Pre-mixing of omega-3 fatty acid-containing liposomes enhances the drug release rate and therapeutic efficacy of anticancer drugs loaded in liposomes.

The therapeutic efficacy of anticancer drugs loaded in liposomes composed of rigid phosphatidylcholine (PC) is hindered by the limited release of these drugs at the tumor site, which in turn hampers delivery of the drug to its intracellular target. In an attempt to improve the therapeutic efficacy of liposomal anticancer drugs, we here explored the use of empty liposomes as "trigger" vehicles to induce drug release from drug-loaded liposomes through liposome-liposome interactions. Empty liposomes containing PC in which omega-3 fatty acids comprised both fatty acid strands (Omega-L) showed a triggering effect on drug release from doxorubicin (DOX)-loaded liposomes (Caelyx). The effectiveness of this triggered-release effect was dependent on the Omega-L composition as well as the mixing ratio of Omega-L to Caelyx. Cryo-TEM and differential calorimetry studies revealed that the Omega-L effect was associated with liposome-liposome interactions that led to loosened membrane packing and increased fluidity of Caelyx. In cultured cells, the intracellular/intranuclear DOX uptake and anticancer efficacy of Caelyx was greatly improved by Omega-L pre-mixing. Intravenous injection of rats with Caelyx, premixed with Omega-L, decreased the area under the plasma concentration-time curve from time zero to time infinity and increased clearance without significantly changing the mean residence time or terminal half-life of DOX compared with Caelyx alone. Ex vivo bioimaging showed that DOX fluorescence in tumors, but not in other organs, was significantly increased by Omega-L premixing. In the mouse xenograft model, premixing of Omega-L with Caelyx suppressed tumor growth 2.5-fold compared with Caelyx. Collectively, the data provide preliminary evidence that the Omega-L-triggered drug release that occurs before and after dosing, particularly at tumor site, improved the therapeutic efficacy of Caelyx. The simple approach described here could enhance the therapeutic value of Caelyx and other anticancer drug-loaded liposomes.

Effects of Isosakuranetin on Pharmacokinetic Changes of Tofacitinib in Rats with N-Dimethylnitrosamine-Induced Liver Cirrhosis.

Tofacitinib, a Janus kinase 1 and 3 inhibitor, is used to treat rheumatoid arthritis. It is mainly metabolized by the cytochromes p450 (CYP) 3A1/2 and CYP2C11 in the liver. Chronic inflammation eventually leads to cirrhosis in patients with rheumatoid arthritis. Isosakuranetin (ISN), a component of Citrus aurantium L., has hepatoprotective effects in rats. This study was performed to determine the effects of ISN on the pharmacokinetics of tofacitinib in rats with N-dimethylnitrosamine-induced liver cirrhosis (LC). After intravenous administration of 10 mg/kg tofacitinib to control (CON), LC, and LC treated with ISN (LC-ISN) rats, the total area under the plasma concentration-time curves (AUC) from time zero to infinity increased by 158% in LC rats compared to those in CON rats; however, the AUC of LC-ISN rats decreased by 35.1% compared to that of LC rat. Similar patterns of AUC changes were observed in the LC and LC-ISN rats after oral administration of 20 mg/kg tofacitinib. These results can be attributed to decreased non-renal clearance (CLNR) and intestinal intrinsic clearance (CLint) in the LC rats and increased intestinal and hepatic CLint in the LC-ISN rats. Our findings imply that ISN treatment in LC rats restored the decrease in either CLNR or CLint, or both, through increased hepatic and intestinal expression of CYP3A1/2 and CYP2C11, which is regulated by the induction of pregnane X receptor (PXR) and constitutive androstane receptor (CAR).

Effects of Dextran Sulfate Sodium-Induced Ulcerative Colitis on the Disposition of Tofacitinib in Rats.

Tofacitinib, a Janus kinase 1 and 3 inhibitor, is mainly metabolized by CYP3A1/2 and CYP2C11 in the liver. The drug has been approved for the chronic treatment of severe ulcerative colitis, a chronic inflammatory bowel disease. This study investigated the pharmacokinetics of tofacitinib in rats with dextran sulfate sodium (DSS)-induced ulcerative colitis. After 1-min of intravenous infusion of tofacitinib (10 mg/kg), the area under the plasma concentration-time curves from time zero to time infinity (AUC) of tofacitinib significantly increased by 92.3%. The time-averaged total body clearance decreased significantly by 47.7% in DSS rats compared with control rats. After the oral administration of tofacitinib (20 mg/kg), the AUC increased by 85.5% in DSS rats. These results could be due to decreased intrinsic clearance of the drug caused by the reduction of CYP3A1/2 and CYP2C11 in the liver and intestine of DSS rats. In conclusion, ulcerative colitis inhibited CYP3A1/2 and CYP2C11 in the liver and intestines of DSS rats and slowed the metabolism of tofacitinib, resulting in increased plasma concentrations of tofacitinib in DSS rats.

The role of fluorodeoxyglucose-PET/computed tomography as a predictor of breast cancer characteristics and prognosis.

PURPOSE: Fluorodeoxyglucose-PET/computed tomography (FDG-PET/CT) affects the management of patients with breast cancer. Our study aimed to determine the predictive ability of characteristics such as lymph node involvement or subtype and the prognostic value of pretreatment FDG-PET/CT in breast cancer. METHOD: A total of 270 patients who were confirmed with breast cancer histopathologically and underwent pretreatment FDG-PET/CT were enrolled in the study. Nuclear medicine specialists obtained the readings and measured the maximum standardized uptake value (SUVmax) of the images. Tumor and lymph node SUVmax were evaluated according to lymph node metastasis and subtype status. Survival outcomes were analyzed by the Kaplan-Meier method. RESULTS: The lymph node SUVmax and the lymph node/tumor SUVmax ratio were significantly higher in the subgroup of patients with lymph node metastasis than in those without lymph node metastasis. High cutoff lymph node SUVmax value and lymph node/tumor SUVmax ratio were confirmed as significant predictive factors in multivariate analysis. In a comparison of the tumor SUVmax values, the more biological aggressive subtype showed higher tumor SUVmax values. In survival analysis, tumor SUVmax and lymph node SUVmax were significant predisposing factors for disease-free survival in breast cancer. In subgroup analysis, tumor SUVmax was a more significant prognostic factor in patients who had breast cancer with tumor sizes of ≤2 cm. The lymph node SUVmax was more a significant prognostic factor in patients who had breast cancer with lymph node metastasis. CONCLUSION: In this study, we showed that the SUVmax of FDG-PET/CT was a useful predictor of lymph node metastasis and breast cancer prognosis.

Slower Elimination of Tofacitinib in Acute Renal Failure Rat Models: Contribution of Hepatic Metabolism and Renal Excretion.

Tofacitinib is a Jak inhibitor developed as a treatment for rheumatoid arthritis. Tofacitinib is metabolized mainly through hepatic CYP3A1/2, followed by CYP2C11. Rheumatoid arthritis tends to increase renal toxicity due to drugs used for long-term treatment. In this study, pharmacokinetic changes of tofacitinib were evaluated in rats with gentamicin (G-ARF) and cisplatin-induced acute renal failure (C-ARF). The time-averaged total body clearance (CL) of tofacitinib in G-ARF and C-ARF rats after 1-min intravenous infusion of 10 mg/kg was significantly decreased by 37.7 and 62.3%, respectively, compared to in control rats. This seems to be because the time-averaged renal clearance (CLR) was significantly lower by 69.5 and 98.6%, respectively, due to decreased creatinine clearance (CLCR). In addition, the time-averaged nonrenal clearance (CLNR) was also significantly lower by 33.2 and 57.4%, respectively, due to reduction in the hepatic CYP3A1/2 and CYP2C11 subfamily in G-ARF and C-ARF rats. After oral administration of tofacitinib (20 mg/kg) to G-ARF and C-ARF rats, both CLR and CLNR were also significantly decreased. In conclusion, an increase in area under plasma concentration-time curves from time zero to time infinity (AUC) of tofacitinib in G-ARF and C-ARF rats was due to the significantly slower elimination of tofacitinib contributed by slower hepatic metabolism and urinary excretion of the drug.

Imidazole Antifungal Drugs Inhibit the Cell Proliferation and Invasion of Human Breast Cancer Cells.

Breast cancer is currently the most prevalent cancer in women, and its incidence increases every year. Azole antifungal drugs were recently found to have antitumor efficacy in several cancer types. They contain an imidazole (clotrimazole and ketoconazole) or a triazole (fluconazole and itraconazole) ring. Using human breast adenocarcinoma cells (MCF-7 and MDA-MB-231), we evaluated the effects of azole drugs on cell proliferation, apoptosis, cell cycle, migration, and invasion, and investigated the underlying mechanisms. Clotrimazole and ketoconazole inhibited the proliferation of both cell lines while fluconazole and itraconazole did not. In addition, clotrimazole and ketoconazole inhibited the motility of MDA-MB-231 cells and induced G1-phase arrest in MCF-7 and MDA-MB-231 cells, as determined by cell cycle analysis and immunoblot data. Moreover, Transwell invasion and gelatin zymography assays revealed that clotrimazole and ketoconazole suppressed invasiveness through the inhibition of matrix metalloproteinase 9 in MDA-MB-231 cells, although no significant changes in invasiveness were observed in MCF-7 cells. There were no significant changes in any of the observed parameters with fluconazole or itraconazole treatment in either breast cancer cell line. Taken together, imidazole antifungal drugs showed strong antitumor activity in breast cancer cells through induction of apoptosis and G1 arrest in both MCF-7 and MDA-MB-231 cells and suppression of invasiveness via matrix metalloproteinase 9 inhibition in MDA-MB-231 cells. Imidazole drugs have well-established pharmacokinetic profiles and known toxicity, which can make these generic drugs strong candidates for repositioning as antitumor therapies.

Ligand-Mediated Folding of the OmpA Periplasmic Domain from Acinetobacter baumannii.

The periplasmic domain of OmpA from Acinetobacter baumannii (AbOmpA-PD) binds to diaminopimelate and anchors the outer membrane to the peptidoglycan layer in the cell wall. Although the crystal structure of AbOmpA-PD with its ligands has been reported, the mechanism of ligand-mediated folding of AbOmpA remains elusive. Here, we report that in vitro refolded apo-AbOmpA-PD in the absence of ligand exists as a mixture of two partially folded forms in solution: mostly unfolded (apo-state I) and hololike (apo-state II) states. Binding of the diaminopimelate or glycine ligand induced complete folding of AbOmpA-PD. The apo-state I was highly flexible and contained some secondary structural elements, whereas the apo-state II closely resembled the holo-state in terms of both structure and backbone dynamics, except for the ligand-binding region. 15N-relaxation-dispersion analyses for apo-state II revealed substantial motion on a millisecond timescale of residues in the H3 helix near the ligand-binding site, with this motion disappearing upon ligand binding. These results provide an insight into the ligand-mediated folding mechanism of AbOmpA-PD in solution.

Screening of Pre-miRNA-155 Binding Peptides for Apoptosis Inducing Activity Using Peptide Microarrays.

MicroRNA-155, one of the most potent miRNAs that suppress apoptosis in human cancer, is overexpressed in numerous cancers, and it displays oncogenic activity. Peptide microarrays, constructed by immobilizing 185 peptides containing the C-terminal hydrazide onto epoxide-derivatized glass slides, were employed to evaluate peptide binding properties of pre-miRNA-155 and to identify its binding peptides. Two peptides, which were identified based on the results of peptide microarray and in vitro Dicer inhibition studies, were found to inhibit generation of mature miRNA-155 catalyzed by Dicer and to enhance expression of miRNA-155 target genes in cells. In addition, the results of cell experiments indicate that peptide inhibitors promote apoptotic cell death via a caspase-dependent pathway. Finally, observations made in NMR and molecular modeling studies suggest that a peptide inhibitor preferentially binds to the upper bulge and apical stem-loop region of pre-miRNA-155, thereby suppressing Dicer-mediated miRNA-155 processing.

Prediction of RNA 1H and 13C chemical shifts: a structure based approach.

The use of NMR-derived chemical shifts in protein structure determination and prediction has received much attention, and, as such, many methods have been developed to predict protein chemical shifts from three-dimensional (3D) coordinates. In contrast, little attention has been paid to predicting chemical shifts from RNA coordinates. Using the random forest machine learning approach, we developed RAMSEY, which is capable of predicting both (1)H and protonated (13)C chemical shifts from RNA coordinates. In this report, we introduce RAMSEY, assess its accuracy, and demonstrate the sensitivity of RAMSEY-predicted chemical shifts to RNA 3D structure.

A distal mutation perturbs dynamic amino acid networks in dihydrofolate reductase.

Correlated networks of amino acids have been proposed to play a fundamental role in allostery and enzyme catalysis. These networks of amino acids can be traced from surface-exposed residues all the way into the active site, and disruption of these networks can decrease enzyme activity. Substitution of the distal Gly121 residue in Escherichia coli dihydrofolate reductase results in an up to 200-fold decrease in the hydride transfer rate despite the fact that the residue is located 15 Å from the active-site center. In this study, nuclear magnetic resonance relaxation experiments are used to demonstrate that dynamics on the picosecond to nanosecond and microsecond to millisecond time scales are changed significantly in the G121V mutant of dihydrofolate reductase. In particular, picosecond to nanosecond time scale dynamics are decreased in the FG loop (containing the mutated residue at position 121) and the neighboring active-site loop (the Met20 loop) in the mutant compared to those of the wild-type enzyme, suggesting that these loops are dynamically coupled. Changes in methyl order parameters reveal a pathway by which dynamic perturbations can be propagated more than 25 Å across the protein from the site of mutation. All of the enzyme complexes, including the model Michaelis complex with folate and nicotinamide adenine dinucleotide phosphate bound, assume an occluded ground-state conformation, and we do not observe sampling of a higher-energy closed conformation by (15)N R2 relaxation dispersion experiments. This is highly significant, because it is only in the closed conformation that the cofactor and substrate reactive centers are positioned for reaction. The mutation also impairs microsecond to millisecond time scale fluctuations that have been implicated in the release of product from the wild-type enzyme. Our results are consistent with an important role for Gly121 in controlling protein dynamics critical for enzyme function and further validate the dynamic energy landscape hypothesis of enzyme catalysis.

Replication protein A 32 interacts through a similar binding interface with TIPIN, XPA, and UNG2.

The 32kDa subunit of replication protein A (RPA32) is involved in various DNA repair systems such as nucleotide excision repair, base excision repair, and homologous recombination. In these processes, RPA32 interacts with different binding partners via its C-terminal domain (RPA32C; residues 172-270). It has been reported recently that RPA32C also interacts with TIPIN during the intra-S checkpoint. To determine the significance of the interaction of RPA32C with TIPIN, we have examined the interaction mode using NMR spectroscopy and an in silico modeling approach. Here, we show that TIPIN(185-218), which shares high sequence similarity with XPA(10-43) and UNG2(56-89), is less ordered in the free state and then forms a longer alpha-helix upon binding to RPA32C. The binding interface between TIPIN(185-218) and RPA32C is similar to those of XPA and UNG2, but its mode of interaction is different. The results suggest that RPA32 is an exchange point for multiple proteins involved in DNA repair, homologous recombination, and checkpoint processes and that it binds to different partners with comparable binding affinity using a single site.

Prion proteins with pathogenic and protective mutations show similar structure and dynamics.

Conformational change in the prion protein (PrP) is thought to be responsible for a group of rare but fatal neurodegenerative diseases of humans and other animals, including Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. However, little is known about the mechanism by which normal cellular PrPs initiate and propagate the conformational change. Here, we studied backbone dynamics of the inherited pathogenic mutants (P101L and H186R), protective mutants (Q167R and Q218K), and wild-type mouse PrP(89-230) at pH 5.5 and 3.5. Mutations result in minor chemical shift changes around the mutation sites except that H186R induces large chemical shift changes at distal regions. At lower pH values, the C-terminal half of the second helix is significantly disordered for the wild-type and all mutant proteins, while other parts of the protein are essentially unaffected. This destabilization is accompanied by protonation of the partially exposed histidine H186 in the second helix of the wild-type protein. This region in the mutant protein H186R is disordered even at pH 5.5. The wild-type and mutant proteins have similar microsecond conformational exchange near the two beta-strands and have similar nanosecond internal motions in several regions including the C-terminal half of the second helix, but only wild type and P101L have extensive nanosecond internal motions throughout the helices. These motions mostly disappear at lower pH. Our findings raise the possibility that the pathogenic or dominant negative mutations exert their effects on some non-native intermediate form such as PrP* after conversion of cellular PrP (PrP(C)) into the pathogenic isoform PrP(Sc) has been initiated; additionally, formation of PrP(Sc) might begin within the C-terminal folded region rather than in the disordered N-terminal region.

Prediction of the rotational tumbling time for proteins with disordered segments.

For well-structured, rigid proteins, the prediction of rotational tumbling time (tau(c)) using atomic coordinates is reasonably accurate, but is inaccurate for proteins with long unstructured sequences. Under physiological conditions, many proteins contain long disordered segments that play important regulatory roles in fundamental biological events including signal transduction and molecular recognition. Here we describe an ensemble approach to the boundary element method that accurately predicts tau(c) for such proteins by introducing two layers of molecular surfaces whose correlated velocities decay exponentially with distance. Reliable prediction of tau(c) will help to detect intra- and intermolecular interactions and conformational switches between more ordered and less ordered states of the disordered segments. The method has been extensively validated using 12 reference proteins with 14 to 103 disordered residues at the N- and/or C-terminus and has been successfully employed to explain a set of published results on a system that incorporates a conformational switch.

Structural and dynamics study of DNA dodecamer duplexes that contain un-, hemi-, or fully methylated GATC sites.

Methylation of DNA plays a regulatory role in DNA metabolism. The Escherichia coli DNA adenine methyltransferase methylates the N6 positions of adenines in the sequence 5'-GATC-3', which exists in the fully methylated state during most of the cell cycle. Just after DNA replication, however, the GATC sites transiently become hemimethylated, a condition that is indispensable for various cellular processes, such as negative modulation of replication initiation at oriC by SeqA. The lack of structural and dynamic information on DNA duplexes that contain fully methylated GATC sites makes it difficult to explain how hemimethylated GATC sites are recognized in vivo by proteins in a sea of fully methylated ones. Here, we used NMR spectroscopy to characterize the solution structure of a dodecamer DNA duplex that contained a fully methylated GATC site and the dynamics of the unmethylated, hemimethylated, and fully methylated GATC duplexes. Only the hemimethylated GATC duplex displays a unique major groove conformation, which is optimized for entrance into the cleft structure of SeqA. The apparent equilibrium constants for base-pair opening of the three differentially methylated GATC duplexes revealed that N6-methylation of the adenine residue affects the thermodynamics and kinetics of its own and neighboring base pairs. The equilibrium constants for base-pair opening of three GATC duplexes were determined using proton exchange catalyzed by TRIS. The two G.C base pairs of the hemimethylated GATC duplex displayed a faster base-pair opening rate and required less energy for the base-pair opening reaction than did those of the fully methylated one.

Structure of the nucleoid-associated protein Cnu reveals common binding sites for H-NS in Cnu and Hha.

Cnu is a nucleoid protein that has a high degree of sequence homology with Hha/YmoA family proteins, which bind to chromatin and regulate the expression of Escherichia coli virulence genes in response to changes in temperature or ionic strength. Here, we determined its solution structure and dynamic properties and mapped H-NS binding sites. Cnu consists of three alpha helices that are comparable with those of Hha, but it has significant flexibility in the C-terminal region and lacks a short alpha helix present in Hha. Upon increasing ionic strength, the helical structure of Cnu is destabilized, especially at the ends of the helices. The dominant H-NS binding sites, located at helix 3 as in Hha, reveal a common structural platform for H-NS binding. Our results may provide structural and dynamic bases for the similarity and dissimilarity between Cnu and Hha functions.

NMR relaxation study of the complex formed between CBP and the activation domain of the nuclear hormone receptor coactivator ACTR.

Overexpression of the p160 steroid receptor coactivator ACTR is associated with breast and ovarian cancers. Complex formation between ACTR and the general transcriptional coactivators CBP and p300 plays a key role in the nuclear receptor-dependent regulation of gene transcription and was the first reported example of mutual synergistic folding of two disordered polypeptide chains. In order to investigate the structure and dynamics of the free domains and complex, we measured and analyzed 15N longitudinal and transverse relaxation rates and [1H]-15N heteronuclear Overhauser effects of the backbone amides of the free and bound forms of human ACTR (residues 1041-1088) and mouse CBP (residues 2059-2117). Secondary chemical shifts for the free and bound forms were well correlated with the extent of backbone flexibility. The free ACTR domain has no residual secondary structure and shows all of the characteristics of a completely unfolded polypeptide chain. The free CBP domain retains most of the alpha-helical content seen in the complex but is significantly more flexible. Despite the disordered nature of the free individual domains, the complex has the motional characteristics of a completely folded protein complex and has no significant residual backbone fluctuation that might compensate for the massive loss of conformational entropy upon complex formation.

Structural and dynamic basis of a supercoiling-responsive DNA element.

In both eukaryotes and prokaryotes, negative supercoiling of chromosomal DNA acts locally to regulate a variety of cellular processes, such as transcription, replication, recombination and response to environmental stresses. While studying the interaction between the Hin recombinase and mutated versions of its cognate DNA-binding site, we identified a mutated DNA site that binds Hin only when the DNA is supercoiled. To understand the mechanism of this supercoiling-responsive DNA site, we used NMR spectroscopy and fluorescence resonance energy transfer to determine the solution structures and dynamics of three related DNA oligonucleotides. The supercoiling-responsive DNA site formed a partially unwound and stretched helix and showed significant flexibility and base pair opening kinetics. The single CAG/CTG triplet contained in this DNA sequence displayed the same characteristics as do multiple CAG/CTG repeats, which are associated with several hereditary neuromuscular diseases. It is known that short DNA sequence motifs that have either very high or low bending flexibility occur preferentially at supercoiling-sensitive bacterial and eukaryotic promoters. From our results and these previous data, we propose a model in which supercoiling utilizes the intrinsic flexibility of a short DNA site to switch the local DNA structure from an inefficient conformation for protein binding to an efficient one, or vice versa.

Cnu, a novel oriC-binding protein of Escherichia coli.

We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

Synthesis and characterization of tri(ethylene oxide)-attached poly(amidoamine) dendrimer layers on gold.

This paper describes the synthesis of a tri(ethylene oxide)-attached fourth-generation poly(amidoamine) dendrimer (EO3-dendrimer) and the characterization of its layers on gold. NMR analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that about 61 amine groups of a G4 PAMAM dendrimer were covalently conjugated with tri(ethylene oxide) units, accounting for a 95% modification level. Layers of the EO3-dendrimer were formed on gold, and the resulting surface was characterized by infrared reflection absorption spectroscopy, ellipsometry, and contact angle goniometry. The EO3-dendrimer resulted in more hydrophilic and less compact layers with no substantial deformation of the molecule during layer formation by virtue of the EO3 units, compared to a PAMAM dendrimer. Interestingly, the specific binding of avidin to the biotinylated layers of the EO3-dendrimer approached a surface density of 5.2 +/- 0.2 ngmm-2, showing about 92% of full surface coverage. The layers of the EO3-dendrimer were found to be more resistant to nonspecific adsorption of proteins than PAMAM dendrimer layers when bovine serum albumin and serum proteins were tested.

Structure and dynamics of hemimethylated GATC sites: implications for DNA-SeqA recognition.

DNA methylation occurs in most organisms from bacteria to mammals and provides a mechanism for epigenetic control of a variety of cellular processes. In Escherichia coli, most of the N6 positions in adenines found in the sequence GATC are methylated by DNA adenine methyltransferase. After DNA replication, the GATC sites exist transiently in the hemimethylated state, and the specific recognition of these hemimethylated GATC sites is essential for several processes, including sequestration of the site of replication initiation by the SeqA protein, strand discrimination in DNA mismatch repair by the MutH protein, and transcription of several genes. Here, we characterize the solution structure and dynamics of two dodecamer DNA duplexes that each contains a single GATC site in either unmethylated or hemimethylated state. We found that the N6-methylated adenine of a hemimethylated GATC site undergoes a slow trans-cis interconversion. The release of a tightly bound cation from hemimethylated DNA explains the instability of this structure. In addition, quantitative structural analysis revealed that hemimethylated DNA has unusual backbone structures and a remarkably narrow major groove. These dynamic and structural features provide insights into the specific recognition of hemimethylated GATC sites by the SeqA protein.