Use of abasic site-containing DNA strands for nucleobase recognition in water

Nucleobase recognition in water is successfully achieved by the use of an abasic site (AP site) as the molecular recognition field. We intentionally construct the AP site in DNA duplex so as to orient the AP site toward a target nucleobase and examine the complexation of 2-amino-7-methylnaphthyridine (AMND) with nucleobases at the AP site. AMND is found to selectively bind to cytosine (C) base with a 1:1 binding constant of >106 M-1, accompanied by remarkable quenching of its fluorescence. In addition to hydrogen bonding, a stacking interaction with nucleobases flanking the AP site seems responsible for the binding properties of AMND at the AP site. Possible use of AMND is also presented for selective and visible detection of a single-base alternation related to the cytosine base.     

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe.     

Configurational specificity of stacking interactions in DNA base pairs: A computational analysis

A theoretical study of stacking patterns of various hydrogen-bonded base pair complexes has been undertaken. Modified Rayleigh-Schrodinger perturbation theory for intermediate range interactions, has been employed to evaluate the stacking interactions using multicentered-multipole expansion method. Net atomic charge and corresponding dipole components located at each of the atomic centers have been computed by CNDO/2 method. An analysis of the intermolecular forces involved in the stable formation of the various base pair complexes, has been presented and the results have been discussed in the light of experimental as well as other theoretical observations. The possibility of relative preference of the left-handed configuration for alternating sequences has been quantitatively explored.     

Detection of single base mismatches and abasic sites using phenanthridinium as an artificial DNA base and charge donor

Combining the fluorescence properties of phenanthridinium as an artificial DNA base together with DNA-mediated charge transfer processes allows the homogeneous detection of DNA base mismatches and abasic sites.     

Theoretical investigation on DNA/RNA base pairs mediated by copper, silver, and gold cations

B3LYP density functional based computations were performed in order to characterize the interactions present in some Cu(+), Ag(+), and Au(+) metal ion-mediated DNA and RNA base pairs from both structural and electronic points of view. Examined systems involve as ligands canonical Watson-Crick, Hoogsteen and Wobble base pairs. Two artificial Hoogsteen base pairs were also taken into account. Binding energy values indicate that complexes involving silver cations are less stable than those in which copper or gold are present, and propose a similar behaviour for these two latter ions. The nature of the bond linking metal ions and bases was described by the NBO analysis that suggests metal coordinative interactions to be covalent. An evaluation of the dispersion contributions for the investigated systems was performed with the B3LYP-D3 functional.     

Stabilization by extra-helical thymines of a DNA duplex with Hoogsteen base pairs

We present the crystal structure of the DNA duplex formed by d(ATATATCT). The crystals contain seven stacked antiparallel duplexes in the asymmetric unit with A.T Hoogsteen base pairs. The terminal CT sequences bend over so that the thymines enter the minor groove and form a hydrogen bond with thymine 2 of the complementary strand in the Hoogsteen duplex. Cytosines occupy extra-helical positions; they contribute to the crystal lattice through various kinds of interactions, including a unique CAA triplet. The presence of thymine in the minor groove apparently contributes to the stability of the DNA duplex in the Hoogsteen conformation. These observations open the way toward finding under what conditions the Hoogsteen duplex may be stabilized in vivo. The present crystal structure also confirms the tendency of A.T-rich oligonucleotides to crystallize as long helical stacks of duplexes.     

Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein

Background

Single-stranded DNA binding proteins (SSB) are essential for DNA replication, repair, and recombination in all organisms. SSB works in concert with a variety of DNA metabolizing enzymes such as DNA polymerase.

Results

We have cloned and purified SSB from Bacillus anthracis (SSB_BA). In the absence of DNA, at concentrations ??100 ??g/ml, SSB_BA did not form a stable tetramer and appeared to resemble bacteriophage T4 gene 32 protein. Fluorescence anisotropy studies demonstrated that SSB_BA bound ssDNA with high affinity comparable to other prokaryotic SSBs. Thermodynamic analysis indicated both hydrophobic and ionic contributions to ssDNA binding. FRET analysis of oligo(dT)₇₀ binding suggested that SSB_BA forms a tetrameric assembly upon ssDNA binding. This report provides evidence of a bacterial SSB that utilizes a novel mechanism for DNA binding through the formation of a transient tetrameric structure.

Conclusions

Unlike other prokaryotic SSB proteins, SSB_BA from Bacillus anthracis appeared to be monomeric at concentrations ??100 ??g/ml as determined by SE-HPLC. SSB_BA retained its ability to bind ssDNA with very high affinity, comparable to SSB proteins which are tetrameric. In the presence of a long ssDNA template, SSB_BA appears to form a transient tetrameric structure. Its unique structure appears to be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to perturbation of stable dimer and tetramer formation. The structural features of SSB_BA could promote facile assembly and disassembly of the protein-DNA complex required in processes such as DNA replication.     

Charge localization in stacked radical cation DNA base pairs and the benzene dimer studied by self-interaction corrected density-functional theory

The incomplete cancellation of the electron self-interaction can be a serious shortcoming of density-functional theory especially when treating odd-electron systems. In this work, several popular and potentially viable correction schemes are applied in order to characterize the electronic structure of stacked molecular pairs, consisting of a neutral molecule and adjacent radical cation, as a function of separation distance. The unphysical sharing of the positive charge between adjacent molecules separated by 6-7 A is corrected for by applying a new empirical scheme proposed by VandeVondele and Sprik [Phys. Chem. Chem. Phys. 2005, 7, 1363] with a unique choice of parameters. This method is subsequently applied to characterize the electronic structure of two neighboring guanines excised from a canonical Arnott B-DNA structure and will be used in future investigations of certain model DNA fibers.     

Long-distance radical cation hopping in DNA: the effect of thymine-Hg(II)-thymine base pairs

Thymine-Hg(II)-thymine base pairs have been incorporated in an oligonucleotide duplex to study their effect on DNA-mediated charge transport. The introduction of a formally charged Hg atom inside the DNA base core does not significantly alter the charge hopping and trapping properties, as discussed in this paper. Hg(II) replaces the protons normally found on thymines within the complex and acts like a "big proton" in terms of its role in DNA charge transport.     

Recognition of ten base pairs of DNA by head-to-head hairpin dimers

Hairpin polyamides coupled head-to head with alkyl linkers of varying lengths were synthesized, and their DNA binding properties were determined. The DNA binding affinities of six-ring hairpin dimers Im-Im-Py-(R)[Im-Im-Py-(R)(HNCO(CH))(n)(CO)gamma-Py-Py-Py-beta-Dp](NH)gamma-Im-Py-Py-beta-Dp (1-4) (where n = 1-4) for their 10-bp, 11-bp, and 12-bp match sites 5'-TGGCATACCA-3', 5'-TGGCATTACCA-3', and 5'-TGGCATATACCA-3' were determined by quantitative DNase I footprint titrations. The most selective dimer Im-Im-Py-(R)[Im-Im-Py-(R)(HNCO(CH)(2))(2)(CO)gamma-Py-Py-Py-beta-Dp](NH)gamma-Im-Py-Py-beta-Dp (2) binds the 10-bp site match site with an equilibrium association constant of K(a) = 7.5 x 10(10) M(-1) and displays 25- and 140-fold selectivity over the 11-bp and 12-bp match sites, respectively. The affinity toward single base pair mismatched sequences is 4- to 8-fold lower if one hairpin module of the dimer is affected, but close to 200-fold lower if both hairpin modules face a single mismatch base pair. The head-to-head hairpin dimer motif expands the binding site size of DNA sequences targetable with polyamides.     

Binding of metal ions by pyrimidine base pairs in DNA duplexes

Pyrimidine base pairs in DNA duplexes selectively capture metal ions to form metal ion-mediated base pairs, which can be evaluated by thermal denaturation, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. In this critical review, we discuss the metal ion binding of pyrimidine bases (thymine, cytosine, 4-thiothymine, 2-thiothymine, 5-fluorouracil) in DNA duplexes. Thymine-thymine (T-T) and cytosine-cytosine (C-C) base pairs selectively capture Hg(II) and Ag(I) ions, respectively, and the metallo-base pairs, T-Hg(II)-T and C-Ag(I)-C, are formed in DNA duplexes. The metal ion binding properties of the pyrimidine-pyrimidine pairs can be changed by small chemical modifications. The binding selectivity of a metal ion to a 5-fluorouracil-5-fluorouracil pair in a DNA duplex can be switched by changing the pH of the solution. Two silver ions bind to each thiopyrimidine-thiopyrimidine pair in the duplexes, and the duplexes are largely stabilized. Oligonucleotides containing these bases are commercially available and can readily be applied in many scientific fields (86 references).     

Designed compounds for recognition of 10 base pairs of DNA with two at binding sites

Short AT base pair sequences that are separated by a small number of GCs are common in eukaryotic parasite genomes. Cell-permeable compounds that bind effectively and selectively to such sequences present an attractive therapeutic approach. Compounds with linked, one or two amidine-benzimidazole-phenyl (ABP) motifs were designed, synthesized, and evaluated for binding to adjacent AT sites by biosensor-surface plasmon resonance (SPR). A surprising feature of the linked ABP motifs is that a set of six similar compounds has three different minor groove binding modes with the target sequences. Compounds with one ABP bind independently to two separated AT sites. Unexpectedly, compounds with two ABP motifs can bind strongly either as monomers or as cooperative dimers to the full site. The results are supported by mass spectrometry and circular dichroism, and models to explain the different binding modes are presented.     

Direct visualization of abasic sites on a single DNA molecule using fluorescence microscopy

A new method was developed to allow direct visualization of damaged sites on individual DNA molecules. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labeled with a biotinylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. The light emitted by a single fluorochrome-DNA complex was calibrated. The number of abasic sites on the DNA molecule was estimated by counting each fluorochrome-DNA complex. The present study directly visualized and characterized the abasic sites of single DNA molecules.     

A novel pyrrolidinyl PNA showing high sequence specificity and preferential binding to DNA over RNA

A novel conformationally constrained pyrrolidinyl peptide nucleic acid (PNA) carrying an D-aminopyrrolidine carboxylic acid (D-Apc) spacer was synthesized, and its interactions with complementary oligo- and polynucleotides were studied by UV and CD spectroscopy. The decathymine PNA formed very stable PNA-DNA complexes with poly(dA) and (dA)(10) by a sequence-specific A-T pairing. The interaction with poly(rA) gave the corresponding PNA-RNA complex with much lower stability.     

Enhancement in fluorescence response by a quencher for amiloride upon binding to thymine opposite an abasic site in a DNA duplex

By using iodide (I⁻) as a quencher, we successfully improve the fluorescence response of amiloride when binding to thymine opposite an AP site in a 21-meric DNA duplex. From fluorescence measurements, as compared to the NaCl solutions, the addition of NaI as a quencher as well as salt to adjust the ionic strength effectively suppresses the background fluorescence from unbound amiloride in a solution. The Stern-Volmer analysis shows that the bound amiloride to the nucleobase at the AP site is unexposed to NaI quencher. Therefore the high signal-to-background fluorescence response of amiloride is obtained. Such enhancement in fluorescence response of amiloride by using the quencher can provide the significant improvement of the detection limit for DNA duplexes carrying T target base. The method presented in this study is simple and effective. The present method could be applicable to other detection system where microenvironment of fluorophores changes at a recognition event.     

Conformational analysis of trinucleotides containing a single abasic residue; mimics of DNA damage

The conformations of three deoxytrinucleotide analogues [d(TpXpT), where X = T, tetrahydrofuran (THF) or propyl (Pr)] were investigated using ¹H NMR spectroscopy as part of our studies of DNA‐base damage. The phosphorus‐decoupled ¹H NMR spectrum of each compound was simulated and values for the vicinal proton–proton coupling constants of the sugar ring hydrogens were extracted at several different temperatures, for use in conformational analyses. It was found that the south‐pucker preference of the sugar 3′ to the modification is increased whereas that of the 5′ is decreased relative to the puckers observed for the non‐modified system. The conformational change is <25%. This subtle effect may be sufficient for recognition by DNA repair enzymes. Copyright © 2003 John Wiley & Sons, Ltd.     

Multiple proton-transfer reactions in DNA base pairs by coordination of pt complex

The possibility of multiple proton-transfer reactions in DNA base pairs because of coordination of cisplatin is theoretically elucidated by density functional theory (DFT) and by quantum mechanics/molecular mechanics (QM/MM) methods with an ONIOM method. From the energetics of two base pairs with the cisplatin, it is theoretically confirmed that the Pt complex is likely to bind in the order cis-(CG)-Pt-(GC), cis-(CG)-Pt-(AT), cis-(TA)-Pt-(AT), where G, C, A, and T are guanine, cytosine, adenine, and thymine, respectively, and the Pt atom bonds to the N7 site of G and A. This result supports the experimental evidence, where the structure cis-A-Pt-A is seldom observed at room temperature. The single proton-transfer reaction occurs in one of the two GC pairs. No simultaneous single proton-transfer reaction can occur in both base pairs. Two different single proton-transferred structures (cis-(CG*)(d)-Pt-(GC)(p) and cis-(CG)(d)-Pt-(G*C)(p), where the asterisk means a proton donor of G) are as stable as the original structures (CG)(d)-Pt-(GC)(p). The same tendency was observed with cis-(CG*)-Pt-(AT). In contrast to cisplatin, multiple single proton-transfer reactions occur in the system consisting of two base pairs with transplatin. The optimized structure agrees with the experimental data for Pt-G coordination except for the hydrogen-bonding length.