded CpG and 5MeCpG are 5.52 eV and 5.51 eV respectively and they reflect the nuclear reorganization that takes place after the abstraction of the electron. The aqueous adiabatic ionization energy values that correspond to the CpG+. radical cation and the hydrated electron, e-,, being at infinite distance, adIPaq+Vo, are 3.92 eV and 3.91 eV respectively with (Vo=-1.6 eV) Analysis of data suggest that the HOMO-LUMO energy gap in the hard/soft-acid/base (HSAB) concept cannot be used a priori to determine the effect of cytosine methylation on the guanine enhanced oxidative damage in DNA.

Structure of native four-repeat satellite III sequence with non-canonical base interactions.

Tandem-repetitive DNA (where two or more DNA bases are repeated numerous times) can adopt non-canonical secondary structures. Many of these structures are implicated in important biological processes. Human Satellite III (HSat3) is enriched for tandem repeats of the sequence ATGGA and is located in pericentromeric heterochromatin in many human chromosomes. Here, we investigate the secondary structure of the four-repeat HSat3 sequence 5'-ATGGA ATGGA ATGGA ATGGA-3' using X-ray crystallography, NMR, and biophysical methods. Circular dichroism spectroscopy, thermal stability, native PAGE, and analytical ultracentrifugation indicate that this sequence folds into a monomolecular hairpin with non-canonical base pairing and B-DNA characteristics at concentrations below 0.9 mM. NMR studies at 0.05-0.5 mM indicate that the hairpin is likely folded-over into a compact structure with high dynamics. Crystallographic studies at 2.5 mM reveal an antiparallel self-complementary duplex with the same base pairing as in the hairpin, extended into an infinite polymer. The non-canonical base pairing includes a G-G intercalation sandwiched by sheared A-G base pairs, leading to a cross-strand four guanine stack, so called guanine zipper. The guanine zippers are spaced throughout the structure by A-T/T-A base pairs. Our findings lend further insight into recurring structural motifs associated with the HSat3 and their potential biological functions.

Formation and Nanomechanical Properties of Silver-Mediated Guanine DNA Duplexes in Aqueous Solution.

Silver cations can mediate base pairing of guanine (G) DNA oligomers, yielding linear parallel G-Ag+-G duplexes with enhanced stabilities compared to those of canonical DNA duplexes. To enable their use in programmable DNA nanotechnologies, it is critical to understand solution-state formation and the nanomechanical stiffness of G-Ag+-G duplexes. Using temperature-controlled circular dichroism (CD) spectroscopy, we find that heating mixtures of G oligomers and silver salt above 50 °C fully destabilizes G-quadruplex structures and converts oligomers to G-Ag+-G duplexes. Electrospray ionization mass spectrometry supports that G-Ag+-G duplexes form at stoichiometries of 1 Ag+ per base pair, and CD spectroscopy suggests that as the Ag+/base stoichiometry increases further, G-Ag+-G duplexes undergo additional morphological changes. Using liquid-phase atomic force microscopy, we find that this excess Ag+ enables assembly of long fiberlike structures with ∼2.5 nm heights equivalent to a single DNA duplex but with lengths that far exceed a single duplex. Finally, using the conditions established to form single G-Ag+-G duplexes, we use a surface forces apparatus (SFA) to compare the solution-phase stiffness of single G-Ag+-G duplexes with dG-dC Watson-Crick-Franklin duplexes. SFA shows that G-Ag+-G duplexes are 1.3 times stiffer than dG-dC duplexes, confirming gas-phase ion mobility spectrometry measurements and computational predictions. These findings may guide the development of structural DNA nanotechnologies that rely on silver-mediated base pairing.

Structure of a DNA G-quadruplex that Modulates SP1 Binding Sites Architecture in HIV-1 Promoter.

Nucleic acid sequences containing guanine tracts are able to form non-canonical DNA or RNA structures known as G-quadruplexes (or G4s). These structures, based on the stacking of G-tetrads, are involved in various biological processes such as gene expression regulation. Here, we investigated a G4 forming sequence, HIVpro2, derived from the HIV-1 promoter. This motif is located 60 nucleotides upstream of the proviral Transcription Starting Site (TSS) and overlaps with two SP1 transcription factor binding sites. Using NMR spectroscopy, we determined that HIVpro2 forms a hybrid type G4 structure with a core that is interrupted by a single nucleotide bulge. An additional reverse-Hoogsteen AT base pair is stacked on top of the tetrad. SP1 transcription factor is known to regulate transcription activity of many genes through the recognition of Guanine-rich duplex motifs. Here, the formation of HIVpro2 G4 may modulate SP1 binding sites architecture by competing with the formation of the canonical duplex structure. Such DNA structural switch potentially participates to the regulation of viral transcription and may also interfere with HIV-1 reactivation or viral latency.

Best method to determine DNA G-quadruplex folding: The 1H-13C HSQC NMR experiment.

NMR spectroscopy is the major method for G-quadruplex structure determination under physiologically relevant solution conditions. Unlike duplex B-DNA, in which all nucleotides adopt an anti glycosidic conformation, the core tetrad-guanines in a G-quadruplex can adopt anti or syn glycosidic conformation depending on the folding structure. An experimental method that can clearly and unambiguously determine syn and anti tetrad-Gs in a G-quadruplex is highly desirable and necessary. In the present study, we exploit the advantages of the 1H-13C HSQC experiment to determine tetrad-G's glycosidic conformation and thus folding topology of G-quadruplexes. We use several examples to demonstrate the clear and straightforward determination of the guanine glycosidic conformations and G-quadruplex folding structures. Moreover, 1H-13C HSQC data can readily identify adenine H2 resonances as well as determine unusual syn conformation in loop and flanking sequences, a challenging task by standard 2D NOESY.

Sensing of DNA modifications by pAgo proteins in vitro.

Many prokaryotic Argonaute (pAgo) proteins act as programmable nucleases that use small guide DNAs for recognition and cleavage of complementary target DNA. Recent studies suggested that pAgos participate in cell defense against invader DNA and may also be involved in other genetic processes, including DNA replication and repair. The ability of pAgos to recognize specific targets potentially make them an invaluable tool for DNA manipulations. Here, we demonstrate that DNA-guided DNA-targeting pAgo nucleases from three bacterial species, DloAgo from Dorea longicatena, CbAgo from Clostridium butyricum and KmAgo from Kurthia massiliensis, can sense site-specific modifications in the target DNA, including 8-oxoguanine, thymine glycol, ethenoadenine and pyrimidine dimers. The effects of DNA modifications on the activity of pAgos strongly depend on their positions relative to the site of cleavage and are comparable to or exceed the effects of guide-target mismatches at corresponding positions. For all tested pAgos, the strongest effects are observed when DNA lesions are located at the cleavage position. The results demonstrate that DNA cleavage by pAgos is strongly affected by DNA modifications, thus making possible their use as sensors of DNA damage.

Base flipping mechanism and binding strength of methyl-damaged DNA during the interaction with AGT.

Methyl damage to DNA bases is common in the cell nucleus. O6-alkylguanine-DNA alkyl transferase (AGT) may be a promising candidate for direct damage reversal in methylated DNA (mDNA) at the O6 point of the guanine. Indeed, atomic-level investigations in the contact region of AGT-DNA complex can provide an in-depth understanding of their binding mechanism, allowing to evaluate the silico-drug nature of AGT and its utility in removing methyl damage in DNA. In this study, molecular dynamics (MD) simulation was utilized to examine the flipping of methylated nucleotide, the binding mechanism between mDNA and AGT, and the comparison of binding strength prior and post methyl transfer to AGT. The study reveals that methylation at the O6 atom of guanine weakens the hydrogen bond (H-bond) between guanine and cytosine, permitting for the flipping of such nucleotide. The formation of a H-bond between the base pair of methylated nucleotide (i.e., cytosine) and the intercalated arginine of AGT also forces the nucleotide to rotate. Following that, electrostatics and van der Waals contacts as well as hydrogen bonding contribute to form the complex of DNA and protein. The stronger binding of AGT with DNA before methyl transfer creates the suitable condition to transfer methyl adduct from DNA to AGT.

Multiscale simulations reveal the role of PcrA helicase in protecting against spontaneous point mutations in DNA.

Proton transfer across hydrogen bonds in DNA can produce non-canonical nucleobase dimers and is a possible source of single-point mutations when these forms mismatch under replication. Previous computational studies have revealed this process to be energetically feasible for the guanine-cytosine (GC) base pair, but the tautomeric product (G[Formula: see text]C[Formula: see text]) is short-lived. In this work we reveal, for the first time, the direct effect of the replisome enzymes on proton transfer, rectifying the shortcomings of existing models. Multi-scale quantum mechanical/molecular dynamics (QM/MM) simulations reveal the effect of the bacterial PcrA Helicase on the double proton transfer in the GC base pair. It is shown that the local protein environment drastically increases the activation and reaction energies for the double proton transfer, modifying the tautomeric equilibrium. We propose a regime in which the proton transfer is dominated by tunnelling, taking place instantaneously and without atomic rearrangement of the local environment. In this paradigm, we can reconcile the metastable nature of the tautomer and show that ensemble averaging methods obscure detail in the reaction profile. Our results highlight the importance of explicit environmental models and suggest that asparagine N624 serves a secondary function of reducing spontaneous mutations in PcrA Helicase.

Quantitative Structure-Activity Relationship in the Series of 5-Ethyluridine, N2-Guanine, and 6-Oxopurine Derivatives with Pronounced Anti-Herpetic Activity.

A quantitative analysis of the relationship between the structure and inhibitory activity against the herpes simplex virus thymidine kinase (HSV-TK) was performed for the series of 5-ethyluridine, N2-guanine, and 6-oxopurines derivatives with pronounced anti-herpetic activity (IC50 = 0.09 ÷ 160,000 µmol/L) using the GUSAR 2019 software. On the basis of the MNA and QNA descriptors and whole-molecule descriptors using the self-consistent regression, 12 statistically significant consensus models for predicting numerical pIC50 values were constructed. These models demonstrated high predictive accuracy for the training and test sets. Molecular fragments of HSV-1 and HSV-2 TK inhibitors that enhance or diminish the anti-herpetic activity are considered. Virtual screening of the ChEMBL database using the developed QSAR models revealed 42 new effective HSV-1 and HSV-2 TK inhibitors. These compounds are promising for further research. The obtained data open up new opportunities for developing novel effective inhibitors of TK.

Effect of Guanine Adduct Size, Shape, and Linker Type on the Conformation of Adducted DNA: A DFT and Molecular Dynamics Study.

DNA is damaged through various exogenous sources (e.g., automobile exhaust, tobacco smoke, and processed foods), which can yield diverse C8-dG bulky aryl adducts. Adducts are known to induce structural changes to DNA that can lead to various biological outcomes, ranging from cell death to diseases such as cancer. Unfortunately, the relationship between the chemical composition of the damaged product, the adducted DNA structure, and the biological consequences is not well understood, which limits the development of disease detection and prevention strategies. The present study uses density functional theory (DFT) calculations and quintuplicate 1 µs molecular dynamics (MD) simulations to characterize the structure of DNA containing 21 model C8-dG adducts that systematically differ in size (phenyl to pyrenyl), shape (α (2,3), ß (3,4) fusion, or ring substitution), and nucleobase-aryl group linkage (N, O, and C-linked). DFT calculations reveal that the inherent structural features of the G nucleobase adducts are impacted by linker type and bulky moiety shape, but not size, with the conformational flexibility reducing with α-ring fusion and linker composition as N > O > C. These structural properties are maintained in nucleoside models, which also reveal an increased propensity for anti-to-syn rotation about the glycosidic bond with N < O < C linker type. Although these diverse chemical features do not influence the global structure of adducted DNA, the adducts differentially impact the conformation local to the adducted site, including the relative populations of structures with the bulky moiety in the major groove (B conformer) and intercalated (stacked) into the helix (S conformer). Specifically, while the smallest phenyl adducts favor the B conformation and the largest pyrenyl-derived adducts stabilize the S conformation, the B/S ratio decreases with an increase in ring size and N > O > C linker composition. The shape and size (length) of the adduct can further finetune the B/S ratio, with ß-fused naphthyl or α-fused phenanthryl N-linked adducts and O or C-linked adducts containing ring substitution increasing the prevalence of the S adducted DNA conformation. Overall, this work uncovers the significant effect of bulky moiety size and linker type, as well as the lesser impact of aryl group shape, on adducted DNA structure, which suggests differential replication and repair outcomes, and thereby represents an important step toward rationalizing connections between the structure and biological consequences of diverse DNA adducts.