Electronic and Nuclear Quantum Effects on Proton Transfer Reactions of Guanine-Thymine (G-T) Mispairs Using Combined Quantum Mechanical/Molecular Mechanical and Machine Learning Potentials.

Rare tautomeric forms of nucleobases can lead to Watson-Crick-like (WC-like) mispairs in DNA, but the process of proton transfer is fast and difficult to detect experimentally. NMR studies show evidence for the existence of short-time WC-like guanine-thymine (G-T) mispairs; however, the mechanism of proton transfer and the degree to which nuclear quantum effects play a role are unclear. We use a B-DNA helix exhibiting a wGT mispair as a model system to study tautomerization reactions. We perform ab initio (PBE0/6-31G*) quantum mechanical/molecular mechanical (QM/MM) simulations to examine the free energy surface for tautomerization. We demonstrate that while the ab initio QM/MM simulations are accurate, considerable sampling is required to achieve high precision in the free energy barriers. To address this problem, we develop a QM/MM machine learning potential correction (QM/MM-ΔMLP) that is able to improve the computational efficiency, greatly extend the accessible time scales of the simulations, and enable practical application of path integral molecular dynamics to examine nuclear quantum effects. We find that the inclusion of nuclear quantum effects has only a modest effect on the mechanistic pathway but leads to a considerable lowering of the free energy barrier for the GT*⇌G*T equilibrium. Our results enable a rationalization of observed experimental data and the prediction of populations of rare tautomeric forms of nucleobases and rates of their interconversion in B-DNA.

Comprehensive analysis of the editing window of C-to-T TALE base editors.

One of the most recent advances in the genome editing field has been the addition of "TALE Base Editors", an innovative platform for cell therapy that relies on the deamination of cytidines within double strand DNA, leading to the formation of an uracil (U) intermediate. These molecular tools are fusions of transcription activator-like effector domains (TALE) for specific DNA sequence binding, split-DddA deaminase halves that will, upon catalytic domain reconstitution, initiate the conversion of a cytosine (C) to a thymine (T), and an uracil glycosylase inhibitor (UGI). We developed a high throughput screening strategy capable to probe key editing parameters in a precisely defined genomic context in cellulo, excluding or minimizing biases arising from different microenvironmental and/or epigenetic contexts. Here we aimed to further explore how target composition and TALEB architecture will impact the editing outcomes. We demonstrated how the nature of the linker between TALE array and split DddAtox head allows us to fine tune the editing window, also controlling possible bystander activity. Furthermore, we showed that both the TALEB architecture and spacer length separating the two TALE DNA binding regions impact the target TC editing dependence by the surrounding bases, leading to more restrictive or permissive editing profiles.

Novel Nucleic Acid-Assisted Ion-Responsive ECL Biosensor Based on Hollow AuAg Nanoboxes with Excellent SPR and Effective Coreaction Acceleration.

Novel hollow AuAg nanoboxes (AuAg NBs) were designed for an innovative electrochemiluminescence (ECL) sensor to ultrasensitively detect Pb2+ and Hg2+ with the aid of DNAzyme and "thymine-Hg2+-thymine" ("T-Hg2+-T") structure. AuAg NBs are employed as an excellent surface plasma resonance (SPR) source, as well as an effective coreaction accelerator for the CoNi NFs/S2O82- system to greatly improve ECL performance. To detect Pb2+, the DNAzyme catalyzes the cleavage of ribonucleic acid targets into numerous small nucleic acid fragments, leading to an ECL signal. When Hg2+ is added, the thymine-thymine (T-T) mismatches of the Hg2+ aptamer bind Hg2+ to form the "T-Hg2+-T" structure, which not only inhibits the SPR process but also produces a large steric hindrance, thus quenching the ECL signal and allowing quantification of Hg2+. The novel ECL sensor quantifies Pb2+ in the range of 0.1 fM to 0.1 µM with a limit of detection of 0.07 fM and Hg2+ in the range of 10 pM to 1 µM with a LOD of 4.07 pM.

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.

Isotope-Edited Variable Temperature Infrared Spectroscopy for Measuring Transition Temperatures of Single A-T Watson-Crick Base Pairs in DNA Duplexes.

Experimental methods to determine transition temperatures for individual base pair melting events in DNA duplexes are lacking despite intense interest in these thermodynamic parameters. Here, we determine the dimensions of the thymine (T) C2═O stretching vibration when it is within the DNA duplex via isotopic substitutions at other atomic positions in the structure. First, we determined that this stretching state was localized enough to specific atoms in the molecule to make submolecular scale measurements of local structure and stability in high molecular weight complexes. Next, we develop a new isotope-edited variable temperature infrared method to measure melting transitions at various locations in a DNA structure. As an initial test of this "sub-molecular scale thermometer", we applied our T13C2 difference infrared signal to measure location-dependent melting temperatures (TmL) in a DNA duplex via variable temperature attenuated total reflectance Fourier transform infrared (VT-ATR-FTIR) spectroscopy. We report that the TmL of a single Watson-Crick A-T base pair near the end of an A-T rich sequence (poly T) is ∼34.9 ± 0.7°C. This is slightly lower than the TmL of a single base pair near the middle position of the poly T sequence (TmL ∼35.6±0.2°C). In addition, we also report that the TmL of a single Watson-Crick A-T base pair near the end of a 50% G-C sequence (12-mer) is ∼52.5 ± 0.3°C, which is slightly lower than the global melting Tm of the 12-mer sequence (TmL ∼54.0±0.9°C). Our results provide direct physical evidence for end fraying in DNA sequences with our novel spectroscopic methods.

Gene identification and enzymatic characterization of the initial enzyme in pyrimidine oxidative metabolism, uracil-thymine dehydrogenase.

Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP+ or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD+, NADP+, flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors.

Ultrafast and Multichannel Generation of the Triplet State in DNA-Intercalated Gilvocarcin V for an Enhanced Photoaddition Reaction.

Gilvocarcin V (GV) is a natural antibiotic exhibiting excellent antitumor activities and remarkably low toxicity in near-ultraviolet or visible light-dependent treatment. Notwithstanding, the [2 + 2] cycloaddition reaction between GV and thymine has been proven to be the key for its function in photodynamic therapy, and crucial mechanistic details about such a reaction are poorly understood. In this study, the electronic relaxation pathways and photoaddition reaction are characterized by femto- to nanosecond time-resolved spectroscopy combined with quantum chemical calculation. Our results reveal that ultrafast intersystem crossing (<3 ps) leads to the population of a local triplet excited state in DNA-intercalated GV. Such a state can further induce the formation of a biradical state, which is identified as the important reactive precursor for photoaddition between GV and thymine. The overall photoaddition quantum efficiency is determined to be 11.57 ± 1.0%. These results are essential to the elucidation of the DNA photoaddition mechanism of C-aryl glycoside-based artificial photocytotoxic agents and could help further development of those medicines.

Label-free fluorescence detection of mercury ions based on thymine-mercury-thymine structure and CRISPR-Cas12a.

In this work, we developed a novel label-free fluorescent sensor for the highly sensitive detection of mercury ions (Hg2+) based on the coordination chemistry of thymine-Hg2+-thymine (T-Hg2+-T) structures and the properties of CRISPR-Cas12a systems. Most notably, two T-rich sequences (a blocker and an activator) were designed to form stable double-stranded structures in the presence of Hg2+ via the T-Hg2+-T base pairing. The formation of T-T mismatched double-stranded DNA between the blocker and the activator prevented the cleavage of G-rich sequences by Cas12a, allowing them to fold into G-quadruplex-thioflavin T complexes, resulting in significantly enhanced fluorescence. Under the optimized conditions, the developed sensor showed an excellent response for Hg2+ detection in the linear range of 0.05 to 200 nM with a detection limit of 23 pM. Moreover, this fluorescent sensor exhibited excellent selectivity and was successfully used for the detection of Hg2+ in real samples of Zhujiang river water and tangerine peel, demonstrating its potential in environmental monitoring and food safety applications.

Electron interaction with DNA constituents in aqueous phase.

Electron driven chemistry of biomolecules in aqueous phase presents the realistic picture to study molecular processes. In this study we have investigated the interactions of electrons with the DNA constituents in their aqueous phase in order to obtain the quantities useful for DNA damage assessment. We have computed the inelastic mean free path (IMFP), mass stopping power (MSP) and absorbed dose (D) for the DNA constituents (Adenine, Cytosine, Guanine, Thymine and Uracil) in the aqueous medium from ionisation threshold to 5000 eV. We have modified complex optical potential formalism to include band gap of the systems to calculate inelastic cross sections which are used to estimate these entities. This is the maiden attempt to report these important quantities for the aqueous DNA constituents. We have compared our results with available data in gas and other phase and have observed explicable accord for IMFP and MSP. Since these are the first results of absorbed dose (D) for these compounds, we have explored present results vis-a-vis dose absorption in water.

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.

Harnessing non-Watson-Crick's base pairing to enhance CRISPR effectors cleavage activities and enable gene editing in mammalian cells.

Genomic DNA of the cyanophage S-2L virus is composed of 2-aminoadenine (Z), thymine (T), guanine (G), and cytosine (C), forming the genetic alphabet ZTGC, which violates Watson-Crick base pairing rules. The Z-base has an extra amino group on the two position that allows the formation of a third hydrogen bond with thymine in DNA strands. Here, we explored and expanded applications of this non-Watson-Crick base pairing in protein expression and gene editing. Both ZTGC-DNA (Z-DNA) and ZUGC-RNA (Z-RNA) produced in vitro show detectable compatibility and can be decoded in mammalian cells, including Homo sapiens cells. Z-crRNA can guide CRISPR-effectors SpCas9 and LbCas12a to cleave specific DNA through non-Watson-Crick base pairing and boost cleavage activities compared to A-crRNA. Z-crRNA can also allow for efficient gene and base editing in human cells. Together, our results help pave the way for potential strategies for optimizing DNA or RNA payloads for gene editing therapeutics and give insights to understanding the natural Z-DNA genome.

Photoelectrochemical Sensor with a Z-Scheme Fe2O3/CdS Heterostructure for Sensitive Detection of Mercury Ions.

Mercury (Hg2+) is a highly toxic element and can seriously affect human health. This work proposed a photoelectrochemical (PEC) sensor with a Z-scheme Fe2O3/CdS heterostructure and two thymine-rich DNA strands (DNA-1 and Au@DNA-2) for sensitive detection of Hg2+. The light excitation of the Fe2O3/CdS composite accelerated the electron transfer among Fe2O3, CdS, and the electrode to produce a stable photocurrent response. Upon the recognition of Hg2+ to thymine bases (T) in two DNA strands to form a stable T-Hg2+-T biomimetic structure, the photocurrent response increased with the increasing concentration of Hg2+ due to the opening of electronic transmission channels from Au nanoparticles to Fe2O3/CdS nanocomposite. Under the optimal conditions screened by the Box-Behnken experiments, the proposed PEC sensor showed excellent analytical performance for Hg2+ detection with high sensitivity, a detection limit of 0.20 pM at a signal-to-noise ratio of 3, high selectivity, a detectable concentration range of 1 pM-100 nM, and acceptable stability. The good recovery and low relative standard deviation for the analysis of Hg2+ in lake and tap water samples demonstrated the potential application of the designed Z-scheme Fe2O3/CdS heterostructure in the PEC detection of heavy metal ions.

Short-Range Charge Transfer in DNA Base Triplets: Real-Time Tracking of Coherent Fluctuation Electron Transfer.

The short-range charge transfer of DNA base triplets has wide application prospects in bioelectronic devices for identifying DNA bases and clinical diagnostics, and the key to its development is to understand the mechanisms of short-range electron dynamics. However, tracing how electrons are transferred during the short-range charge transfer of DNA base triplets remains a great challenge. Here, by means of ab initio molecular dynamics and Ehrenfest dynamics, the nuclear-electron interaction in the thymine-adenine-thymine (TAT) charge transfer process is successfully simulated. The results show that the electron transfer of TAT has an oscillating phenomenon with a period of 10 fs. The charge density difference proves that the charge transfer proportion is as high as 59.817% at 50 fs. The peak position of the hydrogen bond fluctuates regularly between -0.040 and -0.056. The time-dependent Marcus-Levich-Jortner theory proves that the vibrational coupling between nucleus and electron induces coherent electron transfer in TAT. This work provides a real-time demonstration of the short-range coherent electron transfer of DNA base triplets and establishes a theoretical basis for the design and development of novel biological probe molecules.

Highly sensitive surface acoustic wave biosensor for the detection of Hg2+ based on the thymine-Hg2+-thymine structure.

The detection of mercury ions (Hg2+) with an ultralow concentration is of great significance for the treatment of heavy metal pollution in industrial waste water and the monitoring of drinking water quality. In this study, a simple, sensitive, and portable surface acoustic wave (SAW) biosensor for the determination of Hg2+ concentration was developed. First, a DNA fragment with a specific binding capacity for Hg2+ was chemically adsorbed on to the sensitive region through forming Au-S bonds with the SAW biosensor. The DNA probe could then form a thymine-Hg2+-thymine (T-Hg2+-T) complex with strong affinity for Hg2+ after immersion in a test solution containing Hg2+, resulting in a significant change in the response frequency of the device. The linear detection range of the device was 10 pM to 1 nM, while the limit of detection (LOD) was as low as 6.3 pM. Furthermore, the SAW biosensor exhibited excellent selectivity to Hg2+ compared with that of interfering ions, e.g., Ag+, Ba2+, Cu2+, Zn2+, Mn2+, Fe3+, Ca2+, and Na+. The results provide a new strategy for the preparation of portable devices that can monitor toxic heavy metal ions with high sensitivity and selectivity.

Register-Shifted Structures in Base-Flipped Uracil-Damaged DNA.

We report the occurrence of register-shifted structures in simulations of uracil-containing dsDNA. These occur when the 3' base vicinal to uracil is thymine in U:A base-paired DNA. Upon base flipping of uracil, this 3' thymine hydrogen bonds with the adenine across the uracil instead of its complementary base. The register-shifted structure is persistent and sterically blocks re-entry of uracil into the helix stack. Register shifting might be important for DNA repair since the longer exposure of the lesion in register-shifted structures could facilitate enzymatic recognition and repair.

Reductive Photocycloreversion of Cyclobutane Dimers Triggered by Guanines.

The quest for simple systems achieving the photoreductive splitting of four-membered ring compounds is a matter of interest not only in organic chemistry but also in biochemistry to mimic the activity of DNA photorepair enzymes. In this context, 8-oxoguanine, the main oxidatively generated lesion of guanine, has been shown to act as an intrinsic photoreductant by transferring an electron to bipyrimidine lesions and provoking their cycloreversion. But, in spite of appropriate photoredox properties, the capacity of guanine to repair cyclobutane pyrimidine dimer is not clearly established. Here, dyads containing the cyclobutane thymine dimer and guanine or 8-oxoguanine are synthesized, and their photoreactivities are compared. In both cases, the splitting of the ring takes place, leading to the formation of thymine, with a quantum yield 3.5 times lower than that for the guanine derivative. This result is in agreement with the more favored thermodynamics determined for the oxidized lesion. In addition, quantum chemistry calculations and molecular dynamics simulations are carried out to rationalize the crucial aspects of the overall cyclobutane thymine dimer photoreductive repair triggered by the nucleobase and its main lesion.

Thymine-Modified Silicones: A Bioinspired Approach to Cross-Linked, Recyclable Silicone Polymers.

In DNA, thymine typically forms hydrogen bonds with adenine to hold two complementary strands together and to preserve the genetic code. While thymine is typically absent in RNA, a thymine-thymine hydrogen bonding structure is reminiscent of the wobble region in tRNA recognition, where noncanonical base pairing can occur. This noncanonical base pairing can be applied to synthetic polymer systems, where thymine is free to hydrogen bond with itself. In this work, the natural hydrogen bonding capacity of thymine was used to produce silicone polymer systems designed to be cross-linked by hydrogen bonds. Backbone and end-group-modified silicones were synthesized with differing concentrations of thymine, which facilitated the cross-linking of the polymeric strands. Removing the hydrogen on N3─which is typically involved in hydrogen bonding─resulted in systems with similar viscosities to the starting material and that were devoid of any apparent cross-links. Differential scanning calorimetry (DSC) studies of the thymine-modified polymers displayed thermal absorptions and releases, indicative of bond breaking and reformation, around 100 and 60 °C, respectively. The cycle of bond breaking and formation could be repeated without any noticeable degradation of the chemical structure of the polymers. These polymeric materials could be readily recycled and remolded by heating them at 110 °C for 5 min, followed by cooling to room temperature, confirming their thermoplastic nature.

Mechanisms and energetics of benzophenone photosensitized thymine damage and repair from Paternò-Büchi cycloaddition.

Here, we report the detailed mechanisms of benzophenone (BZP) photosensitized thymine damage and repair by Paternò-Büchi (PB) cycloaddition. It was found that the head-to-head and head-to-tail PB cycloadditions lead to the formation of the C-O bonds in the 3(nπ*) state and the 3(ππ*) state, respectively. The conical intersection occurs before the head-to-tail C-O bonding. Then, the C-C bonds are formed via intersystem crossing (ISC). The C-O bonding is the rate-determining step of PB cycloaddition. For the cycloreversion reactions, the ring-opening processes completely occur in the singlet excited states of oxetanes. The head-to-head oxetane goes through a conical intersection before cycloreversion with a little energy barrier of 1.8 kcal mol-1. The head-to-tail oxetane splits without a barrier. Then, the ISC processes take place to restore thymine. Throughout the ring-closing and ring-opening processes, ISC plays an important role. These findings are in good agreement with the available experimental findings. We hope that this comprehensive work can provide a deeper understanding of photosensitive DNA damage and repair.

Enhancement of the thermal stability of G-quadruplex structures by urea.

The solute urea has been used extensively as a denaturant in protein folding studies; double-stranded nucleic acid structures are also destabilized by urea, but comparatively less than proteins. In previous research, the solute has been shown to strongly destabilize folded G-quadruplex DNA structures. This contribution demonstrates the stabilizing effect of urea on the G-quadruplex formed by the oligodeoxyribonucleotide (ODN), G3T (d[5'-GGGTGGGTGGGTGGG-3']), and related sequences in the presence of sodium or potassium cations. Stabilization is observed up to 7 M urea, which was the highest concentration we investigated. The folded structure of G3T has three G-tetrads and three loops that consist of single thymine residues. ODNs related to G3T, in which the thymine residues in the loop are substituted by adenosine residues, also exhibit enhanced stability in the presence of molar concentrations of urea. The circular dichroism (CD) spectra of these ODNs in the presence of urea are consistent with that of a G-quadruplex. As the urea concentration increases, the spectral intensities of the peaks and troughs change, while their positions change very little. The heat-induced transition from the folded to unfolded state, Tm, was measured by monitoring the change in the UV absorption as a function of temperature. G-quadruplex structures with loops containing single bases exhibited large increases in Tm with increasing urea concentrations. These data imply that the loop region play a significant role in the thermal stability of tetra-helical DNA structures in the presence of the solute urea.

Coupling of conformation and CPD damage in nucleosomal DNA.

UV-light can cause photodimerization and hence damages in DNA. Most frequent are cyclobutane pyrimidine dimer (CPD) damages, which predominantly form at TpT (thymine-thymine) steps. It is well known that CPD damage probability is different for single-stranded or double stranded DNA and depends on the sequence context. However, DNA deformation due to packing in nucleosomes can also influence CPD formation. Quantum mechanical calculations and Molecular Dynamics simulations indicate little CPD damage probability for DNA's equilibrium structure. We find that DNA needs to be deformed in a specific way to allow the HOMO → LUMO transition required for CPD damage formation. The simulation studies further show that the periodic CPD damage patterns measured in chromosomes and nucleosomes can be directly explained by the periodic deformation pattern of the DNA in the nucleosome complex. It supports previous findings on characteristic deformation patterns found in experimental nucleosome structures that relate to CPD damage formation. The result may have important implications for our understanding of UV-induced DNA mutations in human cancers.