The human AP-endonuclease 1 (APE1) is a DNA G-quadruplex structure binding protein and regulates KRAS expression in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive types of cancer, is characterized by aberrant activity of oncogenic KRAS. A nuclease-hypersensitive GC-rich region in KRAS promoter can fold into a four-stranded DNA secondary structure called G-quadruplex (G4), known to regulate KRAS expression. However, the factors that regulate stable G4 formation in the genome and KRAS expression in PDAC are largely unknown. Here, we show that APE1 (apurinic/apyrimidinic endonuclease 1), a multifunctional DNA repair enzyme, is a G4-binding protein, and loss of APE1 abrogates the formation of stable G4 structures in cells. Recombinant APE1 binds to KRAS promoter G4 structure with high affinity and promotes G4 folding in vitro. Knockdown of APE1 reduces MAZ transcription factor loading onto the KRAS promoter, thus reducing KRAS expression in PDAC cells. Moreover, downregulation of APE1 sensitizes PDAC cells to chemotherapeutic drugs in vitro and in vivo. We also demonstrate that PDAC patients' tissue samples have elevated levels of both APE1 and G4 DNA. Our findings unravel a critical role of APE1 in regulating stable G4 formation and KRAS expression in PDAC and highlight G4 structures as genomic features with potential application as a novel prognostic marker and therapeutic target in PDAC.

The volume changes of unfolding of dsDNA.

High hydrostatic pressure can have profound effects on the stability of biomacromolecules. The magnitude and direction (stabilizing or destabilizing) of this effect is defined by the volume changes in the system, ΔV. Positive volume changes will stabilize the starting native state, whereas negative volume changes will lead to the stabilization of the final unfolded state. For the DNA double helix, experimental data suggested that when the thermostability of dsDNA is below 50°C, increase in hydrostatic pressure will lead to destabilization; i.e., helix-to-coil transition has negative ΔV. In contrast, the dsDNA sequences with the thermostability above 50°C showed positive ΔV values and were stabilized by hydrostatic pressure. In order to get insight into this switch in the response of dsDNA to hydrostatic pressure as a function of temperature, first we further validated this trend using experimental measurements of ΔV for 10 different dsDNA sequences using pressure perturbation calorimetry. We also developed a computational protocol to calculate the expected volume changes of dsDNA unfolding, which was benchmarked against the experimental set of 50 ΔV values that included, in addition to our data, the values from the literature. Computation predicts well the experimental values of ΔV. Such agreement between computation and experiment lends credibility to the computation protocol and provides molecular level rational for the observed temperature dependence of ΔV that can be traced to the hydration. Difference in the ΔV value for A/T versus G/C basepairs is also discussed.

Increased Flexibility between Stems of Intramolecular Three-Way Junctions by the Insertion of Bulges.

Intramolecular junctions are a ubiquitous structure within DNA and RNA; three-way junctions in particular have high strain around the junction because of the lack of flexibility, preventing the junctions from adopting conformations that would allow for optimal folding. In this work, we used a combination of calorimetric and spectroscopic techniques to study the unfolding of four intramolecular three-way junctions. The control three-way junction, 3H, has the sequence d(GAAATTGCGCT5GCGCGTGCT5GCACAATTTC), which has three arms of different sequences. We studied three other three-way junctions in which one (2HS1H), two (HS12HS1), and three (HS1HS1HS1) cytosine bulges were placed at the junction to allow the arms to adopt a wider range of conformations that may potentially relieve strain. Through calorimetric studies, it was concluded that bulges produce only minor effects on the enthalpic and thermal stability at physiological salt concentrations for 2HS1H and HS1HS1HS1. HS12HS1 displays the strongest effect, with the GTGC stem lacking a defined transition. In addition to unfolding thermodynamics, the differential binding of counterions, water, and protons was determined. It was found that with each bulge, there was a large increase in the binding of counterions; this correlated with a decrease in the immobilization of structural water molecules. The increase in counterion uptake upon folding likely displaces binding of structural water, which is measured by the osmotic stress method, in favor of electrostricted waters. The cytosine bulges do not affect the binding of protons; this finding indicates that the bulges are not forming base-triplet stacks. These results indicate that bulges in junctions do not affect the unfolding profile or the enthalpy of oligonucleotides but do affect the number and amount of molecules immobilized by the junction.

Thermodynamic Stability of DNA Duplexes Comprising the Simplest T → dU Substitutions.

Members of the uracil-DNA glycosylase (UDG) enzyme family recognize and bind uracil, sequestering it within the binding site pocket and catalyzing the cleavage of the base from the deoxyribose, leaving an abasic site. The recognition and binding are passive and rely on innate dynamic motions of DNA wherein base pairs undergo thermally induced breakage and conformational fluctuations. Once the uracil breaks from its base pair, it can be recognized and bound by the enzyme, which then alters its conformation for sequestration and catalysis. Our results suggest that the thymine to uracil substitution, which differs only by a single methyl group, causes a destabilization of the duplex thermodynamics, which would lead to an increase in the population of the extrahelical state and increase the probability of uracil being recognized and excised from DNA by UDG. This destabilization is dependent on the identity of the nearest-neighbor base-pair stacks; a G·C nearest neighbor leads to thermal and enthalpic destabilization that is weaker that that seen with two A·T neighbors. In addition, uracil substitution yields a nearest-neighbor increase in the counterion uptake of the duplexes but decreases the level of immobilization of structural water for all substituted duplexes regardless of the neighbor identity or number of substitutions.

Effect of GCAA stabilizing loops on three- and four-way intramolecular junctions.

Tetraloops are a common way of changing the melting behavior of a DNA or RNA structure without changing the sequence of the stem. Because of the ubiquitous nature of tetraloops, our goal is to understand the effect a GCAA tetraloop, which belongs to the GNRA family of tetraloops, has on the unfolding thermodynamics of intramolecular junctions. Specifically, we have described the melting behavior of intramolecular three-way and four-way junctions where a T5 loop has been replaced with a GCAA tetraloops in different positions. Their thermodynamic profiles, including ΔnNa+ and ΔnW, were analyzed based on the position of the tetraloop. We obtained between -16.7 and -27.5 kcal mol-1 for all junctions studied. The experimental data indicates the influence of the GCAA tetraloop is primarily dictated by the native unfolding of the junction; if the tetraloop is placed on a stem that unfolds as a single domain when the tetraloop is not present, it will unfold as a single domain when the tetraloop is present but with a higher thermal stability. Conversely, if the tetraloop is placed on a stem which unfolds cooperatively with other stems when the tetraloop is not present, the tetraloop will increase the thermal stability of all the stems in the melting domain. The oligonucleotide structure and not the tetraloop itself affects ion uptake; three-way junctions do not gain an increase in ion uptake, but four-way junctions do. This is not the case for water immobilization, where the position of the tetraloop dictates the amount of water immobilized.

Investigation of the Melting Behavior of DNA Three-Way Junctions in the Closed and Open States.

Intramolecular three-way junctions are commonly found in both DNA and RNA. These structures are functionally relevant in ribozymes, riboswitches, rRNA, and during replication. In this work, we present a thermodynamic description of the unfolding of DNA intramolecular three-way junctions. We used a combination of spectroscopic and calorimetric techniques to investigate the folding/unfolding thermodynamics of two three-way junctions with a closed (Closed-J) or open (Open-J) junction and their appropriate control stem-loop motifs (GAAATT-Hp, CTATC-Hp, and Dumbbell). The overall results show that both junctions are stable over a wide range of salt concentrations. However, Open-J is more stable due to a higher enthalpy contribution from the formation of a higher number of basepair stacks whereas Closed-J has a defined structure and retains the basepair stacking of all three stems. The comparison of the experimental results of Closed-J and Open-J with those of their component stem-loop motifs allowed us to be more specific about their cooperative unfolding. For instance, Closed-J sacrifices thermal stability of the Dumbbell structure to maintain an overall folded state. At higher salt concentration, the simultaneous unfolding of the above domains is lost, resulting in the unfolding of the three separate stems. In contrast, the junction of Open-J in low salt retains the thermal and enthalpic stability of the Dumbbell structure although sacrificing stability of the CTATC stem. The relative stability of Dumbbell is the primary reason for the higher ΔG°(5), or free energy, value seen for Open-J at low salt. Higher salt not only maintains thermal stability of the Dumbbell structure in Open-J but causes the CTATC stem to fully fold.

Structural Insight into the Unbound State of the DNA Analogue of the PreQ1 Riboswitch: A Thermodynamic Approach.

The preQ1 riboswitch aptamer domain is very dynamic in its unbound state with the ability to form multiple structures: a hairpin, kissing hairpins, and pseudoknot-like structure. The aim of this study is to determine whether the DNA analogue (PreQ1) is able to form structures similar to that of the reported RNA aptamer. Using a thermodynamic approach, we report on structural determination using differential scanning calorimetry under different salt conditions. Further analysis of the primary sequence allowed us to design modified molecules to determine what potential structures are forming in this single-stranded DNA analogue. We found, in a 16 mM Na+ solution, PreQ1 has three transitions with TM values of 14.8, 19.4, and 26.2 °C and a total ΔH of -44.7 kcal/mol. With the increase in salt concentration to 116 mM, there are TM values of 22.3, 28.7, and 38.9 °C and a ΔH of -69.1 kcal/mol, while at 216 mM, the three transitions have TM values of 24.4, 31.6, and 42.9 °C with a total ΔH of -71.5 kcal/mol. Therefore, the increase in enthalpy is due to the formation of additional base-pair stacks. The modified molecules, which would inhibit pseudoknot formation, kissing hairpins, and internal loop interactions, were fully characterized and compared to the native DNA analogue. The analysis of the enthalpy and differential binding of counterions allows us to conclude this single-stranded DNA analogue under physiological conditions is not forming a pseudoknot-like structure. Instead, two potential structures, Compact-Hairpin and Kissing-Complex, are more likely and could be in equilibrium.

Application of differential scanning calorimetry to measure the differential binding of ions, water and protons in the unfolding of DNA molecules.

BACKGROUND: The overall stability of DNA molecules globally depends on base-pair stacking, base-pairing, polyelectrolyte effect and hydration contributions. In order to understand how they carry out their biological roles, it is essential to have a complete physical description of how the folding of nucleic acids takes place, including their ion and water binding. SCOPE OF REVIEW: To investigate the role of ions, water and protons in the stability and melting behavior of DNA structures, we report here an experimental approach i.e., mainly differential scanning calorimetry (DSC), to determine linking numbers: the differential binding of ions (Δnion), water (ΔnW) and protons (ΔnH(+)) in the helix-coil transition of DNA molecules. GENERAL SIGNIFICANCE: We use DSC and temperature-dependent UV spectroscopic techniques to measure the differential binding of ions, water, and protons for the unfolding of a variety of DNA molecules: salmon testes DNA (ST-DNA), one dodecamer, one undecamer and one decamer duplexes, nine hairpin loops, and two triplexes. These methods can be applied to any conformational transition of a biomolecule. MAJOR CONCLUSIONS: We determined complete thermodynamic profiles, including all three linking numbers, for the unfolding of each molecule. The favorable folding of a DNA helix results from a favorable enthalpy-unfavorable entropy compensation. DSC thermograms and UV melts as a function of salt, osmolyte and proton concentrations yielded releases of ions and water. Therefore, the favorable folding of each DNA molecule results from the formation of base-pair stacks and uptake of both counterions and water molecules. In addition, the triplex with C(+)GC base triplets yielded an uptake of protons. Furthermore, the folding of a DNA duplex is accompanied by a lower uptake of ions and a similar uptake of four water molecules as the DNA helix gets shorter. In addition, the oligomer duplexes and hairpin thermodynamic data suggest ion and water binding depends on the DNA sequence rather than DNA composition.

Melting Behavior of a DNA Four-Way Junction Using Spectroscopic and Calorimetric Techniques.

Intramolecular four-way junctions are structures present during homologous recombination, repair of double stranded DNA breaks, and integron recombination. Because of the wide range of biological processes four-way junctions are involved in, understanding how and under what conditions these structures form is critical. In this work, we used a combination of spectroscopic and calorimetric techniques to present a complete thermodynamic description of the unfolding of a DNA four-way junction (FWJ) and its appropriate control stem-loop motifs (Dumbbell, GAAATT-Hp, CTATC-Hp, GTGC-Hp, and GCGC-Hp). The overall results show that the four-way junction increases the cooperative unfolding of its stems, although the reason for this is unclear, as the arms do not unfold as coaxial stacks, and thus its melting behavior cannot be accurately described by its control molecules. This is in contrast to what has been seen for two- and three-way junctions. In addition, the lack of base stacking and the ΔHvH/ΔHcal ratio seen at low salt indicate the four-way junction exists as a mixture of conformations, one of which is most likely the open-X structure which has unpaired bases at the junction. This was confirmed by single value decomposition of CD and UV spectra. This indicates that at low salt there is a third spectroscopically distinct species, while at higher salt there are only two species, folded and unfolded. Based on the enthalpy, Δnion, and ΔnW, the dominant folded structure at high salt is most likely the antiparallel stacked-X structure.

Looking for Waldo: a potential thermodynamic signature to DNA damage.

DNA in its simplest form is an ensemble of nucleic acids, water, and ions, and the conformation of DNA is dependent on the relative proportions of all three components. When DNA is covalently damaged by endogenous or exogenous reactive species, including those produced by some anticancer drugs, the ensemble undergoes localized changes that affect nucleic acid structure, thermodynamic stability, and the qualitative and quantative arrangement of associated cations and water molecules. Fortunately, the biological effects of low levels of DNA damage are successfully mitigated by a large number of proteins that efficiently recognize and repair DNA damage in the midst of a vast excess of canonical DNA. In this Account, we explore the impact of DNA modifications on the high resolution and dynamic structure of DNA, DNA stability, and the uptake of ions and water and explore how these changes may be sensed by proteins whose function is to initially locate DNA lesions. We discuss modifications on the nucleobases that are located in the major and minor grooves of DNA and include lesions that are observed in vivo, including oxidized bases, as well as some synthetic nucleobases that allow us to probe how the location and nature of different substituents affect the thermodynamics and structure of the DNA ensemble. It is demonstrated that disruption of a cation binding site in the major groove by modification of the N7-position on the purines, which is the major site for DNA alkylation, is enthalpically destabilizing. Accordingly, tethering a cationic charge in the major groove is enthalpically stabilizing. The combined structural and thermodynamic studies provide a detailed picture of how different DNA lesions affect the dynamics of DNA and how modified bases interact with their environment. Our work supports the hypothesis that there is a "thermodynamic signature" to DNA lesions that can be exploited in the initial search that requires differentiation between canonical DNA and DNA with a lesion. The differentiation between a lesion and a cognate lesion that is a substrate for a particular enzyme involves another layer of thermodynamic and kinetic factors.

Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function.

Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein, Eps15 homology domain 1 (EHD1). We have demonstrated this interaction by glutathione S-transferase-pulldown, yeast two-hybrid assay, isothermal calorimetry and co-immunoprecipitation, and found that the binding occurs between the EH domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps26), suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as overexpression of a wild-type Rank-5-small interfering RNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.

Thermodynamic profiles and nuclear magnetic resonance studies of oligonucleotide duplexes containing single diastereomeric spiroiminodihydantoin lesions.

The spiroiminodihydantoins (Sp) are highly mutagenic oxidation products of guanine and 8-oxo-7,8-dihydroguanine in DNA. The Sp lesions have recently been detected in the liver and colon of mice infected with Helicobacter hepaticus that induces inflammation and the development of liver and colon cancers in murine model systems [Mangerich, A., et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109, E1820-E1829]. The impact of Sp lesions on the thermodynamic characteristics and the effects of the diastereomeric Sp-R and Sp-S lesions on the conformational features of double-stranded 11-mer oligonucleotide duplexes have been studied by a combination of microcalorimetric methods, analysis of DNA melting curves, and two-dimensional nuclear magnetic resonance methods. The nonplanar, propeller-like shapes of the Sp residues strongly diminish the extent of local base stacking interactions that destabilize the DNA duplexes characterized by unfavorable enthalpy contributions. Relative to that of an unmodified duplex, the thermally induced unfolding of the duplexes with centrally positioned Sp-R and Sp-S lesions into single strands is accompanied by a smaller release of cationic counterions (Δn(Na⁺) = 0.6 mol of Na⁺/mol of duplex) and water molecules (Δn(w) = 17 mol of H2O/mol of duplex). The unfolding parameters are similar for the Sp-R and Sp-S lesions, although their orientations in the duplexes are different. The structural disturbances radiate one base pair beyond the flanking C:G pair, although Watson-Crick hydrogen bonding is maintained at all flanking base pairs. The observed relatively strong destabilization of B-form DNA by the physically small Sp lesions is expected to have a significant impact on the processing of these lesions in biological environments.

The human ITPA polymorphic variant P32T is destabilized by the unpacking of the hydrophobic core.

Inosine triphosphate pyrophosphatase (ITPA), a key enzyme involved in maintaining the purity of cellular nucleoside triphosphate pools, specifically recognizes inosine triphosphate and xanthosine triphosphate (including the deoxyribose forms) and detoxifies them by catalyzing the hydrolysis of a phosphoanhydride bond, releasing pyrophosphate. This prevents their inappropriate use as substrates in enzymatic reactions utilizing (d)ATP or (d)GTP. A human genetic polymorphism leads to the substitution of Thr for Pro32 (P32T) and causes ITPA deficiency in erythrocytes, with heterozygotes having on average 22.5% residual activity, and homozygotes having undetectable activity. This polymorphism has been implicated in modulating patients' response to mercaptopurines and ribavirin. Human fibroblasts containing this variant have elevated genomic instability upon treatment with base analogs. We find that the wild-type and P32T forms are dimeric in solution and in the crystal structure. This abolishes the previous speculation that the P32T change disrupts dimerization as a mechanism of inactivation. The only difference in structure from the wild-type protein is that the area surrounding Thr32 is disrupted. Phe31 is flipped from the hydrophobic core out into the solvent, leaving a hole in the hydrophobic core of the protein which likely accounts for the reduced thermal stability of P32T ITPA and ultimately leads to its susceptibility to degradation in human cells. Circular dichroism and thermal denaturation studies confirm these structural results. We propose that the dimer of P32T variant subunit with wild-type subunit is degraded in cells similarly to the P32T homodimer explaining the level of loss of ITPA activity in heterozygotes.

Characterization of DNA with an 8-oxoguanine modification.

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2'-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2-8 kcal mol(-1) over a 16-116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoG•C is fundamentally different than that found at G•C. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoG•C pairing and those base pairs that are 5' of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.

Prevention of orthopedic device-associated osteomyelitis using oxacillin-containing biomineral-binding liposomes.

PURPOSE: To develop novel biomineral-binding liposomes (BBL) for the prevention of orthopedic implant associated osteomyelitis. METHODS: A biomineral-binding lipid, alendronate-tri(ethyleneglycol)-cholesterol conjugate (ALN-TEG-Chol), was synthesized through Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (a versatile click reaction). Mixing with other excipients, the new lipid was used to develop BBL. Thermodynamic behavior was studied by differential scanning calorimetry (DSC). In vitro biomineral-binding potential and kinetics were evaluated on hydroxyapatite (HA, a widely used material for orthopedic implant devices) particles. Oxacillin was encapsulated into BBL and used for in vitro evaluation in preventing Staphylococcus aureus biofilm formation. RESULTS: DSC analysis showed that ALN-TEG-Chol could inhibit the phase transition of liposomes by reducing its cooperativity, yielding liposomes with thermodynamic stability similar to liposomes containing regular cholesterol. BBL showed fast and strong binding ability to HA. Oxacillin-loading BBL demonstrated significantly better preventive efficacy against bacteria colonization when challenged with S. aureus isolate, implying its potential in preventing orthopedic implant associated osteomyelitis. CONCLUSIONS: In this proof of concept study, novel BBL has been successfully developed and validated for reducing the frequency of implantable device-related infections.

Probing the Temperature Unfolding of a Variety of DNA Secondary Structures Using the Fluorescence Properties of 2-aminopurine.

The fluorescence probe 2-aminopurine (2AP) is widely used to monitor the molecular environment, including the local solvent environment, and overall dynamics of nucleic acids and nucleic acid-ligand complexes. This work reports on the temperature-induced conformational flexibility of a variety of secondary structures of nucleic acids using optical and calorimetric melting techniques, and evaluates the usefulness of fluorescence melting curves obtained from monitoring the fluorescence changes of 2AP as a function of temperature. Furthermore, the base stacking properties of 2AP are examined in these structures for a first time. Specifically, we incorporated single A → 2AP substitutions into a variety of DNA structures, such as a single strand (SS), a dodecamer duplex (Duplex), a hairpin loop (Hairpin), a G-quadruplex (G2), and an intramolecular triplex (Triplex). A combination of fluorescence, UV, and circular dichroism spectroscopies, and differential scanning calorimetric (DSC) techniques is used to investigate their temperature-induced unfolding. The melting curves of each molecule show monophasic transitions with similar TMs and van't Hoff enthalpies indicating that all transitions are two-state and that the fluorescence changes for the unstacking of 2AP follow the unfolding of the whole molecule. The DSC thermodynamic profiles of each 2AP modified molecule, relative to their unmodified control molecules, yielded folding ΔΔG°s of 1.6 kcal/mol (Duplex), 3.1 kcal/mol (Hairpin), 1.6 kcal/mol (Triplex), and -1.7 kcal/mol (G2). These ΔΔG°s are driven by unfavorable differential enthalpies (Duplex and Hairpin), favorable differential enthalpy (G2), and by a favorable differential entropy term for Triplex. These enthalpy effects are explained in terms of stacking and hydration contributions, that are associated with the local environment that 2AP is experiencing. For example, the lower ΔΔHcal value of 8.7 kcal/mol (Hairpin), relative to Duplex, is due to weaker base-pair stacks and higher hydration state of the stem of Hairpin. We conclude that the incorporation of 2AP in nucleic acids is a useful tool to monitor their temperature-induced unfolding; especially, when these sensitive fluorescent moieties are placed in the proper molecular environment of the nucleic acid.

Introduction of cationic charge into DNA near the major groove edge of a guanine x cytosine base pair: characterization of oligodeoxynucleotides substituted with 7-aminomethyl-7-deaza-2'-deoxyguanosine.

The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c(7)G) alters the electronic properties of the heterocyclic base and eliminates a potential major groove cation binding site, which affects the organization of salts and water in the major groove. This has a destabilizing effect on DNA. We report herein the characterization of DNA oligomers containing 7-(aminomethyl)-7-deazaguanine (1) residues using a variety of spectroscopic and thermodynamic approaches. 1 is an intramolecular model for the major groove binding of cations and basic amino acid residues to G. In contrast to c(7)G, the tethering of a cation in the major groove using 1 affords DNA that is as, or more, stable than the corresponding unmodified DNA. The stabilization is associated with the folding enthalpy and hydration.

Energetic and hydration contributions of the removal of methyl groups from thymine to form uracil in G-quadruplexes.

A combination of spectroscopic and calorimetric techniques is used to investigate the unfolding of two G-quadruplexes: d(G2U2G2UGUG2U2G2), G2-U, and d(G2T2G2TGTG2T2G2), G2. The comparisons of their thermodynamic data allow us to elucidate the role of methylation on the energetic and hydration properties accompanying their stable formation. The favorable formation of each G-quadruplex results from the characteristic enthalpy-entropy compensation, uptake of ions, and release of water molecules. The loops of G2-U and G2 contribute favorably to their formation, and the absence of methyl groups stabilizes the G-quadruplex. The unfolding of G2-U produces a larger DeltaV, indicating a difference in the hydration states of the two oligonucleotides, while the opposite signs between DeltaDeltaG with the DeltaDeltaV suggest that the differential hydration reflects structural, or hydrophobic, water is involved in the unfolding of G-quadruplexes.

Unfolding thermodynamics of intramolecular G-quadruplexes: base sequence contributions of the loops.

G-quadruplexes are a highly studied DNA motif with a potential role in a variety of cellular processes and more recently are considered novel targets for drug therapy in aging and anticancer research. In this work, we have investigated the thermodynamic contributions of the loops on the stable formation of G-quadruplexes. Specifically, we use a combination of UV, circular dichroism (CD) and fluorescence spectroscopies, and differential scanning calorimetry (DSC) to determine thermodynamic profiles, including the differential binding of ions and water, for the unfolding of the thrombin aptamer: d(GGT2GGTGTGGT2GG) that is referred to as G2. The sequences in italics, TGT and T2, are known to form loops. Other sequences examined contained base substitutions in the TGT loop (TAT, TCT, TTT, TAPT, and UUU), in the T2 loops (T4, U2), or in both loops (UGU and U2, UUU and U2). The CD spectra of all molecules show a positive band centered at 292 nm, which corresponds to the "chair" conformation. The UV and DSC melting curves of each G-quadruplex show monophasic transitions with transition temperatures (T(M)s) that remained constant with increasing strand concentration, confirming their intramolecular formation. These G-quadruplexes unfold with T(M)s in the range from 43.2 to 56.5 degrees C and endothermic enthalpies from 22.9 to 37.2 kcal/mol. Subtracting the contribution of a G-quartet stack from each experimental profile indicated that the presence of the loops stabilize each G-quadruplex by favorable enthalpy contributions, larger differential binding of K+ ions (0.1-0.6 mol K+/ mol), and a variable uptake/release of water molecules (-6 to 8 mol H2O/mol). The thermodynamic contributions for these specific base substitutions are discussed in terms of loop stacking (base-base stacking within the loops) and their hydration effects.

A study of 7-deaza-2'-deoxyguanosine 2'-deoxycytidine base pairing in DNA.

The incorporation of 7-deazaguanine modifications into DNA is frequently used to probe protein recognition of H-bonding information in the major groove of DNA. While it is generally assumed that 7-deazaguanine forms a normal Watson-Crick base pair with cytosine, detailed thermodynamic and structural analyses of this modification have not been reported. The replacement of the 7-N atom on guanine with a C-H, alters the electronic properties of the heterocycle and eliminates a major groove cation-binding site that could affect the organization of salts and water in the major groove. We report herein the characterization of synthetic DNA oligomers containing 7-deazaguanine using a variety of complementary approaches: UV thermal melting, differential scanning calorimetry (DSC), circular dichroism (CD), chemical probing and NMR. The results indicate that the incorporation of a 7-deazaguanine modification has a significant effect on the dynamic structure of the DNA at the flanking residue. This appears to be mediated by changes in hydration and cation organization.