Hydration regulates thermodynamics of G-quadruplex formation under molecular crowding conditions

The effect of molecular crowding on the structure and stability of biomolecules has become a subject of increasing interest because it can clarify how biomolecules behave under cell-mimicking conditions. Here, we quantitatively analyzed the effects of molecular crowding on the thermodynamics of antiparallel G-quadruplex formation via Hoogsteen base pairs and of antiparallel hairpin-looped duplex (HP duplex) formation via Watson-Crick base pairs. The free energy change at 25 degrees C for G-quadruplex formation decreased from -3.5 to -5.5 kcal mol(-1) when the concentration of poly(ethylene glycol) 200 was increased from 0 to 40 wt %, whereas that of duplex formation increased from -9.8 to -6.9 kcal mol(-1). These results showed that the antiparallel G-quadruplex is stabilized under molecular crowding conditions, but that the HP duplex is destabilized. Moreover, plots of stability (ln K(obs)) of the DNA structures versus water activity (ln a(w)) demonstrated that the ln K(obs) for G-quadruplex formation decreased linearly as the ln a(w) increased, whereas that for duplex formation increased linearly with the increase in ln a(w), suggesting that the slope approximately equals the number of water molecules released or taken up during the formation of these structures. Thus, molecular crowding affects the thermodynamics of DNA structure formation by altering the hydration of the DNA. The stabilization of the DNA structures with Hoogsteen base pairs and destabilization of DNA structures with Watson-Crick base pairs under molecular crowding conditions lead to structural polymorphism of DNA sequences regulated by the state of hydration.     

Structural,Electronic, and Optical Properties of Metallo Base Pairs in Duplex DNA: A Theoretical Insight

Using density functional theory calculations, we investigated the structural, energetic, electronic, and optical properties of recently synthesized duplex DNA containing metal‐mediated base pairs. The studied duplex DNA consists of three imidazole (Im) units linked through metal (Im‐M‐Im, M=metal) and four flanking A:T base pairs (two on each side). We examined the role of artificial base pairing in the presence of two distinctive metal ions, diamagnetic Ag⁺ and magnetic Cu²⁺ ions, on the stability of duplex DNA. We found that metal‐mediated base pairs form stable duplex DNA by direct metal ion coordination to the Im bases. Our results suggest a higher binding stability of base pairing mediated by Cu²⁺ ions than by Ag⁺ ions, which is attributed to a larger extent of orbital hybridization. We furthermore found that DNA modified with Im‐Ag⁺‐Im shows the low‐energy optical absorption characteristic of π–π*orbital transition of WC A:T base pairs. On the other hand, we found that the low‐energy optical absorption peaks for DNA modified with Im‐Cu²⁺‐Im originate from spin–spin interactions. Additionally, this complex exhibits weak ferromagnetic coupling between Cu²⁺ ions and strong spin polarization, which could be used for memory devices. Moreover, analyzing the role of counter ions (Na⁺) and the presence of explicit water molecules on the structural stability and electronic properties of the DNA duplex modified with Im‐Ag⁺‐Im, we found that the impact of these two factors is negligible. Our results are fruitful for understanding the experimental data and suggest a potential route for constructing effective metal‐mediated base pairs in duplex DNA for optoelectronic applications.     

On the structure and dynamics of duplex GNA

Glycol nucleic acid (GNA), with a nucleotide backbone comprising of just three carbons and the stereocenter derived from propylene glycol (1,2-propanediol), is a structural analog of nucleic acids with intriguing biophysical properties, such as formation of highly stable antiparallel duplexes with high Watson-Crick base pairing fidelity. Previous crystallographic studies of double stranded GNA (dsGNA) indicated two forms of backbone conformations, an elongated M-type (containing metallo-base pairs) and the condensed N-type (containing brominated base pairs). A herein presented new crystal structure of a GNA duplex at 1.8 ? resolution from self-complementary 3'-CTC(Br)UAGAG-2' GNA oligonucleotides reveals an N-type conformation with alternating gauche-anti torsions along its (O3'-C3'-C2'-O2') backbone. To elucidate the conformational state of dsGNA in solution, molecular dynamic simulations over a period of 20 ns were performed with the now available repertoire of structural information. Interestingly, dsGNA adopts conformational states in solution intermediate between experimentally observed backbone conformations: simulated dsGNA shows the all-gauche conformation characteristic of M-type GNA with the higher helical twist common to N-type GNA structures. The so far counterintuitive, smaller loss of entropy upon duplex formation as compared to DNA can be traced back to the conformational flexibility inherent to dsGNA but missing in dsDNA. Besides extensive interstrand base stacking and conformational preorganization of single strands, this flexibility contributes to the extraordinary thermal stability of GNA.     

Monitoring G-quadruplex structures and G-quadruplex-ligand complex using 2-aminopurine modified oligonucleotides

The fluorescent base 2-aminopurine (2Ap) was incorporated into the human telomeric DNA sequence d[AGGG(TTAGGG)₃]. The substitution of 2Ap for A in the TTA loops did not affect the G-quadruplex stability. Interestingly, a significant change in the fluorescence intensity of 2Ap between the G-quadruplex and duplex was observed. Therefore, we demonstrated that 2Ap can be used to monitor the duplex to quadruplex conformational change in the human telomeric DNA sequence. This mechanism is explained by the difference in the base stacking in the TTA loop region. Moreover, these probes distinguished between the basket-type and propeller-type G-quadruplexes. We also demonstrated the detection of the telomerase inhibitor agent, such as TMPyP4, using a 2Ap modified telomeric DNA. The formation of the G-quadruplex-ligand complex was observed by the fluorescence titration of TMPyP4.     

Understanding the role of base stacking in nucleic acids. MD and QM analysis of tandem GA base pairs in RNA duplexes

Preceding NMR experiments show that the conformation of tandem GA base pairs, an important recurrent non-canonical building block in RNA duplexes, is context dependent. The GA base pairs adopt "sheared" N3(G)-N6(A), N2(G)-N7(A) geometry in the r(CGAG)(2) and r(iGGAiC)(2) contexts while switching to "imino" N1(G)-N1(A), O6(G)-N6(A) geometry in the r(GGAC)(2) and r(iCGAiG)(2) contexts (iC and iG stand for isocytosine and isoguanine, respectively). As base stacking is likely to be one of the key sources of the context dependence of the conformation of GA base pairs, we calculated base stacking energies in duplexes containing such base pairs, to see if this dependence can be predicted by stacking energy calculations. When investigating the context dependence of the GA geometry two different conformations of the same duplex were compared (imino vs. sheared). The geometries were generated via explicit solvent MD simulations of the respective RNA duplexes, while the subsequent QM energy calculations focused on base stacking interactions of the four internal base pairs. Geometrical relaxation of nucleobase atoms prior to the stacking energy computations has a non-negligible effect on the results. The stacking energies were derived at the DFT-D/6-311++G(3df,3pd) level. We show a rather good correspondence between the intrinsic gas-phase stacking energies and the NMR-determined GA geometries. The conformation with more favorable gas-phase stacking is in most cases the one observed in experiments. This correlation is not improved when including solvent effects via the COSMO method. On the other side, the stacking calculations do not predict the relative thermodynamic stability of duplex formation for different sequences.     

Intra- and intermolecular interactions between cyclic-AMP receptor protein and DNA: ab initio fragment molecular orbital study

The ab initio fragment molecular orbital (FMO) calculations were performed for the cAMP receptor protein (CRP) complexed with a cAMP and DNA duplex to elucidate their sequence-specific binding and the stability of the DNA duplex, as determined by analysis of their inter- and intramolecular interactions. Calculations were performed with the AMBER94 force field and at the HF and MP2 levels with several basis sets. The interfragment interaction energies (IFIEs) were analyzed for interactions of CRP-cAMP with each base pair, DNA duplex with each amino acid residue, and each base pair with each residue. In addition, base-base interactions were analyzed including hydrogen bonding and stacking of DNA. In the interaction between DNA and CRP-cAMP, there was a significant charge transfer (CT) from the DNA to CRP, and this CT interaction played an important role as well as the electrostatic interactions. It is necessary to apply a quantum mechanical approach beyond the "classical" force-field approach to describe the sequence specificity. In the DNA intramolecular interaction, the dispersion interactions dominated the stabilization of the base-pair stacking interactions. Strong, attractive 1,2-stacking interactions and weak, repulsive 1,3-stacking interactions were observed. Comparison of the intramolecular interactions of free and complexed DNA revealed that the base-pairing interactions were stronger, and the stacking interactions were weaker, in the complexed structure. Therefore, the DNA duplex stability appears to change due to both the electrostatic and the CT interactions that take place under conditions of DNA-CRP binding.     

Locked nucleic acid (LNA) recognition of RNA: NMR solution structures of LNA:RNA hybrids

Locked nucleic acids (LNAs) containing one or more 2'-O,4'-C-methylene-linked bicyclic ribonucleoside monomers possess a number of the prerequisites of an effective antisense oligonucleotide, e.g. unprecedented helical thermostability when hybridized with cognate RNA and DNA. To acquire a detailed understanding of the structural features of LNA giving rise to its remarkable properties, we have conducted structural studies by use of NMR spectroscopy and now report high-resolution structures of two LNA:RNA hybrids, the LNA strands being d(5'-CTGAT(L)ATGC-3') and d(5'-CT(L)GAT(L)AT(L)GC-3'), respectively, T(L) denoting a modified LNA monomer with a thymine base, along with the unmodified DNA:RNA hybrid. In the structures, the LNA nucleotides are positioned as to partake in base stacking and Watson-Crick base pairing, and with the inclusion of LNA nucleotides, we observe a progressive change in duplex geometry toward an A-like duplex structure. As such, with the inclusion of three LNA nucleotides, the hybrid adopts an almost canonical A-type duplex geometry, and thus it appears that the number of modifications has reached a saturation level with respect to structural changes, and that further incorporations would furnish only minute changes in the duplex structure. We attempt to rationalize the conformational steering induced by the LNA nucleotides by suggesting that the change in electronic density at the brim of the minor groove, introduced by the LNA modification, is causing an alteration of the pseudorotational profile of the 3'-flanking nucleotide, thus shifting this sugar equilibrium toward N-type conformation.     

NMR study on the B-Z junction formation of DNA duplexes induced by Z-DNA binding domain of human ADAR1

Z-DNA is produced in a long genomic DNA by Z-DNA binding proteins, through formation of two B-Z junctions with the extrusion of one base pair from each junction. To answer the question of how Z-DNA binding proteins induce B-Z transitions in CG-rich segments while maintaining the B-conformation of surrounding segments, we investigated the kinetics and thermodynamics of base-pair openings of a 13-bp DNA in complex with the Z-DNA binding protein, Zα(ADAR1). We also studied perturbations in the backbone of Zα(ADAR1) upon binding to DNA. Our study demonstrates the initial contact conformation as an intermediate structure during B-Z junction formation induced by Zα(ADAR1), in which the Zα(ADAR1) protein displays unique backbone conformational changes, but the 13-bp DNA duplex maintains the B-form helix. We also found the unique structural features of the 13-bp DNA duplex in the initial contact conformation: (i) instability of the AT-rich region II and (ii) longer lifetime for the opening state of the CG-rich region I. Our findings suggest a three-step mechanism of B-Z junction formation: (i) Zα(ADAR1) specifically interacts with a CG-rich DNA segment maintaining B-form helix via a unique conformation; (ii) the neighboring AT-rich region becomes very unstable, and the CG-rich DNA segment is easily converted to Z-DNA; and (iii) the AT-rich regions are base-paired again, and the B-Z junction structure is formed.     

Solution structure of a DNA duplex containing a biphenyl pair

Hydrogen-bonding and stacking interactions between nucleobases are considered to be the major noncovalent interactions that stabilize the DNA and RNA double helices. In recent work we found that one or multiple biphenyl pairs, devoid of any potential for hydrogen bond formation, can be introduced into a DNA double helix without loss of duplex stability. We hypothesized that interstrand stacking interactions of the biphenyl residues maintain duplex stability. Here we present an NMR structure of the decamer duplex d(GTGACXGCAG) d(CTGCYGTCAC) that contains one such X/Y biaryl pair. X represents a 3',5'-dinitrobiphenyl- and Y a 3',4'-dimethoxybiphenyl C-nucleoside unit. The experimentally determined solution structure shows a B-DNA duplex with a slight kink at the site of modification. The biphenyl groups are intercalated side by side as a pair between the natural base pairs and are stacked head to tail in van der Waals contact with each other. The first phenyl rings of the biphenyl units each show tight intrastrand stacking to their natural base neighbors on the 3'-side, thus strongly favoring one of two possible interstrand intercalation structures. In order to accommodate the biphenyl units in the duplex the helical pitch is widened while the helical twist at the site of modification is reduced. Interestingly, the biphenyl rings are not static in the duplex but are in dynamic motion even at 294 K.     

A breathing wormlike chain model on DNA denaturation and bubble: effects of stacking interactions

DNA stably exists as a double-stranded structure due to hydrogen-bonding and stacking interactions between bases. The stacking interactions are strengthened when DNA is paired, which results in great enhancement of bending rigidity. We study the effects of this stacking-induced stiffness difference on DNA denaturation and bubble formations. To this end, we model double-stranded DNA as a duplex of two semiflexible chains whose persistence length varies depending on the base-pair distance. Using this model, we perform the Langevin dynamics simulation to examine the characteristics of the denaturation transition and the statistics of the bubbles. We find that the inclusion of the stacking interactions causes the denaturation transition to be much sharper than otherwise. At physiological temperature, the stacking interactions prohibit the initiation of bubble formation but promote bubbles, once grown, to retain the large size.     

Site-selective RNA cleavage by DNA bearing a base pair-mimic nucleoside

We have synthesized the deoxyadenosine derivative tethering a phenyl group (X), which mimics the Watson-Crick A/T base pair. The RNA/DNA hybrid duplexes containing X in the middle of the DNA sequence showed a similar thermal stability regardless of the ribonucleotide species (A, G, C, or U) opposite to X, probably because of the phenyl group stacking inside of the duplex accompanied by the opposite ribonucleotide base flipped in an extrahelical position. The RNA strand hybridized with the DNA strand bearing X was cleaved on the 3'-side of the ribonucleotide opposite to X in the presence of MgCl2, and the RNA sequence to be cleaved was not restricted. The site-specific RNA hydrolysis suggests that the DNA strand bearing X has the advantage of the site-selective base flipping in the target sequence and the development of a "universal deoxyribozyme" to exclusively cleave a target RNA sequence.     

Cyclohexyl "base pairs" stabilize duplexes and intensify pyrene fluorescence by shielding it from natural base pairs

In this study, we investigated the stability and structure of artificial base pairs that contain cyclohexyl rings. The introduction of a single pair of isopropylcyclohexanes into the middle of DNA slightly destabilized the duplex. Interestingly, as the number of the "base pairs" increased, the duplex was remarkably stabilized. A duplex with six base pairs was even more stable than one containing six A-T pairs. Thermodynamic analysis revealed that changes in entropy and not enthalpy contributed to duplex stability, demonstrating that hydrophobic interactions between isopropyl groups facilitated the base pairing, and thus stabilized the duplex. NOESY of a duplex containing an isopropylcyclohexane-methylcyclohexane pair unambiguously demonstrated its "pairing" in the duplex because distinct NOEs between the protons of cyclohexyl moieties and imino protons of both of the neighboring natural base pairs were observed. CD spectra of duplexes tethering cyclohexyl moieties also showed a positive-negative couplet that is characteristic of the B-form DNA duplex. Taken together, these results showed that cyclohexyl moieties formed base pairs in the DNA duplex without severely disturbing the helical structure of natural DNA. Next, we introduced cyclohexyl base pairs between pyrene and nucleobases as an "insulator" that suppresses electron transfer between them. We found a massive increase in the quantum yield of pyrene due to the efficient shielding of pyrene from nucleobases. The cyclohexyl base pairs reported here have the potential to prepare highly fluorescent labeling agents by multiplying fluorophores and insulators alternately into DNA duplexes.     

Theoretical determination of one-electron redox potentials for DNA bases, base pairs, and stacks

Electron affinities, ionization potentials, and redox potentials for DNA bases, base pairs, and N-methylated derivatives are computed at the DFT/M06-2X/6-31++G(d,p) level of theory. Redox properties of a guanine-guanine stack model are explored as well. Reduction and oxidation potentials are in good agreement with the experimental ones. Electron affinities of base pairs were found to be negative. Methylation of canonical bases affects the ionization potentials the most. Base pair formation and base stacking lower ionization potentials by 0.3 eV. Pairing of guanine with the 5-methylcytosine does not seem to influence the redox properties of this base pair much.     

Robust charge transport in DNA double crossover assemblies

BACKGROUND: Multiple-stranded DNA assemblies, encoded by sequence, have been constructed in an effort to self-assemble nanodevices of defined molecular architecture. Double-helical DNA has been probed also as a molecular medium for charge transport. Conductivity studies suggest that DNA displays semiconductor properties, whereas biochemical studies have shown that oxidative damage to B-DNA at the 5'-G of a 5'-GG-3' doublet can occur by charge transport through DNA up to 20 nm from a photo-excited metallointercalator. The possible application of DNA assemblies, in particular double crossover (DX) molecules, in electrical nanodevices prompted the design of a DNA DX assembly with oxidatively sensitive guanine moieties and a tethered rhodium photo-oxidant strategically placed to probe charge transport. RESULTS: DX assemblies support long-range charge transport selectively down the base stack bearing the intercalated photo-oxidant. Despite tight packing, no electron transfer (ET) crossover to the adjacent base stack is observed. Moreover, the base stack of a DX assembly is well-coupled and less susceptible than duplex DNA to stacking perturbations. Introducing a double mismatch along the path for charge transport entirely disrupts long-range ET in duplex DNA, but only marginally decreases it in the analogous stack within DX molecules. CONCLUSIONS: The path for charge transport in a DX DNA assembly is determined directly by base stacking. As a result, the two closely packed stacks within this assembly are electronically insulated from one another. Therefore, DX DNA assemblies may serve as robust, insulated conduits for charge transport in nanoscale devices.     

Construction of photoresponsive RNA for photoswitching RNA hybridization

By introducing azobenzenes into RNA using d-threoninol as a scaffold, a photoresponsive RNA was constructed for efficiently photoswitching the formation and dissociation of RNA/RNA duplexes. The difference in melting temperature (T(m)) between the trans and cis forms was so large that efficient photoregulation of RNA hybridization became possible, irrespective of the sequence adjacent to the introduced azobenzene. Compared to the corresponding photoresponsive DNA, the photoregulatory efficiency of azobenzene-modified RNA was even higher due to the drastic destabilization by cis-azobenzene. Structural analysis by NMR and molecular modeling indicated that the planar trans-azobenzene could not stabilize the RNA/RNA duplex with a rigid A-form structure by base pair stacking. However, the large steric hindrance caused by nonplanar cis-azobenzene was quite effective at distorting and destabilizing the duplex structure. We also discuss the effect of methylation of azobenzene at the ortho positions on photoregulation of RNA/RNA duplex formation. This newly constructed photoresponsive RNA has promising applications such as photoswitching of RNA functions.     

Solution structure of xDNA: a paired genetic helix with increased diameter

We describe the structure in aqueous solution of an extended-size DNA-like duplex with base pairs that are approximately 2.4 A longer than those of DNA. Deoxy-lin-benzoadenosine (dxA) was employed as a dA analogue to form hydrogen-bonded base pairs with dT. The 10mer self-complementary extended oligodeoxynucleotide 5'-d(xATxAxATxATTxAT) forms a much more thermodynamically stable duplex than the corresponding DNA sequence, 5'-d(ATAATATTAT). NMR studies show that this extended DNA (xDNA) retains many features of natural B-form DNA, but with a few structural alterations due to its increased helical diameter. The results give insight into the structural plasticity of the natural DNA backbone and lend insight into the evolutionary origins of the natural base pairs. Finally, this structural study confirms the hypothesis that extended nucleobase analogues can form stable DNA-like structures, suggesting that alternative genetic systems might be viable for storage and transfer of genetic information.     

Spatial Structure of a DNA Duplex d(CGTTTATT)p-Net:d(AATAAACG) with an Unusual Localization of a Covalently Bound Netropsin (Net) Analog According to Two-Dimensional NMR Spectroscopy

Two-dimensional ¹H NMR (NOESY, COSY) spectroscopy was used to study the spatial structure of a DNA complex CGTTTATTp-Net:AATAAACG with a netropsin analog bound at the terminal 3-phosphate group in aqueous solution. The positions of the proton NMR signals of the duplex are compared with those for the unmodified duplex and the free ligand. A mean conformation of the duplex is constructed by the molecular mechanics technique using the obtained NMR limitations on the interproton distances. The data obtained indicate that the aromatic pyrrole rings of the netropsin analog are involved in a stacking interaction with the terminal AT base pair of the duplex; this is generally not typical of ordinary minor-groove netropsin type ligands.     

yDNA versus xDNA pyrimidine nucleobases: computational evidence for dependence of duplex stability on spacer location

The structural and binding properties of the natural and x- and y-pyrimidines were compared using computational methods. Our calculations show that although the x-pyrimidines favor different orientations about the glycosidic bond compared to the natural pyrimidines, which could have implications for the formation and resulting stability of xDNA duplexes and jeopardize the selectivity of expanded nucleobases, y-pyrimidines have rotational profiles more similar to the natural bases. Increasing the pyrimidine size using a benzene spacer leads to relatively minor changes in the hydrogen-bond strength of isolated Watson-Crick base pairs. However, differences in the anomeric carbon distances in pairs composed of x- or y-pyrimidines suggest yDNA may yield a more optimal expanded structure. By stacking two monomers via their centers of mass, we find that the expanded nucleobases stack much stronger than the natural bases. Additionally, although replacing xT by yT changes the stacking energy by less than 5 kJ mol (-1), replacing xC by yC significantly strengthens complexes with the natural nucleobases (by up to 30%). Calculations on larger duplex models composed of four nucleobases reveal that x- and y-pyrimidines can increase duplex stability of natural helices by strengthening both the intra and interstrand stacking interactions. Furthermore, when the total stability (sum of all hydrogen-bonding and (intrastrand and interstrand) stacking interactions) of the larger models is considered, y-pyrimidines lead to more stable complexes than x-pyrimidines for all but three duplex sequences. Thus, through analysis of a variety of properties, our calculations suggest that the location of the benzene spacer affects the properties of expanded nucleobases and the stability of expanded duplexes, and therefore should be carefully considered when designing future expanded analogues.     

Synthesis of abasic locked nucleic acid and two seco-LNA derivatives and evaluation of their hybridization properties compared with their more flexible DNA counterparts

To investigate the structural basis of the unique hybridization properties of LNA (locked nucleic acid) three novel LNA derivatives with modified carbohydrate parts were synthesized and evaluated with respect to duplex stabilities. The abasic LNA monomer (X(L), Figure 1) with the rigid carbohydrate moiety of LNA but no nucleobase attached showed no enhanced duplex stabilities compared to its more flexible abasic DNA counterpart (X, Figure 1). These results suggest that the exceptional hybridization properties of LNA primarily originate from improved intrastrand nucleobase stacking and not backbone preorganization. Two monocyclic seco-LNA derivatives, obtained by cleavage of the C1'-O4' bond of an LNA monomer or complete removal of the O4'-furanose oxygen atom (Z(L) and dZ(L), respectively, Figure 1), were compared to their acyclic DNA counterpart (Z, Figure 1). Even though they are more constrained than Z, the seco-LNA derivatives Z(L) and dZ(L) destabilize duplex formation even more than the flexible seco-DNA monomer Z.