Base Triplets Research Articles

The Development of Non-natural Type Nucleoside to Stabilize Triplex DNA Formation against CG and TA Inversion Site.

Based on the sequence-specific recognition of target duplex DNA by triplexforming oligonucleotides (TFOs) at the major groove side, the antigene strategy has been exploited as a gene-targeting tool with considerable attention. Triplex DNA is formed via the specific base triplets by the Hoogsteen or reverse Hoogsteen hydrogen bond interaction between TFOs and the homo-purine strand from the target duplex DNA, leading to the established sequence-specificity. However, the presence of inversion sites, which are known as non-natural nucleosides that can form satisfactory interactions with 2'- deoxythymidine (dT) and 2'-deoxycytidine (dC) in TA and CG base pairs in the target homo-purine DNA sequences, drastically restricts the formation of classically stable base triplets and even the triplex DNA. Therefore, the design of non-natural type nucleosides, which can effectively recognize CG or/and TA inversion sites with satisfactory selectivity, should be of great significance to expanding the triplex-forming sequence. Here, this review mainly provides a comprehensive review of the current development of novel nonnatural nucleosides to recognize CG or/and TA inversion sites in triplex DNA formation against double-strand DNA (dsDNA).

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.

Triplex DNA Helix Sensor Based on Reduced Graphene Oxide and Electrodeposited Gold Nanoparticles for Sensitive Lead(II) Detection.

A triplex DNA electrochemical sensor based on reduced graphene oxide (rGO) and electrodeposited gold nanoparticles (EAu) was simply fabricated for Pb2+ detection. The glass carbon electrode (GCE) sequentially electrodeposited with rGO and EAu was further modified with a triplex DNA helix that consisted of a guanine (G)-rich circle and a stem of triplex helix based on T-A•T base triplets. With the existence of Pb2+, the DNA configuration which was formed via the Watson-Crick and Hoogsteen base pairings was split and transformed into a G-quadruplex. An adequate electrochemical response signal was provided by the signal indicator methylene blue (MB). The proposed sensor demonstrated a linear relationship between the differential pulse voltammetry (DPV) peak currents and the logarithm of Pb2+ concentrations from 0.01 to 100.00 μM with a detection limit of 0.36 nM. The proposed sensor was also tested with tap water, river and medical wastewater samples with qualified recovery and accuracy and represented a promising method for Pb2+ detection.

Non-Canonical Amino Acids in Analyses of Protease Structure and Function.

All known organisms encode 20 canonical amino acids by base triplets in the genetic code. The cellular translational machinery produces proteins consisting mainly of these amino acids. Several hundred natural amino acids serve important functions in metabolism, as scaffold molecules, and in signal transduction. New side chains are generated mainly by post-translational modifications, while others have altered backbones, such as the β- or γ-amino acids, or they undergo stereochemical inversion, e.g., in the case of D-amino acids. In addition, the number of non-canonical amino acids has further increased by chemical syntheses. Since many of these non-canonical amino acids confer resistance to proteolytic degradation, they are potential protease inhibitors and tools for specificity profiling studies in substrate optimization and enzyme inhibition. Other applications include in vitro and in vivo studies of enzyme kinetics, molecular interactions and bioimaging, to name a few. Amino acids with bio-orthogonal labels are particularly attractive, enabling various cross-link and click reactions for structure-functional studies. Here, we cover the latest developments in protease research with non-canonical amino acids, which opens up a great potential, e.g., for novel prodrugs activated by proteases or for other pharmaceutical compounds, some of which have already reached the clinical trial stage.

Structural insights into translation regulation by the THF-II riboswitch.

In bacteria, expression of folate-related genes is controlled by the tetrahydrofolate (THF) riboswitch in response to specific binding of THF and its derivatives. Recently, a second class of THF riboswitches, named THF-II, was identified in Gram-negative bacteria, which exhibit distinct architecture from the previously characterized THF-I riboswitches found in Gram-positive bacteria. Here, we present the crystal structures of the ligand-bound THF-II riboswitch from Mesorhizobium loti. These structures exhibit a long rod-like fold stabilized by continuous base pair and base triplet stacking across two helices of P1 and P2 and their interconnecting ligand-bound binding pocket. The pterin moiety of the ligand docks into the binding pocket by forming hydrogen bonds with two highly conserved pyrimidines in J12 and J21, which resembles the hydrogen-bonding pattern at the ligand-binding site FAPK in the THF-I riboswitch. Using small-angle X-ray scattering and isothermal titration calorimetry, we further characterized the riboswitch in solution and reveal that Mg2+ is essential for pre-organization of the binding pocket for efficient ligand binding. RNase H cleavage assay indicates that ligand binding reduces accessibility of the ribosome binding site in the right arm of P1, thus down-regulating the expression of downstream genes. Together, these results provide mechanistic insights into translation regulation by the THF-II riboswitch.

Understanding protein diffusion on force-induced stretched DNA conformation.

DNA morphology is subjected to environmental conditions and is closely coupled with its function. For example, DNA experiences stretching forces during several biological processes, including transcription and genome transactions, that significantly alter its conformation from that of B-DNA. Indeed, a well-defined 1.5 times extended conformation of dsDNA, known as Σ-DNA, has been reported in DNA complexes with proteins such as Rad51 and RecA. A striking feature in Σ-DNA is that the nucleobases are partitioned into triplets of three locally stacked bases separated by an empty rise gap of Å. The functional role of such a DNA base triplet was hypothesized to be coupled with the ease of recognition of DNA bases by DNA-binding proteins (DBPs) and the physical origin of three letters (codon/anti-codon) in the genetic code. However, the underlying mechanism of base-triplet formation and the ease of DNA base-pair recognition by DBPs remain elusive. To investigate, here, we study the diffusion of a protein on a force-induced stretched DNA using coarse-grained molecular dynamics simulations. Upon pulling at the 3' end of DNA by constant forces, DNA exhibits a conformational transition from B-DNA to a ladder-like S-DNA conformation via Σ-DNA intermediate. The resulting stretched DNA conformations exhibit non-uniform base-pair clusters such as doublets, triplets, and quadruplets, of which triplets are energetically more stable than others. We find that protein favors the triplet formation compared to its unbound form while interacting non-specifically along DNA, and the relative population of it governs the ruggedness of the protein-DNA binding energy landscape and enhances the efficiency of DNA base recognition. Furthermore, we analyze the translocation mechanism of a DBP under different force regimes and underscore the significance of triplet formation in regulating the facilitated diffusion of protein on DNA. Our study, thus, provides a plausible framework for understanding the structure-function relationship between triplet formation and base recognition by a DBP and helps to understand gene regulation in complex regulatory processes.

NMR assignment of non-modified tRNAIle from Escherichia coli

tRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3′-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNAIle from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNAIle as a first step towards the analysis of the effect of nucleotide modifications on tRNA’s structure and dynamics.

Pyridazine Nucleobase in Triplex-Forming PNA Improves Recognition of Cytosine Interruptions of Polypurine Tracts in RNA.

Sequence specific recognition of regulatory noncoding RNAs would open new possibilities for fundamental science and medicine. However, molecular recognition of such complex double-stranded RNA (dsRNA) structures remains a formidable problem. Recently, we discovered that peptide nucleic acids (PNAs) form an unusually stable and sequence-specific triple helix with dsRNA. Triplex-forming PNAs could become universal tools for recognition of noncoding dsRNAs but are limited by the requirement of polypurine tracts in target RNAs as only purines form stable Hoogsteen hydrogen bonded base triplets. Herein, we systematically surveyed simple nitrogen heterocycles PN as modified nucleobases for recognition of cytosine in PN*C-G triplets. We found that a 3-pyridazinyl nucleobase formed significantly more stable PN*C-G triplets than other heterocycles including the pyrimidin-2-one previously used by us and others for recognition of cytosine interruptions in polypurine tracts of PNA-dsRNA triplexes. Our results improve triple helical recognition of dsRNA and provide insights for future development of new nucleobases to expand the sequence scope of noncoding dsRNAs that can be targeted by triplex-forming PNAs.

Homozygosity of BACHD rats not only causes strong behavioral deficits in young female rats but also a reduced breeding success

Huntington’s disease is known to be a purely genetic disease based on an expansion of a CAG base triplet repeat in the coding region of the Huntingtin gene. Some years ago, researchers were able to introduce the extensive full-length gene sequence of the mutant huntingtin gene into a rodent model. The resulting BACHD rat is already well characterized for behavioral deficits. So far, all analyses in this preclinical rat model were performed in male hemizygous animals. As homozygosity of transgenic models often causes an amplification of the phenotype and female HD patients present a stronger phenotype compared to men, we established a homozygous breeding colony and tested 2 and 5 months old homozygous male and female BACHD rats in a behavioral test battery. The tests included the grip strength test, Rota Rod, elevated plus maze, passive avoidance, and Barnes maze test. Our results show strong deficits in young female homozygous BACHD rats including increased body weight, motor deficits, muscle weakness, reduced anxiety and hypoactivity, as well as learning and memory deficits. Analysis of male homozygous BACHD rats showed only weak disease symptoms, similar compared to male hemizygous BACHD rats of already published studies. Evaluation of the breeding success showed that homozygous BACHD have a reduced number of pups at the time of birth that even decreases until weaning. Our results suggest that the phenotype of homozygous male BACHD rats barely differs from already published results of hemizygous BACHD rats while female homozygous BACHD rats display strong and early alterations.

RNA structure prediction using positive and negative evolutionary information.

Knowing the structure of conserved structural RNAs is important to elucidate their function and mechanism of action. However, predicting a conserved RNA structure remains unreliable, even when using a combination of thermodynamic stability and evolutionary covariation information. Here we present a method to predict a conserved RNA structure that combines the following three features. First, it uses significant covariation due to RNA structure and removes spurious covariation due to phylogeny. Second, it uses negative evolutionary information: basepairs that have variation but no significant covariation are prevented from occurring. Lastly, it uses a battery of probabilistic folding algorithms that incorporate all positive covariation into one structure. The method, named CaCoFold (Cascade variation/covariation Constrained Folding algorithm), predicts a nested structure guided by a maximal subset of positive basepairs, and recursively incorporates all remaining positive basepairs into alternative helices. The alternative helices can be compatible with the nested structure such as pseudoknots, or overlapping such as competing structures, base triplets, or other 3D non-antiparallel interactions. We present evidence that CaCoFold predictions are consistent with structures modeled from crystallography.

Altered third-party punishment in Huntington's disease: A study using neuroeconomic games.

BackgroundHuntington's disease (HD) is a heritable degenerative brain disease caused by a mutation in the huntingtin gene with excessive repeats of the base triplet cytosine–adenine–guanine (CAG), which codes for the aminoacid glutamine. HD is associated with a broad spectrum of neurocognitive dysfunction, including deficits in social cognition. The appreciation of fairness rules and reciprocity has not been studied in HD. Based on theoretical considerations suggesting that brain regions known to be affected from HD are involved in economic decision‐making, the present study sought to examine HD patients' performance in two neuroeconomic games.MethodsTwenty‐nine manifest HD mutation carriers (20 males, nine females) performed an Ultimatum Game (UG) and a Dictator Game (DG) where third‐party punishment of observed unfairness was required. In addition, patients were tested for neurocognition and the ability to understand other people's mental states (“theory of mind”). For comparison, a clinical control group of 30 patients with chronic schizophrenia, and 30 unaffected healthy controls matched for age and verbal intelligence took part in the study.ResultsPatients with HD had some appreciation of fairness rules, as they tended to reject unfair offers in the UG similar to controls. However, unlike the other two groups, individuals with HD did not punish observed unfairness from a third‐party perspective. This lack of “altruistic punishment” was associated with deficits in executive functioning including working memory, inhibitory control and cognitive flexibility, and to a lesser degree with poor “theory of mind.”ConclusionsHD seems to be associated with impairments in understanding of more complex rules of social exchange. Aside from deficits in executive functioning, this behavior could, in part, be linked to an inability to experience third‐party punishment as rewarding.

The Adipokinetic Peptides in Diptera: Structure, Function, and Evolutionary Trends.

Nineteen species of various families of the order Diptera and one species from the order Mecoptera are investigated with mass spectrometry for the presence and primary structure of putative adipokinetic hormones (AKHs). Additionally, the peptide structure of putative AKHs in other Diptera are deduced from data mining of publicly available genomic or transcriptomic data. The study aims to demonstrate the structural biodiversity of AKHs in this insect order and also possible evolutionary trends. Sequence analysis of AKHs is achieved by liquid chromatography coupled to mass spectrometry. The corpora cardiaca of almost all dipteran species contain AKH octapeptides, a decapeptide is an exception found only in one species. In general, the dipteran AKHs are order-specific- they are not found in any other insect order with two exceptions only. Four novel AKHs are revealed by mass spectrometry: two in the basal infraorder of Tipulomorpha and two in the brachyceran family Syrphidae. Data mining revealed another four novel AKHs: one in various species of the infraorder Culicumorpha, one in the brachyceran superfamily Asiloidea, one in the family Diopsidae and in a Drosophilidae species, and the last of the novel AKHs is found in yet another Drosophila. In general, there is quite a biodiversity in the lower Diptera, whereas the majority of the cyclorraphan Brachycera produce the octapeptide Phote-HrTH. A hypothetical molecular peptide evolution of dipteran AKHs is suggested to start with an ancestral AKH, such as Glomo-AKH, from which all other AKHs in Diptera to date can evolve via point mutation of one of the base triplets, with one exception.

The Pattern of Occurrence of Cytosine in the Genetic Code Minimizes Deleterious Mutations and Favors Proper Function of the Translational Machinery

The standard genetic code consists of 64 combinations of base triplets made from four different bases. The research aim of this study was to investigate the pattern of occurrence of cytosine in the genetic code. By exploring the base composition and sequence of all 64 codons, the author found some important features based on the instability of cytosine. Because cytosine undergoes spontaneous deamination that converts it into uracil, it is evolutionarily favorable to exclude cytosine from codons critical to the initiation and termination of translation. For amino acids that have one to three synonymous codons (also called synonyms), the frequency of occurrence of C in the first and second positions of their mRNA codons is significantly lower than the frequencies of A, U, and G. For mRNA codons that encode amino acids with four synonyms, the trend of base composition is opposite to those encoding amino acids with one to three synonyms; the instability of C could be inhibited or reduced via formation of hydrogen bonds with a G and/or with a protonated C, and the secondary structure of the resultant mRNA could be adjusted via the multiple synonymous alternates at the third position of their codons to facilitate the translation process. The overall pattern of occurrence for C in the genetic code not only minimizes deleterious mutations and favors proper function of the translational machinery by excluding C from certain positions within codons, but also allows the occurrence of genetic diversity via mutation by including C in less-critical positions.

Defining the influence of Rad51 and Dmc1 lineage-specific amino acids on genetic recombination.

The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from Caenorhabditis elegans, an organism without Dmc1, has acquired "Dmc1-like" amino acids. Chimeric C. elegans RAD-51 harboring "canonical" Rad51 amino acids gives rise to toxic recombination intermediates, which must be actively dismantled to permit normal meiotic progression. We propose that Dmc1 lineage-specific amino acids involved in the stabilization of heteroduplex DNA joints with mismatch-containing base triplets may contribute to normal meiotic recombination.

Stability of an RNA•DNA-DNA triple helix depends on base triplet composition and length of the RNA third strand.

Recent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA•DNA–DNA triple helix that regulates gene expression. However, base triplet composition of pyrimidine motif RNA•DNA–DNA triple helices is not well understood beyond the canonical U•A–T and C•G–C base triplets. Using native gel-shift assays, the relative stability of 16 different base triplets at a single position, Z•X–Y (where Z = C, U, A, G and X–Y = A–T, G–C, T–A, C–G), in an RNA•DNA–DNA triple helix was determined. The canonical U•A–T and C•G–C base triplets were the most stable, while three non-canonical base triplets completely disrupted triple-helix formation. We further show that our RNA•DNA–DNA triple helix can tolerate up to two consecutive non-canonical A•G–C base triplets. Additionally, the RNA third strand must be at least 19 nucleotides to form an RNA•DNA–DNA triple helix but increasing the length to 27 nucleotides does not increase stability. The relative stability of 16 different base triplets in DNA•DNA–DNA and RNA•RNA–RNA triple helices was distinctly different from those in RNA•DNA–DNA triple helices, showing that base triplet stability depends on strand composition being DNA and/or RNA. Multiple factors influence the stability of triple helices, emphasizing the importance of experimentally validating formation of computationally predicted triple helices.

A new function of copper zinc superoxide dismutase: as a regulatory DNA-binding protein in gene expression in response to intracellular hydrogen peroxide.

In microorganisms, a number of metalloproteins including PerR are found to regulate gene expression in response to environmental reactive oxygen species (ROS) changes. However, discovery of similar regulatory mechanisms remains elusive within mammalian cells. As an antioxidant metalloenzyme that maintains intracellular ROS homeostasis, copper zinc superoxide dismutase (SOD1) has high affinity for DNA in solution and in cells. Here, we explored the regulatory roles of SOD1 in the expression of genes in response to ROS changes within mammalian cells. SOD1-occupied DNA sites with distinct sequence preference were identified. Changing ROS levels both were found to impact DNA–SOD1 interactions in solution and within HeLa cells. GGA was one of the base triplets that had direct contact with SOD1. DNA–SOD1 interactions were observed to regulate the ROS-responsive expression of functional genes including oncogenes and amyotrophic lateral sclerosis-linked genes in transcriptional phases. Our results confirm another function of SOD1, acting as a H2O2-responsive regulatory protein in the expression of numerous mammalian genes.

Energetics, Ion, and Water Binding of the Unfolding of AA/UU Base Pair Stacks and UAU/UAU Base Triplet Stacks in RNA.

Triplex formation occurs via interaction of a third strand with the major groove of double-stranded nucleic acid, through Hoogsteen hydrogen bonding. In this work, we use a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry to determine complete thermodynamic profiles for the unfolding of polyadenylic acid (poly(rA))·polyuridylic acid (poly(rU)) (duplex) and poly(rA)·2poly(rU) (triplex). Our thermodynamic results are in good agreement with the much earlier work of Krakauer and Sturtevant using only UV melting techniques. The folding of these two helices yielded an uptake of ions, Δ nNa+ = 0.15 mol Na+/mol base pair (duplex) and 0.30 mol Na+/mole base triplet (triplex), which are consistent with their polymer behavior and the higher charge density parameter of triple helices. The osmotic stress technique yielded a release of structural water, Δ nW = 2 mol H2O/mol base pair (duplex unfolding into single strands) and an uptake of structural water, Δ nW = 2 mol H2O/mole base pair (triplex unfolding into duplex and a single strand). However, an overall release of electrostricted waters is obtained for the unfolding of both complexes from pressure perturbation calorimetric experiments. In total, the Δ V values obtained for the unfolding of triplex into duplex and a single strand correspond to an immobilization of two structural waters and a release of three electrostricted waters. The Δ V values obtained for the unfolding of duplex into two single strands correspond to the release of two structural waters and the immobilization of four electrostricted water molecules.

Metal Coordination to Ligand-Modified Peptide Nucleic Acid Triplexes.

A challenging goal in nanotechnology is the precise and programmable arrangement of specific elements in nanosystems in the three-dimensional space. The use of ligand-modified nucleic acids represents an accurate and selective tool to achieve this goal when it comes to metal ion organization. The synthesis of peptide nucleic acid (PNA) monomers that contain ligands instead of nucleobases makes possible the creation of metal-mediated alternative base pairs and triplets at specific locations in PNA duplexes and triplexes, respectively. We report the formation of four- and six-coordinate metal complexes between PNA triplexes modified with 2,2'-bipyridine (Bpy) or 8-hydroxyquinoline (Q) ligands and 3d metal ions. These metal complexes function as alternative base triplets or pairs in that they increase the thermal stability of the triplexes if the stability constants of the metal complexes are relatively high. The increase in the triplex melting temperature correlates with the stability constants of the metal complexes with ligand-containing PNA determined by UV-vis titrations. The metal complexes coordinate two or three ligands although three bidentate ligands are in close proximity of each other within a triplex. Metal coordination to ligand-modified PNA triplexes was further studied by electron paramagnetic resonance (EPR) spectroscopy and circular dichrosim (CD) spectroscopy. EPR spectroscopy indicated the formation of a square planar [CuQ2] complex between Cu2+ and Q-containing PNA triplex. Taken together, the spectroscopic results indicate that in the presence of 1 equiv of Fe2+ or Ni2+ the majority, but not all, of the Bpy-containing PNA triplexes contain [MBpy3] complexes, with a minority of them being metal free. We attribute this behavior to a supramolecular chelate effect exerted by the triplex, which favors the formation of tris-ligand complexes, that is balanced by the steric interactions between the metal complex and the adjacent nucleobase triplets, which decrease the stability of the complex and triplex. In contrast, the very high stability of square planar [MQ2] complexes of Cu2+ and Ni2+ leads to formation of bis-ligand complexes instead of tris-ligand complexes with Q3-containing PNA triplexes. The metal-containing PNA triplexes have a terminal l-lysine and adopt a left-handed chiral structure in solution. The handedness of the PNA triplex determines that of the metal complexes formed with the Bpy-containing PNA triplexes.

Influence of Electron-Holes on DNA Sequence-Specific Mutation Rates.

Biases in mutation rate can influence molecular evolution, yielding rates of evolution that vary widely in different parts of the genome and even among neighboring nucleotides. Here, we explore one possible mechanism of influence on sequence-specific mutation rates, the electron–hole, which can localize and potentially trigger a replication mismatch. A hole is a mobile site of positive charge created during one-electron oxidation by, for example, radiation, contact with a mutagenic agent, or oxidative stress. Its quantum wavelike properties cause it to localize at various sites with probabilities that vary widely, by orders of magnitude, and depend strongly on the local sequence. We find significant correlations between hole probabilities and mutation rates within base triplets, observed in published mutation accumulation experiments on four species of bacteria. We have also computed hole probability spectra for hypervariable segment I of the human mtDNA control region, which contains several mutational hotspots, and for heptanucleotides in noncoding regions of the human genome, whose polymorphism levels have recently been reported. We observe significant correlations between hole probabilities, and context-specific mutation and substitution rates. The correlation with hole probability cannot be explained entirely by CpG methylation in the heptanucleotide data. Peaks in hole probability tend to coincide with mutational hotspots, even in mtDNA where CpG methylation is rare. Our results suggest that hole-enhanced mutational mechanisms, such as oxidation-stabilized tautomerization and base deamination, contribute to molecular evolution.

Effect of dC → d(m5C) substitutions on the folding of intramolecular triplexes with mixed TAT and C+GC base triplets

Oligonucleotide-directed triple helix formation has been recognized as a potential tool for targeting genes with high specificity. Cystosine methylation in the 5′ position is both ubiquitous and a stable regulatory modification, which could potentially stabilize triple helix formation. In this work, we have used a combination of calorimetric and spectroscopic techniques to study the intramolecular unfolding of four triplexes and two duplexes. We used the following triplex control sequence, named Control Tri, d(AGAGAC5TCTCTC5TCTCT), where C5 are loops of five cytosines. From this sequence, we studied three other sequences with dC → d(m5C) substitutions on the Hoogsteen strand (2MeH), Crick strand (2MeC) and both strands (4MeHC). Calorimetric studies determined that methylation does increase the thermal and enthalpic stability, leading to an overall favorable free energy, and that this increased stability is cumulative, i.e. methylation on both the Hoogsteen and Crick strands yields the largest favorable free energy. The differential uptake of protons, counterions and water was determined. It was found that methylation increases cytosine protonation by shifting the apparent pKa value to a higher pH; this increase in proton uptake coincides with a release of counterions during folding of the triplex, likely due to repulsion from the increased positive charge from the protonated cytosines. The immobilization of water was not affected for triplexes with methylated cytosines on their Hoogsteen or Crick strands, but was seen for the triplex where both strands are methylated. This may be due to the alignment in the major groove of the methyl groups on the cytosines with the methyl groups on the thymines which causes an increase in structural water along the spine of the triplex.