Alpha- and beta-stilbenosides as base-pair surrogates in DNA hairpins

The synthesis, structure, and optical spectroscopy of hairpin oligonucleotide conjugates possessing synthetic stilbene C-nucleosides (stilbenosides) are reported. Synthetic methods for selective preparation of both the alpha- and beta-stilbenosides have been developed. Both anomers are effective in stabilizing hairpin structures when used as capping groups at the open end of the hairpin base-pair domain. However, only the beta-anomer effectively stabilizes the hairpin structure when located in the interior of the base-pair domain opposite an abasic site. Similar results are obtained for hairpins possessing two stilbenosides, either adjacent to each other or with one intervening base-pair. Molecular dynamics simulations are employed to obtain averaged structures for these conjugates. The calculated structures for the capped hairpins formed with either anomer show effective pi-stacking with the adjacent base-pair. The calculated structures for the internal stilbenosides show that the alpha- and beta-anomers form extrahelical and intrahelical structures, respectively. The relative orientations of the two stilbenes in the bis-stilbenosides have been studied using a combination of exciton-coupled circular dichroism spectroscopy and molecular modeling.     

Additional base-pair formation in DNA duplexes by a double-headed nucleotide

We have designed and synthesised a double-headed nucleotide that presents two nucleobases in the interior of a dsDNA duplex. This nucleotide recognises and forms Watson-Crick base pairs with two complementary adenosines in a Watson-Crick framework. Furthermore, with judicious positioning in complementary strands, the nucleotide recognises itself through the formation of a T:T base pair. Thus, two novel nucleic acid motifs can be defined by using our double-headed nucleotide. Both motifs were characterised by UV melting experiments, CD and NMR spectroscopy and molecular dynamics simulations. Both motifs leave the thermostability of the native dsDNA duplex largely unaltered. Molecular dynamics calculations showed that the double-headed nucleotides are accommodated in the dsDNA by entirely local perturbations and that the modified duplexes retain an overall B-type geometry with the dsDNA unwound by around 25 or 60°, respectively, in each of the modified motifs. Both motifs can be accommodated twice in a dsDNA duplex without incurring any loss of stability and extrapolating from this observation and the results of modelling, it is conceivable that both can be multiplied several times within a dsDNA duplex. These new motifs extend the DNA recognition repertoire and may form the basis for a complete series of double-headed nucleotides based on all 16 base combinations of the four natural nucleobases. In addition, both motifs can be used in the design of nanoscale DNA structures in which a specific duplex twist is required.     

Dynamics of electron injection in DNA hairpins

The dynamics of electron injection has been investigated in DNA hairpins possessing a stilbenediether electron donor linker by means of femtosecond transient absorption spectroscopy. Ultrafast electron injection and charge recombination are observed with neighboring cytosine or thymine bases; however, guanine-guanine base pairs are not reduced, permitting the investigation of the distance dependence of charge injection.     

Thermodynamic coupling of the loop and stem in unusually stable DNA hairpins closed by CG base pairs

For certain DNA hairpin loops, a CG closing base pair has enhanced stability over other closing base pairs, which cannot be explained by the current nearest-neighbor model. We report the use of three-carbon (C3) spacers to investigate the expandability of DNA hairpin loops and the coupling between the loop and closing base pair. Inserting the C3-spacers at most positions in these model loops produced only a modest stabilization or destabilization except for insertion between the 5' end of the loop and the CG closing base pair, which gave a large destabilization. Further investigation on tetraloops and triloops with other closing base pairs established that this destabilization is specific to the unusually stable CG closing base pair. Studies with the nucleotide analogues 2-aminopurine and 2,6-diaminopurine indicated that this stabilization may be due to coupling between functional groups on the first base of the loop and the CG closing base pair. The C3-spacers provide a simple way to interrupt potential interactions and thereby probe loop/stem coupling.     

Dynamics and energetics of single-step hole transport in DNA hairpins

The dynamics of single-step hole transport processes have been investigated in a number of DNA conjugates possessing a stilbenedicarboxamide electron acceptor, a guanine primary donor, and several secondary donors. Rate constants for both forward and return hole transport between the primary and secondary donor are obtained from kinetic modeling of the nanosecond transient absorption decay profiles of the stilbene anion radical. The kinetic model requires that the hole be localized on either the primary or the secondary donor and not delocalized over both the primary and the secondary donor. Rate constants for hole transport are found to be dependent upon the identity of the secondary donor, the intervening bases, and the location of the secondary donor in the same strand as the primary donor or in the complementary strand. Rate constants for hole transport are much slower than those for the superexchange process used to inject the hole on the primary donor. This difference is attributed to the larger solvent reorganization energy for charge transport versus charge separation. The hole transport rate constants obtained in these experiments are consistent with experimental data for single-step hole transport from other transient absorption studies. Their relevance to long-distance hole migration over tens of base pairs remains to be determined. The forward and return hole transport rate constants provide equilibrium constants and free energies for hole transport equilibria. Secondary GG and GGG donors are found to form very shallow hole traps, whereas the nucleobase deazaguanine forms a relatively deep hole trap. This conclusion is in accord with selected strand cleavage data and thus appears to be representative of the behavior of holes in duplex DNA. Our results are discussed in the context of current theoretical models of hole transport in DNA.     

Dynamics of inter- and intrastrand hole transport in DNA hairpins

The dynamics of photoinduced electron transfer has been investigated in DNA hairpins possessing a stilbenedicarboxamide (Sa) electron acceptor, a guanine (G) primary donor, and two adjacent guanines (GG) as secondary donors. Hole transport from G to GG across a single A is more rapid than across AA or T by factors of 20 +/- 7 and 40 +/- 15, respectively. Intrastrand hole transport across a single A is more rapid than interstrand transport by a factor of 7 +/- 3.     

Charge-transfer and spin dynamics in DNA hairpin conjugates with perylenediimide as a base-pair surrogate

A perylenediimide chromophore (P) was incorporated into DNA hairpins as a base-pair surrogate to prevent the self-aggregation of P that is typical when it is used as the hairpin linker. The photoinduced charge-transfer and spin dynamics of these hairpins were studied using femtosecond transient absorption spectroscopy and time-resolved EPR spectroscopy (TREPR). P is a photooxidant that is sufficiently powerful to quantitatively inject holes into adjacent adenine (A) and guanine (G) nucleobases. The charge-transfer dynamics observed following hole injection from P into the A-tract of the DNA hairpins is consistent with formation of a polaron involving an estimated 3-4 A bases. Trapping of the (A 3-4) (+*) polaron by a G base at the opposite end of the A-tract from P is competitive with charge recombination of the polaron and P (-*) only at short P-G distances. In a hairpin having 3 A-T base pairs between P and G ( 4G), the radical ion pair that results from trapping of the hole by G is spin-correlated and displays TREPR spectra at 295 and 85 K that are consistent with its formation from (1*)P by the radical-pair intersystem crossing mechanism. Charge recombination is spin-selective and produces (3*)P, which at 85 K exhibits a spin-polarized TREPR spectrum that is diagnostic of its origin from the spin-correlated radical ion pair. Interestingly, in a hairpin having no G bases ( 0G), TREPR spectra at 85 K revealed a spin-correlated radical pair with a dipolar interaction identical to that of 4G, implying that the A-base in the fourth A-T base pair away from the P chromophore serves as a hole trap. Our data suggest that hole injection and transport in these hairpins is completely dominated by polaron generation and movement to a trap site rather than by superexchange. On the other hand, the barrier for charge injection from G (+*) back onto the A-T base pairs is strongly activated, so charge recombination from G (or even A trap sites at 85 K) most likely proceeds by a superexchange mechanism.     

Coumarin base-pair replacement as a fluorescent probe of ultrafast DNA dynamics

The design and synthesis of a novel coumarin C-riboside are described, and is based on the well-known photoprobe Coumarin 102. A diastereofacial selective Heck coupling between a furanoid glycal and a coumarin triflate provided a method for glycoside formation. The coumarin C-glycoside was incorporated synthetically into DNA oligomers, and was used to probe ultrafast dynamics of duplex DNA using time-resolved Stokes shift methods.     

Spectroscopic and structural characterization of pascoite

Pascoite mineral having yellow-orange colour of Colorado, USA origin has been characterized by EPR, optical and NIR spectroscopy. The colour dark red-orange to yellow-orange colour of the pascoite indicates that the mineral contain mixed valency of vanadium. The optical spectrum exhibits a number of electronic bands due to presence of VO(II) ions in the mineral. From EPR studies, the parameters of g, A are evaluated and the data confirm that the ion is in distorted octahedron. Optical absorption studies reveal that two sets of VO(II) is in distorted octahedron. The bands in NIR spectra are due to the overtones and combinations of water molecules.     

PNA versus DNA: effects of structural fluctuations on electronic structure and hole-transport mechanisms

The effects of structural fluctuations on charge transfer in double-stranded DNA and peptide nucleic acid (PNA) are investigated. A palindromic sequence with two guanine bases that play the roles of hole donor and acceptor, separated by a bridge of two adenine bases, was analyzed using combined molecular dynamics (MD) and quantum-chemical methods. Surprisingly, electronic structure calculations on individual MD snapshots show significant frontier orbital electronic population on the bridge in approximately 10% of the structures. Electron-density delocalization to the bridge is found to be gated by fluctuations of the covalent conjugated bond structure of the aromatic rings of the nucleic bases. It is concluded, therefore, that both thermal hopping and superexchange should contribute significantly to charge transfer even in short DNA/PNA fragments. PNA is found to be more flexible than DNA, and this flexibility is predicted to produce larger rates of charge transfer.     

Detection of a single DNA base-pair mismatch using an anthracene-tagged fluorescent probe

A novel anthracene-tagged oligonucleotide can discriminate between a fully-matched DNA target sequence and one with a single mismatching base-pair through a remarkable difference in fluorescence emission intensity upon duplex formation.     

Formation and decay of localized contact radical ion pairs in DNA hairpins

The dynamics of charge separation and charge recombination in synthetic DNA hairpins possessing diphenylacetylene-4,4'-dicarboxamide linkers have been investigated by means of femtosecond time-resolved transient absorption spectroscopy. The lowest excited singlet state of the linker is capable of oxidizing nearest neighbor adenine as well as guanine. A large wavelength shift in the transient absorption spectrum accompanies the conversion of the singlet linker to its anion radical, facilitating the investigation of electron-transfer dynamics. The rate constants for charge separation are dependent upon the oxidation potentials of the neighboring nucleobase donors but not upon the identity of nonnearest neighbors. Thus, the charge separation processes yield a contact radical ion pair in which the positive charge is localized on the neighboring nucleobase. Rate constants for charge recombination are dependent upon the identity of the first and second nearest-neighbor nucleobases but not more remote bases. This dependence is attributed to stabilization of the contact radical ion pair by interaction with its nearest neighbor. The absence of charge migration to form a base-pair separated radical ion pair is a consequence of Coulombic attraction in the contact radical ion pair and the low effective dielectric constant (epsilon < 7) experienced by the contact radical ion pair. Photoinduced charge injection to form a base-pair separated radical ion pair is necessary in order to observe charge migration.     

Cationic modified nucleic acids for use in DNA hairpins and parallel triplexes

Non-nucleosidic DNA monomers comprising partially protonated amines at low pH have been designed and synthesized. The modifications were incorporated into DNA oligonucleotides via standard DNA phosphoramidite synthesis. The ability of cationic modifications to stabilize palindromic DNA hairpins and parallel triplexes were evaluated using gel electrophoresis, circular dichroism and thermal denaturation measurements. The non-nucleosidic modifications were found to increase the thermal stability of palindromic hairpins at pH 8.0 as compared with a nucleosidic tetraloop (TCTC). Incorporation of modifications at the 5'-end of a triplex forming oligonucleotide resulted in a significant increase in thermal stability at low pH when the modifications were placed as the 5'-dangling end.     

Development of donor-acceptor modified DNA hairpins for the investigation of charge hopping kinetics in DNA

Investigation of hole or excess electron hopping in DNA is mostly performed based on yield studies, in which an injector modified DNA duplex is irradiated to continuously inject either holes or electrons into the duplex. Observed is a chemical reaction of a "probe" molecule, which can be either one of the two purine bases or a different trap molecule positioned at various distances. The next step in the field will be the direct time resolution of the hole or electron transfer kinetics in DNA. Herein we describe the development of defined donor-DNA-acceptor systems, with properties that may allow time resolved electron and hole transfer studies in stably folded DNA structures.     

Investigation of the pathways of excess electron transfer in DNA with flavin-donor and oxetane-acceptor modified DNA hairpins

Oxetane is a potential intermediate that is enzymatically formed during the repair of (6-4) DNA lesions by special repair enzymes (6-4 DNA photolyases). These enzymes use a reduced and deprotonated flavin to cleave the oxetane by single electron donation. Herein we report synthesis of DNA hairpin model compounds containing a flavin as the hairpin head and two different oxetanes in the stem structure of the hairpin. The data show that the electron moves through the duplex even over distances of 17 A. Attempts to trap the moving electron with N2O showed no reduction of the cleavage efficiency showing that the electron moves through the duplex and not through solution. The electron transfer is sequence dependent. The efficiency is reduced by a factor of 2 in GC rich DNA hairpins.     

Millisecond time-scale folding and unfolding of DNA hairpins using rapid-mixing stopped-flow kinetics

We report stopped-flow kinetics experiments to study the folding and unfolding of 5 base-pair stem and 21 nucleotide polythymidine loop DNA hairpins over various concentrations of NaCl. The reactions occurred on a time scale of milliseconds, considerably longer than the microsecond time scale suggested by previous kinetics studies of similar-sized hairpins. In comparison to a recent fluorescence correlation spectroscopy study (J. Am. Chem. Soc. 2006, 128, 1240-1249), we suggest the microsecond time-scale reactions are due to intermediate states and the millisecond time-scale reactions reported here are due to the formation of the fully folded DNA hairpin. These results support our view that DNA hairpin folding occurs via a minimum three-state mechanism.     

2-Aminopurine: a probe of structural dynamics and charge transfer in DNA and DNA:RNA hybrids

Spectroscopic techniques are employed to probe relationships between structural dynamics and charge transfer (CT) efficiency in DNA duplexes and DNA:RNA hybrids containing photoexcited 2-aminopurine (Ap). To better understand the variety of interactions and reactions, including CT, between Ap and DNA, the fluorescence behavior of Ap is investigated in a full series of redox-inactive as well as redox-active assemblies. Thus, Ap is developed as a dual reporter of structural dynamics and base-base CT reactions in nucleic acid duplexes. CD, NMR, and thermal denaturation profiles are consistent with the family of DNA duplexes adopting a distinct conformation versus the DNA:RNA hybrids. Fluorescence measurements establish that the d(A)-r(U) tract of the DNA:RNA hybrid exhibits enhanced structural flexibility relative to that of the d(A)-d(T) tract of the DNA duplexes. The yield of CT from either G or 7-deazaguanine (Z) to Ap in the assemblies was determined by comparing Ap emission in redox-active G- or Z-containing duplexes to otherwise identical duplexes in which the G or Z is replaced by inosine (I), the redox-inactive nucleoside analogue. Investigations of CT not only demonstrate efficient intrastrand base-base CT in the DNA:RNA hybrids but also reveal a distance dependence of CT yield that is more shallow through the d(A)-r(U) bridge of the A-form DNA:RNA hybrids than through the d(A)-d(T) bridge of the B-form DNA duplexes. The shallow distance dependence of intrastrand CT in DNA:RNA hybrids correlates with the increased conformational flexibility of bases within the hybrid duplexes. Measurements of interstrand base-base CT provide another means to distinguish between the A- and B-form helices. Significantly, in the A-form DNA:RNA hybrids, a similar distance dependence is obtained for inter- and intrastrand reactions, while, in B-DNA, a more shallow distance dependence is evident with interstrand CT reactions. These observations are consistent with evaluations of intra- and interstrand base overlap in A- versus B-form duplexes. Overall, these data underscore the sensitivity of CT chemistry to nucleic acid structure and structural dynamics.