Alternative heterocycles for DNA recognition: the benzimidazole/imidazole pair

Boc-protected benzimidazole-pyrrole, benzimidazole-imidazole, and benzimidazole-methoxypyrrole amino acids were synthesized and incorporated into DNA binding polyamides, comprised of N-methyl pyrrole and N-methyl imidazole amino acids, by means of solid-phase synthesis on an oxime resin. These hairpin polyamides were designed to determine the DNA recognition profile of a side-by-side benzimidazole/imidazole pair for the designated six base pair recognition sequence. Equilibrium association constants of the polyamide-DNA complexes were determined at two of the six base pair positions of the recognition sequence by quantitative DNase I footprinting titrations on DNA fragments each containing matched and single base pair mismatched binding sites. The results indicate that the benzimidazole-heterocycle building blocks can replace pyrrole-pyrrole, pyrrole-imidazole, and pyrrole-hydroxypyrrole constructs while retaining relative site specifities and subnanomolar match site affinities. The benzimidazole-containing hairpin polyamides represent a novel class of DNA binding ligands featuring tunable target recognition sequences combined with the favorable properties of the benzimidazole type DNA minor groove binders.     

Rational engineering of a DNA glycosylase specific for an unnatural cytosine:pyrene base pair

A novel site-specific cytosine DNA glycosylase has been rationally engineered from the active site scaffold of the DNA repair enzyme uracil DNA glycosylase (UDG). UDG, which operates by a nucleotide flipping mechanism, was first converted into a sequence nonspecific cytosine DNA glycosylase (CDG) by altering the base-specific hydrogen bond donor-acceptor groups in the active site. A second mutation that renders UDG defective in nucleotide flipping was then introduced, and the double mutant was rescued using a substrate with a "preflipped" cytosine base. Substrate-assisted flipping was engineered by incorporation of an unnatural pyrene nucleotide wedge (Y) into the DNA strand opposite to the target cytosine. This new enzyme, CYDG, can be used to target cleavage of specific cytosine residues in the context of a C/Y base pair in any DNA fragment.     

Highly efficient sequence-specific DNA interstrand cross-linking by pyrrole/imidazole CPI conjugates

We have developed a novel type of DNA interstrand cross-linking agent by synthesizing dimers of a pyrrole (Py)/imidazole (Im)-diamide-CPI conjugate, ImPyLDu86 (1), connected using seven different linkers. The tetramethylene linker compound, 7b, efficiently produces DNA interstrand cross-links at the nine-base-pair sequence, 5'-PyGGC(T/A)GCCPu-3', only in the presence of a partner triamide, ImImPy. For efficient cross-linking by 7b with ImImPy, one A.T base pair between two recognition sites was required to accommodate the linker region. Elimination of the A.T base pair and insertion of an additional A.T base pair and substitution with a G.C base pair significantly reduced the degree of cross-linking. The sequence specificity of the interstrand cross-linking by 7b was also examined in the presence of various triamides. The presence of ImImIm slightly reduced the formation of a cross-linked product compared to ImImPy. The mismatch partners, ImPyPy and PyImPy, did not produce an interstrand cross-link product with 7b, whereas ImPyPy and PyImPy induced efficient alkylation at their matching site with 7b. The interstrand cross-linking abilities of 7b were further examined using denaturing polyacrylamide gel electrophoresis with 5'-Texas Red-labeled 400- and 67-bp DNA fragments. The sequencing gel analysis of the 400-bp DNA fragment with ImImPy demonstrated that 7b alkylates several sites on the top and bottom strands, including one interstrand cross-linking match site, 5'-PyGGC(T/A)GCCPu-3'. To obtain direct evidence of interstrand cross-linkages on longer DNA fragments, a simple method using biotin-labeled complementary strands was developed, which produced a band corresponding to the interstrand cross-linked site on both top and bottom strands. Densitometric analysis indicated that the contribution of the interstrand cross-link in the observed alkylation bands was approximately 40%. This compound efficiently cross-linked both strands at the target sequence. The present system consisted of a 1:2 complex of the alkylating agent and its partner ImImPy and caused an interstrand cross-linking in a sequence-specific fashion according to the base-pair recognition rule of Py-Im polyamides.     

Exploring both sequence detection and restriction endonuclease cleavage kinetics by recognition site via single-molecule microfluidic trapping

We demonstrate the feasibility of a single-molecule microfluidic approach to both sequence detection and obtaining kinetic information for restriction endonucleases on dsDNA. In this method, a microfluidic stagnation point flow is designed to trap, hold, and linearize double-stranded (ds) genomic DNA to which a restriction endonuclease has been pre-bound sequence-specifically. By introducing the cofactor magnesium, we determine the binding location of the enzyme by the cleavage process of dsDNA as in optical restriction mapping, however here the DNA need not be immobilized on a surface. We note that no special labeling of the enzyme is required, which makes it simpler than our previous scheme using stagnation point flows for sequence detection. Our accuracy in determining the location of the recognition site is comparable to or better than other single molecule techniques due to the fidelity with which we can control the linearization of the DNA molecules. In addition, since the cleavage process can be followed in real time, information about the cleavage kinetics, and subtle differences in binding and cleavage frequencies among the recognition sites, may also be obtained. Data for the five recognition sites for the type II restriction endonuclease EcoRI on λ-DNA are presented as a model system. While the roles of the varying fluid velocity and tension along the chain backbone on the measured kinetics remain to be determined, we believe this new method holds promise for a broad range of studies of DNA-protein interactions, including the kinetics of other DNA cleavage processes, the dissociation of a restriction enzyme from the cleaved substrate, and other macromolecular cleavage processes.     

Altered sequence specificity identified from a library of DNA-binding small molecules

BACKGROUND: The ability to target specific DNA sequences using small molecules has major implications for basic research and medicine. Previous studies revealed that a bis-intercalating molecule containing two 1,4,5,8-napthalenetetracarboxylic diimides separated by a lysine-tris-glycine linker binds to DNA cooperatively, in pairs, with a preference for G + C-rich sequences. Here we investigate the binding properties of a library of bis-intercalating molecules that have partially randomized peptide linkers. RESULTS: A library of bis-intercalating derivatives with varied peptide linkers was screened for sequence specificity using DNase I footprinting on a 231 base pair (bp) restriction fragment. The library mixtures produced footprints that were generally similar to the parent bis-intercalator, which bound within a 15 bp G + C-rich repeat above 125 nM. Nevertheless, subtle differences in cleavage enhancement bands followed by library deconvolution revealed a derivative with novel specificity. A lysine-tris-beta-alanine derivative was found to bind preferentially within a 19 bp palindrome, without substantial loss of affinity. CONCLUSIONS: Synthetically simple changes in the bis-intercalating compounds can produce derivatives with novel sequence specificity. The large size and symmetrical nature of the preferred binding sites suggest that cooperativity may be retained despite modified sequence specificity. Such findings, combined with structural data, could be used to develop versatile DNA ligands of modest molecular weight that target relatively long DNA sequences in a selective manner.     

Inhibition of restriction enzyme's DNA sequence recognition by PUVA treatment

Applying various restriction enzymes on a specially designed 1.5 kb DNA fragment revealed that the inhibitory effects of psoralens + UVA irradiation (PUVA) treatment on restriction endonuclease activities are caused by recognition inhibition. In this study restriction enzymes that have a 5'-TpA sequence at the cleaving site (KpnI, XbaI, PmeI and DraI), and the noncleaving site (PacI) in recognition sites, or have two 5'-TpA sequences at the recognition site, and a nonspecific sequence between the recognition and the cleaving sites (BciVI), were inhibited by PUVA treatment. Most of the other restriction enzymes used in this study, which do not have a 5'-TpA sequence at their restriction site, were not inhibited by PUVA treatment, although a 5'-TpA sequence is located adjacent (SmaI) or very close (BamHI, SacI and PstI) to the recognition and cleaving sites for these enzymes. Because SphI, which does not have 5'-TpA at its restriction site, was strongly inhibited by PUVA treatment, the 5'-CpA sequence is suggested to be a new binding site of psoralens after UVA irradiation.     

Sequence-specific trapping of topoisomerase I by DNA binding polyamide-camptothecin conjugates

Hairpin pyrrole-imidazole polyamides are synthetic ligands that bind in the minor groove of DNA with affinities and specificities comparable to those of DNA binding proteins. Three polyamide-camptothecin conjugates 1-3 with linkers varying in length between 7, 13, and 18 atoms were synthesized to trap the enzyme Topoisomerase I and induce cleavage at predetermined DNA sites. One of these, polyamide-camptothecin conjugate 3 at nanomolar concentration (50 nM) in the presence of Topo I (37 degrees C), induces DNA cleavage between three and four base pairs from the polyamide binding site in high yield (77%).     

EFFICIENT FORMATION OF A CROSSLINKABLE HMT MONOADDUCT AT THE Kpn I RECOGNITION SITE

Abstract The double-stranded Kpn I linker, CGGTACCG, is readily crosslinked by 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT). Under identical conditions, the corresponding Bam HI linker, CGGATCCG, is resistant to modification. The differential reactivity of HMT towards the two sequences is also observed with SV40 DNA where the Kpn I restriction site is ten-fold more susceptible to HMT modification than the Bam HI site. Selective reaction with the Kpn I site is attributed to preferential intercalation of HMT into the TpA sequence. The availability of crosslinked Kpn I linker has allowed us to characterize the kinetics of photoreversal. Upon irradiation at 254 nm, the diadduct reverses at a faster rate than the monoadduct, leading to an accumulation of the latter. The resultant monoadduct reforms crosslink rapidly upon irradiation at 365 nm. DNA oligomers like the Kpn I linker, which can be modified by the above protocol to contain a crosslinkable HMT monoadduct, could he exploited as photoreactive sequence-specific probes.     

Quantitative footprinting analysis of drug-DNA interactions: Fe(III) methidium-propyl-EDTA as a probe

Quantitative footprinting studies involving a 139-base pair restriction fragment from pBR322 DNA, a lexitropsin ligand and two different DNA cleavage agents, the enzyme DNase I and the footprinting reagent Fe(III) methidium-propyl-EDTA (Fe-MPE), are described. The autoradiographic data showed that the ligand, an analogue of netropsin possessing two N-methylimidazole groups, binds to four regions on the 139-mer which are rich in GC. Analysis of the data leading to individual binding constants for each of the four loading events on the 139-mer revealed that Fe-MPE and DNase I report the same binding constants for the lexitropsin bound to its interaction sequences. The fact that the data from both probes can be analyzed using a common model indicates that the DNA cleavage specificity of the probe and not its binding/cleavage mechanism is the important factor in reporting of site loading information in the footprinting experiment. The study also showed that under certain conditions it is possible to gain information on the density of ligand binding sites on carrier DNA by monitoring site loading events on the labeled fragment.     

利用荧光标记引物和DNA自动测序仪确定DNA的断裂位点

建立了利用荧光标记引物和DNA自动测序仪进行DNA断裂位点分析的新方法, 该方法简便易行、灵敏度高、重复性好、数据分析客观性强、结果可靠, 适用于各种因素造成的DNA断裂位点的分析.     

Spectroscopic studies of the interaction of ferrous bleomycin with DNA

Bleomycin is an antibiotic used in cancer chemotherapy for its ability to achieve both single- and double-strand cleavage of DNA through abstraction of the deoxyribose C4'-H. Magnetic circular dichroism (MCD) and X-ray absorption (XAS) spectroscopies have been used to study the interaction of the biologically relevant FeIIBLM complex with DNA. Calf thymus DNA was used as the substrate as well as short oligonucleotides, including one with a preferred 5'-G-pyrimidine-3' cleavage site [d(GGAAGCTTCC)2] and one without [d(GGAAATTTCC)2]. DNA binding to FeIIBLM significantly perturbs the FeII active site, resulting in a change in intensity ratio of the d d transitions and a decrease in excited-state orbital splitting (5Eg). Although this effect is somewhat dependent on length and composition of the oligonucleotide, it is not correlated to the presence of a 5'-G-pyrimidine-3' cleavage site. No effect is observed on the charge-transfer transitions, indicating that the H-bonding recognition between the pyrimidine and guanine base does not perturb Fe-pyrimidine backbonding. Azide binding studies indicate that FeIIBLM bound to either oligomer has the same affinity for N3-. Parallel studies of BLM structural derivatives indicate that FeIIiso-PEPLM, in which the carbamoyl group is shifted on the mannose sugar, forms the same DNA-bound species as FeIIBLM. In contrast, FeIIDP-PEPLM, in which the -aminoalanine group is absent, forms a new species upon DNA binding. These data are consistent with a model in which the primary amine from the -aminoalanine is an FeII ligand and the mannose carbamoyl provides either a ligand to the FeII or significant second-sphere effects on the FeII site; intercalation of the bithiazole tail into the double helix likely brings the metal-bound complex close enough to the DNA to create steric interactions that remove the sugar groups from interaction with the FeII. The fact that the FeII active site is perturbed regardless of DNA sequence is consistent with the fact that cleavage is observed for both 5'-GC-3' and nonspecific oligomers and indicates that different reaction coordinates may be active, depending on orientation of the deoxyribose C4'-H.     

Targeting abasic sites and single base bulges in DNA with metalloinsertors

The site-specific recognition of abasic sites and single base bulges in duplex DNA by sterically expansive rhodium metalloinsertors has been investigated. Through DNA photocleavage experiments, Rh(bpy)2(chrysi)3+ is shown to bind both abasic sites and single base bulges site-specifically and, upon irradiation, to cleave the backbone of the defect-containing DNA. Photocleavage titrations reveal that the metal complex binds DNA containing an abasic site with high affinity (2.6(5) x 106 M-1), comparably to the metalloinsertor and a CC mismatch. The complex binds single base bulge sites with lower affinity (approximately 105 M-1). Analysis of cleavage products and the correlation of affinities with helix destabilization suggest that Rh(bpy)2(chrysi)3+ binds both lesions via metalloinsertion, as observed for Rh binding at mismatched sites, a binding mode in which the mismatched or unpaired bases are extruded from the helix and replaced in the base stack by the sterically expansive ligand of the metalloinsertor.     

Very High Affinity DNA Recognition by Bicyclic and Cross-Linked Oligonucleotides

We report the synthesis and DNA incorporation of a novel C-5 thiopropyne-substituted thymidine derivative which can be used to bring about covalent crosslinks between two noncomplementary DNA strands. This modified thymine pairs normally with adenine in duplex DNA and is shown not to be destabilizing to DNA double helices. Placement of the thiol-nucleotide near the center of opposing pyrimidine strands in pyr·pur·pyr triple helices results in crosslinking of the pyrimidine strands under aerobic conditions. Thermal melting studies at neutral pH show that such crosslinked ligands bind complementary purine strands with higher affinity than is possible with simple Watson-Crick recognition alone. In addition, we describe the construction of a triplex-forming circular oligonucleotide which contains a similar disulfide link across the center. This macrobicyclic ligand binds with extremely high affinity and sequence selectivity to a complementary purine DNA strand. The formation of crosslinks across two noncomplementary strands represents a new strategy for increasing affinity and selectivity of DNA recognition.     

Molecular design of a pyrrole-imidazole hairpin polyamides for effective DNA alkylation

New hairpin polyamide-CPI (CPI = cyclopropylpyrroloindole) conjugates, compounds 12-14, were synthesized and their DNA-alkylating activities compared with the previously prepared hairpin polyamide, compound 1, by high-resolution denaturing gel electrophoresis with 450 base pair (bp) DNA fragments and by HPLC product analysis of the synthetic decanucleotide. In accord with our previous results, alkylation by compound 1 occurred predominantly at the G moiety of the sequence 5'-AGTCAG-3' (site 3). However, compound 12, in which the structure of the alkylating moiety of compound 1 is replaced with segment A of duocarmycin A DU-86 (CPI), did not show any DNA alkylating activity. In clear contrast, the hairpin CPI conjugate 13, which differs from compound 1 in that it lacks one Py unit and possesses a vinyl linker, alkylated the A of 5'-AGTCAG-3' (site 3) efficiently at nanomolar concentrations. Alkylation by compound 14, which has a vinyl linker, occurred at the A of 5'-AGTCCA-3' (site 6) and at several minor alkylation sites, including mismatch alkylation at A of 5'-TCACAA-3' (site 2). The significantly different reactivity of the alkylating hairpin polyamides 1, 12, 13, and 14 was further confirmed by HPLC product analysis by using a synthetic decanucleotide. The results suggest that hairpin polyamide--CPI conjugate 13 alkylates effectively according to Dervan's pairing rule, and with a new mode of recognition in which the Im-vinyl linker (L) pair targets G-C base pairs. These results demonstrate that incorporation of the vinyl-linker pairing with Im dramatically improves the reactivity of hairpin polyamide--CPI conjugates.     

4'-AMINOMETHYL-4,5',8-TRIMETHYLPSORALEN PHOTOCHEMISTRY THE EFFECT OF CONCENTRATION AND UVA FLUENCE ON PHOTOADDUCT FORMATION IN POLY(dAdT) AND CALF THYMUS DNA

Abstract-The photochemistry of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with poly(dA-dT) and calf thymus DNA was studied. The extent of photoadduct formation and the distribution of photoadducts (3,4– and 4',5'-monoadducts and crosslinks) were determined by liquid scintillation analysis and HPLC, respectively. The adducts were characterized on the basis of their UV absorption spectra and mass spectral analysis. The high DNA binding constant for AMT (1.5 x 10⁵ M⁻¹ ) led to a high fraction of intercalated molecules, which contributed to the high level of AMT photoadduct formation, as many as 102 adducts per kilobase pair. In addition, there is a distinct difference in the adduct distribution compared to the previously studied 8-methoxypsoralen (8-MOP). Under the conditions employed for the photochemical studies, virtually all of the AMT molecules in solution are intercalated, occupying 25% of the base pair sites. Under similar conditions, 8-MOP molecules occupied 10 times fewer sites. Thus, for AMT, DNA base pair sites other than 5'TA, the well-characterized strong binding for psoralens in general, are an additional target for photomodification, which results in the formation of a higher percentage of monoadducts. The proportion of photoadducts formed was virtually independent of AMT concentration and UVA (320–400 nm radiation) fluence.     

Enzyme‐Mediated Endogenous and Bioorthogonal Control of a DNAzyme Fluorescent Sensor for Imaging Metal Ions in Living Cells

Bioorthogonal control of metal‐ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18‐base pair recognition site of a homing endonuclease (I‐SceI), which is found by chance only once in 7×10¹⁰ bp of genomic sequences, and can thus form a near bioorthogonal pair with I‐SceI for DNAzyme activation with minimal effect on living cells. Once I‐SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg²⁺ to release the fluorophore‐labeled DNA fragment and produce a fluorescent turn‐on signal for Mg²⁺. Thus I‐SceI bioorthogonally activates the 10–23 DNAzyme for imaging of Mg²⁺ in HeLa cells.     

Investigation of DNA-protein sequence-specific interactions with a ds-DNA array

The sequence specific recognitions between DNAs and proteins play important roles in many biological functions. The use of double-stranded DNA arrays (ds-DNA arrays) for studying sequence specific recognition between DNAs and proteins is a promising method. Here we report the use of a ds-DNA probe with multi operation sites of restriction proteins in the middle sequence to investigate DNA-protein sequence-specific interactions including methylation. We arranged EcoR I site and Rsa I site on the same duplex DNA probe to fabricate ds-DNA arrays. We used the ds-DNA arrays to study DNA-restriction enzyme reactions before and after duplex DNA methylation under different probe concentration and reaction time conditions. Our results indicated that the ds-DNA arrays can be further biochemically modified and made accessible for interactions between DNAs and proteins in complex multi-step gene-regulation processes.     

Gamma and ultraviolet radiation cause DNA crosslinking in the presence of metal ions at high pH.

M-DNA is a novel duplex conformation in which metal ions such as Co2+, Ni2+ or Zn2+ replace the imino protons of every base pair. An ethidium fluorescence assay was used to estimate lesions in M-DNA induced by gamma- and UV radiation. General damage to DNA was assessed from the loss of ethidium fluorescence after irradiation of calf thymus DNA. Crosslinks were measured from the return of ethidium fluorescence after a heating and cooling step. Strand breaks were estimated from the loss of fluorescence in covalently closed circular plasmid DNA after a heating and cooling step. For the Co2+ form of M-DNA, gamma-radiation caused the very efficient formation of crosslinks which was not observed with B-DNA nor with the Ni2+ or Zn2+ forms of M-DNA. The crosslinks occurred in both A-T and G-C base pairs but did not form in the presence of a free radical scavenger. Crosslinks induced by UV radiation also formed at a faster rate in the Co2+, Ni2+ and Zn2+ forms of M-DNA compared to B-DNA; crosslinking occurred in all DNA but was more prominent in AT-rich sequences and was not inhibited by a free radical scavenger. Therefore, the presence of certain metal ions may lead to large increases in the formation of radiation-induced crosslinks in DNA.     

Solution structure of a DNA duplex containing a guanine-difluorotoluene pair: a wobble pair without hydrogen bonding?

The incorporation of synthetic nucleoside analogues into DNA duplexes provides a unique opportunity to probe both structure and function of nucleic acids. We used 1H and 19F NMR and molecular dynamics calculations to determine the solution structures of two similar DNA decamer duplexes, one containing a central G-T mismatched or "wobble" base pair, and one in which the thymine in this base pair is replaced by difluorotoluene (a thymine isostere) creating a G-F pair. Here, we show that the non-hydrogen-bonding G-F pair stacks relatively well into the helix and that the distortions caused by each non-Watson-Crick G-T or G-F base pair are quite localized to a three base pair site around the mismatch. A detailed structural analysis reveals that the absence of hydrogen bonding introduces more dynamic motion into the G-F pair relative to G-T and permits the G-F pair to exhibit stacking and conformational features characteristic of both a Watson-Crick base pair (on the guanine containing strand) and a wobble base pair (on the strand containing the difluorotoluene). We used these results to posit a rationale for recognition and repair of mismatch sites in DNA.