Sugar Pucker Research Articles

A DFT study of energetic and structural properties of a full turn of A-form DNA under relaxed and stretching conditions.

We carried out a first-principles quantum-mechanical study of a set of full-turn A-form DNA oligonucleotides using density functional approximations. The structural features of the fully relaxed model DNA molecules and more importantly, the energetic and structural changes of these molecules under stretching conditions are examined in detail. Special attention is paid to the dependence of the structural properties on the stacking sequences of the constituent DNA base pairs. It appears that DNA oligonucleotides are extremely flexible, and structural properties such as the diameter and the widths of the minor and major grooves change regularly as a function of the stretching rate (stretching length in percentage). Additionally, we found that the length of the hydrogen bonds in the DNA base pairs varies with the DNA stacking sequence. This work indicates that rich information and insights can be gained from first principles studies of model DNA molecules of only O(102-103) atoms.

Conformation-dependent restraints for polynucleotides: the sugar moiety.

Stereochemical restraints are commonly used to aid the refinement of macromolecular structures obtained by experimental methods at lower resolution. The standard restraint library for nucleic acids has not been updated for over two decades and needs revision. In this paper, geometrical restraints for nucleic acids sugars are derived using information from high-resolution crystal structures in the Cambridge Structural Database. In contrast to the existing restraints, this work shows that different parts of the sugar moiety form groups of covalent geometry dependent on various chemical and conformational factors, such as the type of ribose or the attached nucleobase, and ring puckering or rotamers of the glycosidic (χ) or side-chain (γ) torsion angles. Moreover, the geometry of the glycosidic link and the endocyclic ribose bond angles are functionally dependent on χ and sugar pucker amplitude (τm), respectively. The proposed restraints have been positively validated against data from the Nucleic Acid Database, compared with an ultrahigh-resolution Z-DNA structure in the Protein Data Bank, and tested by re-refining hundreds of crystal structures in the Protein Data Bank. The conformation-dependent sugar restraints presented in this work are publicly available in REFMAC, PHENIX and SHELXL format through a dedicated RestraintLib web server with an API function.

Branch site bulge conformations in domain 6 determine functional sugar puckers in group II intron splicing.

Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2′-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3′-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing.

A packing for A-form DNA in an icosahedral virus

Studies on viruses infecting archaea living in the most extreme environments continue to show a remarkable diversity of structures, suggesting that the sampling continues to be very sparse. We have used electron cryo-microscopy to study at 3.7-Å resolution the structure of the Sulfolobus polyhedral virus 1 (SPV1), which was originally isolated from a hot, acidic spring in Beppu, Japan. The 2 capsid proteins with variant single jelly-roll folds form pentamers and hexamers which assemble into a T = 43 icosahedral shell. In contrast to tailed icosahedral double-stranded DNA (dsDNA) viruses infecting bacteria and archaea, and herpesviruses infecting animals and humans, where naked DNA is packed under very high pressure due to the repulsion between adjacent layers of DNA, the circular dsDNA in SPV1 is fully covered with a viral protein forming a nucleoprotein filament with attractive interactions between layers. Most strikingly, we have been able to show that the DNA is in an A-form, as it is in the filamentous viruses infecting hyperthermophilic acidophiles. Previous studies have suggested that DNA is in the B-form in bacteriophages, and our study is a direct visualization of the structure of DNA in an icosahedral virus.

Revealing conformational dynamics of 2′-O-methyl-RNA guanine modified G-quadruplex by replica exchange molecular dynamics

Thrombin-binding DNA aptamer (TBA) can fold into an antiparallel unimolecular G-quadruplex (G4) structure. Different types of modifications lead to various effects on the structure and stability of the G4 structure. Previous study has shown that a modified TBA (mTBA) that 2′-deoxy guanine (dG) at positions 10 and 11 in the TBA sequence were replaced by 2′-O-methyl-RNA guanine (2′OMe-G) can't fold into a well-defined G4 structure. In order to investigate the detailed structural information and probe the instability factors, we successfully employed the replica exchange molecular dynamics (REMD) to characterize the large conformational variations of the mTBA and systemically describe the influences of the 2′OMe-G on the mTBA in terms of conformation variations and the probability distributions of Hoogsteen hydrogen bonds, dihedral, sugar pucker and glycosyl torsion angle. Replacing position 10 with the 2′OMe-G (2′OMe-G10) induced a strong destabilization of the aptamer, while the 2′OMe-G at position 11(2′OMe-G11) was less destabilizing. More importantly, the glycosyl torsion angle and sugar pucker of 2′OMe-G10 were the most critical destabilization factors. These results were in good agreement with the theoretical and experimental results. Moreover, the structure information can be used as guidelines for the further design of modifications on G4 structure.

Molecular Dynamics Study of the Hybridization between RNA and Modified Oligonucleotides.

MicroRNAs (miRNAs) are attractive drug candidates for many diseases as they can modulate the expression of gene networks. Recently, we discovered that DNAs targeting microRNA-22-3p (miR-22-3p) hold the potential for treating obesity and related metabolic disorders (type 2 diabetes mellitus, hyperlipidemia, and nonalcoholic fatty liver disease (NAFLD)) by turning fat-storing white adipocytes into fat-burning adipocytes. In this work, we explored the effects of chemical modifications, including phosphorothioate (PS), locked nucleic acid (LNA), and peptide nucleic acid (PNA), on the structure and energy of DNA analogs by using molecular dynamics (MD) simulations. To achieve a reliable prediction of the hybridization free energy, the AMOEBA polarizable force field and the free energy perturbation technique were employed. The calculated hybridization free energies are generally compatible with previous experiments. For LNA and PNA, the enhanced duplex stability can be explained by the preorganization mechanism, i.e., the single strands adopt stable helical structures similar to those in the duplex. For PS, the S and R isomers (Sp and Rp) have preferences for C2'-endo and C3'-endo sugar puckering conformations, respectively, and therefore Sp is less stable than Rp in DNA/RNA hybrids. In addition, the solvation penalty of Rp accounts for its destabilization effect. PS-LNA is similar to LNA as the sugar puckering is dominated by the locked sugar ring. This work demonstrated that MD simulations with polarizable force fields are useful for the understanding and design of modified nucleic acids.

Structural and Energetic Effects of O2'-Ribose Methylation of Protonated Pyrimidine Nucleosides.

The 2'-substituents distinguish DNA from RNA nucleosides. 2'-O-methylation occurs naturally in RNA and plays important roles in biological processes. Such 2'-modifications may alter the hydrogen-bonding interactions of the nucleoside and thus may affect the conformations of the nucleoside in an RNA chain. Structures of the protonated 2'-O-methylated pyrimidine nucleosides were examined by infrared multiple photon dissociation (IRMPD) action spectroscopy, assisted by electronic structure calculations. The glycosidic bond stabilities of the protonated 2'-O-methylated pyrimidine nucleosides, [Nuom+H]+, were also examined and compared to their DNA and RNA nucleoside analogues via energy-resolved collision-induced dissociation (ER-CID). The preferred sites of protonation of the 2'-O-methylated pyrimidine nucleosides parallel their canonical DNA and RNA nucleoside analogues, [dNuo+H]+ and [Nuo+H]+, yet their nucleobase orientation and sugar puckering differ. The glycosidic bond stabilities of the protonated pyrimidine nucleosides follow the order: [dNuo+H]+ < [Nuo+H]+ < [Nuom+H]+. The slightly altered structures help explain the stabilization induced by 2'-O-methylation of the pyrimidine nucleosides.

Methylation, sugar puckering and Z-form status of DNA from a heavy metal-acclimated freshwater Gordonia sp.

Heavy metal acclimation of bacteria is of particular interest in many aspects. It could add to our understanding of adaptation strategies applied by bacteria, as well as help us in devising ways to use such adaptive bacteria for bioremediation. In this study, we have explored the changes in the DNA of an aquatic Gordonia sp. acclimated to silver, cadmium, and lead. We have measured the changes in the DNA extracted from the acclimated bacteria by using ATR-FTIR coupled with unsupervised and supervised pattern recognition algorithms. Although whole-cell FTIR studies do reveal nucleic acid changes, the special care should be taken when considering marker nucleic acid bands in such spectra, as various other cell or tissue constituents also yield IR bands in the same region. An FTIR study on isolated DNA can be used to avoid this problem. The IR spectral profiles of the DNA molecules revealed significant changes in the backbone and sugar conformations of upon acclimation. We then further analyzed the DNA's global cytosine-methylation patterns of the heavy metal-acclimated bacteria. We aimed to find out whether epigenetic mechanisms operate in bacteria for survival and growth in inhibitory heavy metal concentrations or not. We found hypermethylation in Cd acclimation but hypomethylation for both Pb and Ag in Gordonia sp. Our results imply that changes in the conformational and methylation states of DNA seem to let bacteria to thrive in otherwise inhibitory conditions and mark the involvement of epigenetic modulation in acclimation processes.

Impact of Sodium Cationization on Gas-Phase Conformations of DNA and RNA Cytidine Mononucleotides.

Gas-phase conformations of the sodium-cationized forms of the 2'-deoxycytidine and cytidine mononucleotides, [pdCyd+Na]+ and [pCyd+Na]+, are examined by infrared multiple photon dissociation action spectroscopy. Complimentary electronic structure calculations at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory provide candidate conformations and their respective predicted IR spectra for comparison across the IR fingerprint and hydrogen-stretching regions. Comparisons of the predicted IR spectra and the measured infrared multiple photon dissociation action spectra provide insight into the impact of sodium cationization on intrinsic mononucleotide structure. Further, comparison of present results with those reported for the sodium-cationized cytidine nucleoside analogues elucidates the impact of the phosphate moiety on gas-phase structure. Across the neutral, protonated, and sodium-cationized cytidine mononucleotides, a preference for stabilization of the phosphate moiety and nucleobase orientation is observed, although the details of this stabilization differ with the state of cationization. Several low-energy conformations of [pdCyd+Na]+ and [pCyd+Na]+ involving several different orientations of the phosphate moiety and sugar puckering modes are observed experimentally.

The interplay between molecular flexibility and RNA chemical probing reactivities analyzed at the nucleotide level via an extensive molecular dynamics study.

Determination of the tridimensional structure of ribonucleic acid molecules is fundamental for understanding their function in the cell. A common method to investigate RNA structures of large molecules is the use of chemical probes such as SHAPE (2'-hydroxyl acylation analyzed by primer extension) reagents, DMS (dimethyl sulfate) and CMCT (1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfate), the reaction of which is dependent on the local structural properties of each nucleotide. In order to understand the interplay between local flexibility, sugar pucker, canonical pairing and chemical reactivity of the probes, we performed all-atom molecular dynamics simulations on a set of RNA molecules for which both tridimensional structure and chemical probing data are available and we analyzed the correlations between geometrical parameters and the chemical reactivity. Our study confirms that SHAPE reactivity is guided by the local flexibility of the different chemical moieties but suggests that a combination of multiple parameters is needed to better understand the implications of the reactivity at the molecular level. This is also the case for DMS and CMCT for which the reactivity appears to be more complex than commonly accepted.

Structures and Relative Glycosidic Bond Stabilities of Protonated 2'-Fluoro-Substituted Purine Nucleosides.

The 2'-substituent is the primary distinguishing feature between DNA and RNA nucleosides. Modifications to this critical position, both naturally occurring and synthetic, can produce biologically valuable nucleoside analogues. The unique properties of fluorine make it particularly interesting and medically useful as a synthetic nucleoside modification. In this work, the effects of 2'-fluoro modification on the protonated gas-phase purine nucleosides are examined using complementary tandem mass spectrometry and computational methods. Direct comparisons are made with previous studies on related nucleosides. Infrared multiple photon dissociation action spectroscopy performed in both the fingerprint and hydrogen-stretching regions allows for the determination of the experimentally populated conformations. The populated conformers of protonated 2'-fluoro-2'-deoxyadenosine, [Adofl+H]+, and 2'-fluoro-2'-deoxyguanosine, [Guofl+H]+, are highly parallel to their respective canonical DNA and RNA counterparts. Both N3 and N1 protonation sites are accessed by [Adofl+H]+, stabilizing syn and anti nucleobase orientations, respectively. N7 protonation and anti nucleobase orientation dominates in [Guofl+H]+. Spectroscopically observable intramolecular hydrogen-bonding interactions with fluorine allow more definitive sugar puckering determinations than possible for the canonical systems. [Adofl+H]+ adopts C2'-endo sugar puckering, whereas [Guofl+H]+ adopts both C2'-endo and C3'-endo sugar puckering. Energy-resolved collision-induced dissociation experiments with survival yield analyses provide relative glycosidic bond stabilities. The N-glycosidic bond stabilities of the protonated 2'-fluoro-substituted purine nucleosides are found to exceed those of their canonical analogues. Further, the N-glycosidic bond stability is found to increase with increasing electronegativity of the 2'-substituent, i.e., H < OH < F. The N-glycosidic bond stability is also greater for the adenine nucleoside analogues than the guanine nucleoside analogues.

Long Time-Scale Atomistic Simulations of the Structure and Dynamics of Transcription Factor-DNA Recognition.

Recent years have witnessed an explosion of interest in computational studies of DNA binding proteins, including both coarse-grained and atomistic simulations of transcription factor-DNA recognition, to understand how these transcription factors recognize their binding sites on the DNA with such exquisite specificity. The present study performs microsecond time scale all-atom simulations of the dimeric form of the lactose repressor (LacI), both in the absence of any DNA and in the presence of both specific and nonspecific complexes, considering three different DNA sequences. We examine, specifically, the conformational differences between specific and nonspecific protein-DNA interactions, as well as the behavior of the helix-turn-helix motif of LacI when interacting with the DNA. Our simulations suggest that stable LacI binding occurs primarily to bent A-form DNA, with a loss of LacI conformational entropy and optimization of correlated conformational equilibria across the protein. In addition, binding to the specific operator sequence involves a slightly larger number of stabilizing DNA-protein hydrogen bonds (in comparison to nonspecific complexes), which may account for the experimentally observed specificity for this operator. In doing so, our simulations provide a detailed atomistic description of potential structural drivers for LacI selectivity.

RNA Exclusion Mechanism of the Cytidine Deaminase APOBEC3G

APOBEC3G (A3G) is a potent antiretroviral protein that specifically targets and deaminates cytidine nucleotides to uracil in single stranded DNA (ssDNA) intermediates produced during HIV replication. A3G displays high selectivity for specific polynucleotide contexts targeting 5′‐CC sites for deamination. A3G binds strongly to RNA via the N‐terminal non‐catalytic domain, which is crucial for recruitment of A3G to the virion, yet A3G displays no deamination activity on the cytidines in single stranded RNA. Our recent structure of A3G bound to ssDNA shows the preferred substrate conformation but does not reveal the mechanisms of substrate selection and non‐substrate exclusion. Here, we report NMR and molecular dynamics (MD) simulation based analysis of the interactions between the C‐terminal catalytic domain of A3G (A3Gctd) and multiple substrate and non‐substrate ssDNAs, which are coupled with deamination assays. The extent of interaction with residues located in helix1 and loop1 (T201‐L220) distinguishes substrate ssDNAs from non‐substrate ssDNAs, revealing the difference between catalytic and non‐catalytic ssDNA‐binding modes of A3Gctd. Using fluorine substituted ribonucleotides we show that 2′‐endo sugar pucker of the target cytidine is an essential factor for effective deamination. Furthermore, MD simulations show that ribose cytidine does not stay at catalytic position during 100ns of simulation time whereas deoxy‐ribose cytidine does. Trajectories of the MD simulation suggest that 2′‐hydroxyl group of RNA forms a hydrogen bond with His257 that chelates Zn2+, which results in loss of two hydrogen bonds between the target cytidine and A3Gctd, dislocating cytosine base from the catalytic position. This study provides mechanistic models of the catalytic selectivity of A3G against RNA.Support or Funding InformationInstitute for Molecular Virology Training Program (NIH grant T32 AI83196) University of MinnesotaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of the 2'- and 3'-Sugar Hydroxyl Moieties on Gas-Phase Nucleoside Structure.

Modified nucleosides have been an important target for pharmaceutical development for the treatment of cancer, herpes simplex virus, and the human immunodeficiency virus (HIV). Amongst these nucleoside analogues, those based on 2',3'-dideoxyribose sugars are quite common, particularly in anti-HIV applications. The gas-phase structures of several protonated 2',3'-dideoxyribose nucleosides are examined in this work and compared with those of the analogous protonated DNA, RNA, and arabinose nucleosides to elucidate the influence of the 2'- and combined 2',3'-hydroxyl groups on intrinsic structure. Infrared multiple photon dissociation (IRMPD) action spectra are collected for the protonated 2',3'-dideoxy forms of adenosine, guanosine, cytidine, thymidine and uridine, [ddAdo+H]+, [ddGuo+H]+, [ddCyd+H]+, [ddThd+H]+, and [ddUrd+H]+, in the IR fingerprint and hydrogen-stretching regions. Molecular mechanics conformational searching followed by electronic structure calculations generates low-energy conformers of the protonated 2',3'-dideoxynucleosides and corresponding predicted linear IR spectra to facilitate interpretation of the measured IRMPD action spectra. These experimental IRMPD spectra and theoretical calculations indicate that the absence of the 2'- and 3'-hydroxyls largely preserves the protonation preferences of the canonical forms. The spectra and calculated structures indicate a slight preference for C3'-endo sugar puckering. The presence of the 3'- and further 2'-hydroxyl increases the available intramolecular hydrogen-bonding opportunities and shifts the sugar puckering modes for all nucleosides but the guanosine analogues to a slight preference for C2'-endo over C3'-endo. Graphical Abstract.

Development and Testing of the OPLS-AA/M Force Field for RNA.

Significant improvements have been made to the OPLS-AA force field for modeling RNA. New torsional potentials were optimized based on density functional theory (DFT) scans at the ωB97X-D/6-311++G(d,p) level for potential energy surfaces of the backbone α and γ dihedral angles. In combination with previously reported improvements for the sugar puckering and glycosidic torsion terms, the new force field was validated through diverse molecular dynamics simulations for RNAs in aqueous solution. Results for dinucleotides and tetranucleotides revealed both accurate reproduction of 3 J couplings from NMR and the avoidance of several unphysical states observed with other force fields. Simulations of larger systems with noncanonical motifs showed significant structural improvements over the previous OPLS-AA parameters. The new force field, OPLS-AA/M, is expected to perform competitively with other recent RNA force fields and to be compatible with OPLS-AA models for proteins and small molecules.

A fluorometric study on the effect of DNA methylation on DNA interaction with graphene quantum dots

DNA methylation plays an important role in development process which contributes to genome stability and also regulates gene expression and gene silencing. Detection of genome regions with altered 5-methylcytosine distribution at a genome-wide scale is very important for early detection of gene silencing related diseases. In the present study as a continuation of studies on DNA methylation, the interactions between graphene quantum dots (GQDs) and unmethylated and methylated deoxyribonucleic acid (DNA) fragment were investigated. Based on above interaction a novel GQDs-DNA nanoassembly was developed. Two types of DNA including unmethylated and methylated sequences were interacted with GQDs and contributed to the formation of unmethylated and methylated nanoassemlies. Analysis of the interaction indicated that the GQDs could bind to DNA fragments and led to different fluorescence pattern in two different mechanisms and could provide an efficient biosensing platform for label free and sensitive fluorescent assay of DNA. The excitation and emission wavelengths of experiment were 380 and 480 nm respectively. Fluorescence intensity of unmethylated DNA concentration were detectable from methylated DNA in linear range from 10.0−10M to 10.0–6M and the detection limit was estimated at 7.3 × 10–11 M. Above interaction was not observed in methylated DNA, indicated of distinguished interaction effect. Herein we further showed that GQDs could induce B-DNA to A-DNA form in methylated structure of DNA. The methylation sensitivity of the experiment was also testified by methylation sensitive restriction process. It was assumed that the involvement of methylation alteration in DNA structure could alter not only mechanism of DNA/GDQs interaction but also helical structure of DNA.

Gas-phase structures of protonated arabino nucleosides

Nucleoside modification plays an important role in the native function of DNA and RNA and is also an important synthetic tool for pharmaceuticals. The gas-phase structures of several protonated arabino nucleosides, an important family of modified nucleoside pharmaceuticals, are examined in this work. Infrared multiple photon dissociation action spectra are collected for the protonated forms of the adenine, cytosine, guanine, and uracil arabinosides ([araAdo+H]+, [araCyd+H]+, [araGuo+H]+, and [araUrd+H]+) in the IR fingerprint and hydrogen-stretching regions. Electronic structure calculations are performed to determine low-energy conformers of [araAdo+H]+, [araCyd+H]+, [araGuo+H]+, and [araUrd+H]+, and generate predicted IR spectra for comparison to experiments. Conformers displaying a unique O2′H⋯O5′ hydrogen-bonding interaction are populated in each of these systems. Conformers exhibiting hydrogen-bonding interactions between the nucleobase and O5′ are also found to display good agreement with the measured spectra. Competition between sugar–sugar, and nucleobase–sugar hydrogen bonding reveals a preference for [araCyd+H]+, [araGuo+H]+, and [araUrd+H]+ to stabilize the sugar ring pucker over the nucleobase rotation. N3 protonation of [araAdo+H]+ provides a very strong N3H+⋯O5′ hydrogen-bonding interaction, such that nucleobase–sugar hydrogen-bonding takes energetic preference over stabilization of the sugar puckering. However, conformers exhibiting each mode of hydrogen bonding contribute to the measured spectrum of [araAdo+H]+.

Environmentally benign pH-responsive cytidine-5′-monophosphate molecule-mediated akaganeite (5′-CMP-β-FeOOH) soft supramolecular hydrogels induced by the puckering of ribose sugar with efficient loading/release capabilities

A novel synthetic protocol for environmentally benign 5′-CMP-β-FeOOH soft hydrogels exhibiting a rapid pH-responsive reversible sol–gel transition, efficient adsorption and slow release capabilities is reported.

Influence of 2′-fluoro modification on glycosidic bond stabilities and gas-phase ion structures of protonated pyrimidine nucleosides

The effects of 2′-fluoro substitution on the protonated gas-phase ions of the pyrimidine nucleosides are examined and compared with their previously reported canonical RNA and DNA forms. N-Glycosidic bond cleavage is the only collision-induced dissociation (CID) fragmentation pathway of protonated 2′-fluoro-2′-deoxycytidine, [Cydfl+H]+, and the major pathway of protonated 2′-fluoro-2′-deoxyuridine, [Urdfl+H]+. Based on energy-resolved CID and survival yield analysis, the N-glycosidic bond of [Cydfl+H]+ is more stable than that of [Urdfl+H]+. Further, the N-glycosidic bond stability of protonated pyrimidine nucleosides increases with increasing 2′-substituent electronegativity and follows the order F > OH > H. Gas-phase conformations of [Cydfl+H]+ and [Urdfl+H]+ are studied via infrared multiple photon dissociation (IRMPD) action spectroscopy coupled with theoretical calculations. IRMPD action spectra are measured over the IR fingerprint and hydrogen-stretching regions. Comparisons of theoretical and experimental spectra indicate that the experimentally accessed [Cydfl+H]+ and [Urdfl+H]+ conformers are highly parallel to those populated for their canonical counterparts. Evidence for gas-phase intramolecular O3′H⋯F2′ hydrogen-bonding in the IRMPD spectra of [Cydfl+H]+ and [Urdfl+H]+ allows for more definitive sugar puckering determinations than possible in the analogous canonical nucleosides.

Computational Probing of Watson-Crick/Hoogsteen Breathing in a DNA Duplex Containing N1-Methylated Adenine.

DNA breathing is a local conformational fluctuation spontaneously occurring in double-stranded DNAs. In particular, the possibility of individual base pairs (bps) in duplex DNA to flip between alternate bp modes, i.e., Watson-Crick (WC)-like and Hoogsteen (HG)-like, at relevant time scales has impacted DNA research fields for many years. In this study, to computationally probe effects of chemical modification on the DNA breathing, we present a free energy landscape of spontaneous thermal transitions between WC and HG bps in a free DNA duplex containing N1-methylated adenine (m1A). For the current free energy computation, a variant of well-tempered metadynamics simulation was extensively performed for a total of 40 μs to produce free energy surfaces. The free energy profile indicated that, upon the chemical modification of adenine, the HG bp (m1A·T) was located in the most favorable conformation (96.7%); however, the canonical WC bp (m1A·T) was distorted into two WC-like bps of WC* (2.8%) and WC** (0.5%). The conformational exchange between these two minor WC-like bps occurs with the first hundred nanoseconds. The transition between WC-like and HG bp features multiple transition pathways displaying various extents of base flipping in combination with glycosidic rotation. Analysis of the simulated ensemble showed that the m1A-induced changes of the backbone and sugar pucker were in a reasonable agreement with previous results inferred from NMR experiments. Also, this study revealed that the formation of the stable HG bp upon the mutation alters the characteristics of dynamic fluctuations of the neighboring WC residues of m1A. We expect this simulation approach to be a robust computational scheme to complement and guide future high-resolution experiments on many outstanding issues of duplex DNA breathing.