12-mer Duplex Research Articles

Hydrogen bonding in duplex DNA probed by DNP enhanced solid-state NMR N-H bond length measurements.

Numerous biological processes and mechanisms depend on details of base pairing and hydrogen bonding in DNA. Hydrogen bonds are challenging to quantify by X-ray crystallography and cryo-EM due to difficulty of visualizing hydrogen atom locations but can be probed with site specificity by NMR spectroscopy in solution and the solid state with the latter particularly suited to large, slowly tumbling DNA complexes. Recently, we showed that low-temperature dynamic nuclear polarization (DNP) enhanced solid-state NMR is a valuable tool for distinguishing Hoogsteen base pairs (bps) from canonical Watson-Crick bps in various DNA systems under native-like conditions. Here, using a model 12-mer DNA duplex containing two central adenine-thymine (A-T) bps in either Watson-Crick or Hoogsteen confirmation, we demonstrate DNP solid-state NMR measurements of thymine N3-H3 bond lengths, which are sensitive to details of N-H···N hydrogen bonding and permit hydrogen bonds for the two bp conformers to be systematically compared within the same DNA sequence context. For this DNA duplex, effectively identical TN3-H3 bond lengths of 1.055 ± 0.011Å and 1.060 ± 0.011Å were found for Watson-Crick A-T and Hoogsteen A (syn)-T base pairs, respectively, relative to a reference amide bond length of 1.015 ± 0.010Å determined for N-acetyl-valine under comparable experimental conditions. Considering that prior quantum chemical calculations which account for zero-point motions predict a somewhat longer effective peptide N-H bond length of 1.041Å, in agreement with solution and solid-state NMR studies of peptides and proteins at ambient temperature, to facilitate direct comparisons with these earlier studies TN3-H3 bond lengths for the DNA samples can be readily scaled appropriately to yield 1.083Å and 1.087Å for Watson-Crick A-T and Hoogsteen A (syn)-T bps, respectively, relative to the 1.041Å reference peptide N-H bond length. Remarkably, in the context of the model DNA duplex, these results indicate that there are no significant differences in N-H···N A-T hydrogen bonds between Watson-Crick and Hoogsteen bp conformers. More generally, high precision measurements of N-H bond lengths by low-temperature DNP solid-state NMR based methods are expected to facilitate detailed comparative analysis of hydrogen bonding for a range of DNA complexes and base pairing environments.

Anthracyclines React with Apurinic/Apyrimidinic Sites in DNA.

The combination of doxorubicin (Adriamycin) and cyclophosphamide, referred to as AC chemotherapy, is commonly used for the clinical treatment of breast and other cancers. Both agents target DNA with cyclophosphamide causing alkylation damage and doxorubicin stabilizing the topoisomerase II-DNA complex. We hypothesize a new mechanism of action whereby both agents work in concert. DNA alkylating agents, such as nitrogen mustards, increase the number of apurinic/apyrimidinic (AP) sites through deglycosylation of labile alkylated bases. Herein, we demonstrate that anthracyclines with aldehyde-reactive primary and secondary amines form covalent Schiff base adducts with AP sites in a 12-mer DNA duplex, calf thymus DNA, and MDA-MB-231 human breast cancer cells treated with nor-nitrogen mustard and the anthracycline mitoxantrone. The anthracycline-AP site conjugates are characterized and quantified by mass spectrometry after NaB(CN)H3 or NaBH4 reduction of the Schiff base. If stable, the anthracycline-AP site conjugates represent bulky adducts that may block DNA replication and contribute to the cytotoxic mechanism of therapies involving combinations of anthracyclines and DNA alkylating agents.

The Base Pairs of Isoguanine and 8-Aza-7-deazaisoguanine with 5-Methylisocytosine as Targets for DNA Functionalization.

The isoguanine-isocytosine base pair (isoG-isoC) represents an important expansion of the DNA coding system. The base pair is more stable than the canonical adenine-thymine or guanine-cytosine pairs. However, nothing is known on the functionalization of the noncanonical isoG-isoC pair at the isoguanine site. In this work, functionalization of the isoG-isoC and the isosteric base pair that contains 8-aza-7-deazaisoguanine in place of isoguanine is studied. Short ethynyl, more space demanding octadiynyl, and dendritic tripropargylamine residues attached to the isoG-isoC base pairs were introduced to oligonucleotides. 12-mer duplexes were formed by hybridization with single base pair modification. The use of the two modified nucleobases gave us the freedom to shift nucleobase substituents within the major groove of double helical DNA. Clickable side chains at position-7 stabilize the base pair, whereas 8-substituents reduce its stability strongly. The weak isoguanine-thymine or 8-aza-7-deazaisoguanine-thymine base pairs show a similar sensitivity to the position of nucleobase functionalization as base pair matches formed with 5-methylisocytosine. CD spectra of all modified duplexes display the typical shape of a B-DNA with only marginal changes. Fluorescent pyrene labeled DNA with long, short, and branched linkers was generated using click chemistry. Pyrene click adducts with long linkers are essential to maintain or to increase base pair stability. Labeled duplexes are more fluorescent than corresponding single strands. For the dendritic linker excimer emission was observed for single strands but only monomer emission in duplexes.

Probing Watson-Crick and Hoogsteen base pairing in duplex DNA using dynamic nuclear polarization solid-state NMR spectroscopy

The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.

CTAG vs. GATC: Structural Basis for Representational Differences in Reverse Palindromic DNA Tetranucleotide Sequences

Genomic compositional biases are broadly acknowledged. For example, the tetranucleotide CTAG is extraordinarily rare in almost all bacterial, archaeal, eukaryotic genomes and some phage genomes. Moreover, CTAG is remarkably underrepresented relative to its reverse palindromic sequence GATC. To determine the origin of compositional biases, we analyze structural features of palindromic DNA duplexes derived in all-atom molecular simulations. We answer this question by comparing the structural features of 12-mer DNA duplexes comprising a central tetramer sequence of CTAG, GATC, and CGCG. This study uses unbiased and biased molecular dynamics simulations to compare the torsional angles of dinucleotides in a DNA duplex. Specifically, we use all-atom molecular dynamics and well-tempered metadynamics simulations to produce trajectories of the torsion angles between dinucleotides in the sequence and calculate the free energy surfaces using torsional collective variables. The data show a correlation between the sequence and the degree of the duplex unwinding. More significantly, data show that the CTAG sequence has a more unwinding effect on the DNA duplex than the GATC sequence. This conclusion suggests that the CTAG sequence bases are more accessible from the minor groove surface than the GATC bases. Consequently, the CTAG sequence appears more exposed to chemical modifications which may explain its extreme underrepresentation in the bacterial genome.

A Quencher-Free Linear Probe from Serinol Nucleic Acid with a Fluorescent Uracil Analogue.

With the goal of developing a quencher-free probe composed of an artificial nucleic acid, the fluorescent nucleobase analogue 5-(perylenylethynyl)uracil (Pe U), which was incorporated into totally artificial serinol nucleic acid (SNA) as a substitute for thymine, has been synthesized. In the context of a 12-mer duplex with RNA, these fluorophores reduce duplex stability slightly compared with that of an SNA without Pe U modification; thus suggesting that structural distortion is not induced by the modification. If two Pe Us were incorporated at separate positions in an SNA, the fluorescent emission at λ≈490 nm was clearly enhanced upon hybridization with complementary RNA. A quencher-free SNA linear probe containing three Pe Us, each separated by six nucleobases, has been designed. Detection of target RNA with high sensitivity and discrimination of a single-base mismatch has also been demonstrated.

Anomeric 5-Aza-7-deaza-2'-deoxyguanosines in Silver-Ion-Mediated Homo and Hybrid Base Pairs: Impact of Mismatch Structure, Helical Environment, and Nucleobase Substituents on DNA Stability.

Nucleoside configuration (α-d vs. β-d), nucleobase substituents, and the helical DNA environment of silver-mediated 5-aza-7-deazaguanine-cytosine base pairs have a strong impact on DNA stability. This has been demonstrated by investigations on oligonucleotide duplexes with silver-mediated base pairs of α-d and β-d anomeric 5-aza-7-deaza-2'-deoxyguanosines and anomeric 2'-deoxycytidines incorporated in 12-mer duplexes. To this end, a new synthetic protocol has been developed to access the pure anomers of 5-aza-7-deaza-2'-deoxyguanosine by glycosylation of either the protected nucleobase or its salt followed by separation of the glycosylation products by crystallization and chromatography. Thermal stability measurements were performed on duplexes with α-d/α-d and β-d/β-d homo base pairs or α-d/β-d and β-d/α-d hybrid pairs within two sequence environments, positions 6 or 7, of oligonucleotide duplexes. The respective Tm stability increases observed after silver ion addition differ significantly. Homo base pairs with β-d/β-d or α-d/α-d nucleoside combinations are more stable than α-d/β-d hybrid base pairs. The positional switch of silver-ion-mediated base pairs has a significant impact on stability. Nucleobase substituents introduced at the 5-position of the dC site of silver-mediated base pairs affect base pair stability to a minor extent. Our investigation might lead to applications in the construction of bioinspired nanodevices, in DNA diagnostics, or metal-DNA hybrid materials.

Enhanced Intercellular Delivery of cRGD-siRNA Conjugates by an Additional Oligospermine Modification.

Small interfering RNA (siRNA), consisting a 21-mer duplex molecule, is often modified by conjugation with specific ligands to enhance its capacity for tissue-specific delivery. However, these attempts are hampered by the low permeability of negatively charged RNA molecules to enter the cell membrane. In this study, we designed and synthesized siRNA conjugates modified with cationic oligospermine and cyclic RGD (cRGD) to overcome the low-membrane permeability of siRNA. The siRNA conjugate, which contains 15 spermines and a cRGD peptide, showed sufficient gene-silencing activity at 250 nM final concentration without a transfection reagent. Under these conditions, the cationic oligospermine and cRGD–siRNA conjugate did not show any cytotoxicity.

Trapping of the transport-segment DNA by the ATPase domains of a type II topoisomerase

Type II topoisomerases alter DNA topology to control DNA supercoiling and chromosome segregation and are targets of clinically important anti-infective and anticancer therapeutics. They act as ATP-operated clamps to trap a DNA helix and transport it through a transient break in a second DNA. Here, we present the first X-ray crystal structure solved at 2.83 Å of a closed clamp complete with trapped T-segment DNA obtained by co-crystallizing the ATPase domain of S. pneumoniae topoisomerase IV with a nonhydrolyzable ATP analogue and 14-mer duplex DNA. The ATPase dimer forms a 22 Å protein hole occupied by the kinked DNA bound asymmetrically through positively charged residues lining the hole, and whose mutagenesis impacts the DNA decatenation, DNA relaxation and DNA-dependent ATPase activities of topo IV. These results and a side-bound DNA-ParE structure help explain how the T-segment DNA is captured and transported by a type II topoisomerase, and reveal a new enzyme–DNA interface for drug discovery.

Guanine and 8-Azaguanine in Anomeric DNA Hybrid Base Pairs: Stability, Fluorescence Sensing, and Efficient Mismatch Discrimination with α-d-Nucleosides.

The α-anomers of 8-aza-2'-deoxyguanosine (αGd*) and 2'-deoxyguanosine (αGd) were site-specifically incorporated in 12-mer duplexes opposite to the four canonical DNA constituents dA, dG, dT, and dC. Oligodeoxyribonucleotides containing αGd* display significant fluorescence at slightly elevated pH (8.0). Oligodeoxyribonucleotides incorporating β-anomeric 8-aza-2'-deoxyguanosine (Gd*) and canonical dG were studied for comparison. For αGd* synthesis, an efficient purification of anomeric 8-azaguanine nucleosides was developed on the basis of protected intermediates, and a new αGd* phosphoramidite was prepared. Differences were observed for sugar conformations ( N vs S) and p Ka values of anomeric nucleosides. Duplex stability and mismatch discrimination were studied employing UV-dependent melting and fluorescence quenching. A gradual fluorescence change takes place in duplex DNA when the α-nucleoside αGd* was positioned opposite to the four canonical β-nucleosides. The strongest fluorescence decrease appeared in duplexes incorporating αGd*-Cd base pair matches. Decreasing fluorescence corresponds to increasing Tm values. For mismatch discrimination, the α-anomers αGd* and αGd are more efficient than the corresponding β-nucleosides. Duplexes with single "purine-purine" αGd*-αGd* or αGd-αGd base pairs are significantly more stable than those displaying β-d configuration. CD spectra indicate that single mutations by α-anomeric nucleosides do not affect the global structure of B-DNA.

Structural and Dynamical Impact of a Universal Fluorescent Nucleoside Analogue Inserted Into a DNA Duplex.

Recently, a 3-hydroxychromone based nucleoside 3HCnt has been developed as a highly environment-sensitive nucleoside surrogate to investigate protein-DNA interactions. When it is incorporated in DNA, the probe is up to 50-fold brighter than 2-aminopurine, the reference fluorescent nucleoside. Although the insertion of 3HCnt in DNA was previously shown to not alter the overall DNA structure, the possibility of the probe inducing local effects cannot be ruled out. Hence, a systematic structural and dynamic study is required to unveil the 3HCnt's limitations and to properly interpret the data obtained with this universal probe. Here, we investigated by NMR a 12-mer duplex, in which a central adenine was replaced by 3HCnt. The chemical shifts variations and nOe contacts revealed that the 3HCnt is well inserted in the DNA double helix with extensive stacking interactions with the neighbor base pairs. These observations are in excellent agreement with the steady-state and time-resolved fluorescence properties indicating that the 3HCnt fluorophore is protected from the solvent and does not exhibit rotational motion. The 3HCnt insertion in DNA is accompanied by the extrusion of the opposite nucleobase from the double helix. Molecular dynamics simulations using NMR-restraints demonstrated that 3HCnt fluorophore exhibits only translational dynamics. Taken together, our data showed an excellent intercalation of 3HCnt in the DNA double helix, which is accompanied by localized perturbations. This confirms 3HCnt as a highly promising tool for nucleic acid labeling and sensing.

A Rhodium-Cyanine Fluorescent Probe: Detection and Signaling of Mismatches in DNA.

We report a bifunctional fluorescent probe that combines a rhodium metalloinsertor with a cyanine dye as the fluorescent reporter. The conjugate shows weak luminescence when free in solution or with well matched DNA but exhibits a significant luminescence increase in the presence of a 27-mer DNA duplex containing a central CC mismatch. DNA photocleavage experiments demonstrate that, upon photoactivation, the conjugate cleaves the DNA backbone specifically near the mismatch site on a 27-mer fragment, consistent with mismatch targeting. Fluorescence titrations with the 27-mer duplex containing the CC mismatch reveal a DNA binding affinity of 3.1 × 106 M-1, similar to that of other rhodium metalloinsertors. Fluorescence titrations using genomic DNA extracted from various cell lines demonstrate a clear discrimination in fluorescence between those cell lines that are proficient or deficient in mismatch repair. This differential luminescence reflects the sensitive detection of the mismatchrepair-deficient phenotype.

The effect of S-substitution at the O6-guanine site on the structure and dynamics of a DNA oligomer containing a G:T mismatch.

The effect of S-substitution on the O6 guanine site of a 13-mer DNA duplex containing a G:T mismatch is studied using molecular dynamics. The structure, dynamic evolution and hydration of the S-substituted duplex are compared with those of a normal duplex, a duplex with S-substitution on guanine, but no mismatch and a duplex with just a G:T mismatch. The S-substituted mismatch leads to cell death rather than repair. One suggestion is that the G:T mismatch recognition protein recognises the S-substituted mismatch (GS:T) as G:T. This leads to a cycle of futile repair ending in DNA breakage and cell death. We find that some structural features of the helix are similar for the duplex with the G:T mismatch and that with the S-substituted mismatch, but differ from the normal duplex, notably the helical twist. These differences arise from the change in the hydrogen-bonding pattern of the base pair. However a marked feature of the S-substituted G:T mismatch duplex is a very large opening. This showed considerable variability. It is suggested that this enlarged opening would lend support to an alternative model of cell death in which the mismatch protein attaches to thioguanine and activates downstream damage-response pathways. Attack on the sulphur by reactive oxygen species, also leading to cell death, would also be aided by the large, variable opening.

Anomeric 2'-Deoxycytidines and Silver Ions: Hybrid Base Pairs with Greatly Enhanced Stability and Efficient DNA Mismatch Detection with α-dC.

α-d-Nucleosides are rare in nature but can develop fascinating properties when incorporated into DNA. This work reports on the first silver-mediated base pair constructed from two anomeric nucleosides: α-dC and β-dC. The hybrid base pair was integrated into the DNA and DNA/RNA double helix. A 12-mer duplex with α-dC and β-dC pair exhibits a higher thermal stability (Tm =43 °C) than that incorporating the β-dC-Ag+ -β-dC homo pair (Tm =34 °C). Furthermore, α-dC shows excellent mismatch discrimination for DNA single nucleotide polymorphism (SNP). All four SNPs were identified on the basis of large Tm value differences measured in the presence of silver ions. High resolution melting was not required.

Silver-Mediated Base Pairs in DNA Incorporating Purines, 7-Deazapurines, and 8-Aza-7-deazapurines: Impact of Reduced Nucleobase Binding Sites and an Altered Glycosylation Position.

Formation of silver-mediated DNA was studied with oligonucleotides incorporating 8-aza-7-deazapurine, 7-deazapurine, and purine nucleosides. The investigation was performed on non-self-complementary duplexes with one or two modifications and self-complementary duplexes with an alternating dA-dT motif. Homo base pairs as well as base pair mismatches of dA analogues with dC and Watson-Crick pairs with dT were studied by stoichiometric silver ion titration and Tm measurements. N8 -Glycosylated 8-aza-7-deazaadenine forms silver-ion-mediated base pairs capturing two silver ions (low silver content) whereas regularly glycosylated 8-aza-7-deazapurine, 7-deazapurine (c7 Ad ), and dA do not form comparable structures. Stable silver-mediated "dA-dC" base pair mismatches were detected for all nucleosides. Two silver ions per base pair are bound by 8-aza-7-deazapurine whereas c7 Ad binds only one silver ion. The situation is different when the equivalents of silver ions were increased to the number of total base pairs. Surprisingly, in 12-mer duplexes as well as in related 25-mer duplexes every base pair consumed one silver ion.

Photoinduced Cross-Linking of Short Furan-Modified DNA on Surfaces.

We report for the first time the formation of site-specific interstrand cross-linked (ICL) surface-immobilized furan-modified DNA duplexes via singlet oxygen. 1O2, necessary for effecting furan-mediated ICL formation, was produced in situ using methylene blue or a zinc phthalocyanine derivative (TT1) as a photosensitizer. Via surface plasmon resonance spectroscopy, we show that surface ICL was achieved, and a robust link formed that enhances the stability of the 12-mer duplex even after surface regeneration. The described method represents a novel platform technology based on surfaces with addressable and stable DNA duplexes requiring only short oligonucleotides.

Abstract 1280: The intersection of the PI3K/mTOR and HIPPO pathways: a potential therapeutic target for medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor; it is genetically defined into 4 subgroups which defines the clinical course of the disease. Despite current treatment modalities such as surgical resection, radiation, and chemotherapy, there are still significant morbidities and adverse side-effects. Recently defined genetic pathways could possibly offer a better management and treatment of patients with MB. The HIPPO signaling pathway has recently been shown to intersect with PI3K and Sonic Hedgehog (SHH) pathways to modulate mechanistic target of rapamycin (mTOR), an important regulator in cell growth and migration. The aim of this study is to elucidate the interaction of Hippo and Akt/mTOR signaling pathways with SHH pathways in MB, and define the potential downstream targets of therapy by using combined inhibitors. In order to achieve our goals, we evaluated the expression of activated AKT/mTOR in MB tumors using immunohistochemistry (IHC), along with the expression of transcriptional co-activators yes-associated protein (YAP), a down-stream effector of the HIPPO pathway. Scratch and chemotactic migration were used to assess the motility of MB cells following treatment with inhibitors of the aforementioned pathways. Cell cycle analysis and proliferation were measured using EdU and MTT techniques, respectively. YAP-SiRNA treatment was used to inhibit HIPPO pathway. MicroRNA-29, which mediates the mTOR pathway by modulating PTEN levels, was investigated. Results demonstrated: 1. A significant number of MBs expressed activated pAKTSer473 and pmTORSer2448, along with YAP, the downstream effector of the HIPPO pathway; 2. Treatment with inhibitors of PI3K/AKT (LY294002) and mTOR (rapamycin), given with EGF, significantly reduced cellular motility; 3. SHH inhibitor cyclopamine given with LY294002 or rapamycin significantly reduced cellular motility; 4. Cell proliferation was suppressed by inhibition of the HIPPO pathway using 3 unique 27mer duplexes of YAP1 siRNA treatments; 5. Combined treatment of cyclopamine with either LY294002 or rapamycin caused maximum suppression of cellular growth. In conclusion, these results demonstrate that the Hippo and PI3K/Akt/mTOR pathways are over-expressed in MB, and that the combined targeting of mTOR/HIPPO pathway with SHH inhibitor may provide alternative strategies for the treatment of MB, and offers a targeted therapy for improved prognosis. Citation Format: Jennifer S. Ronecker, Paul Lee, Sudeepta Sridhara, Michael LaBagnara, Raj Murali, Meena Jhanwar-Uniyal. The intersection of the PI3K/mTOR and HIPPO pathways: a potential therapeutic target for medulloblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1280.

Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA)

Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a β-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

4-(2'-Pyridyl)imidazole as an artificial nucleobase in highly stabilizing Ag(I)-mediated base pairs.

A new artificial nucleoside 2 comprising 4-(2'-pyridyl)imidazole as the nucleobase was devised and incorporated into a series of DNA oligonucleotide duplexes. The propensity of 2 to form metal-mediated homo and hetero base pairs was investigated in various 13mer and 26mer duplexes. The artificial nucleoside readily forms Ag(I)-mediated homo base pairs of the type 2-Ag(I)-2, in which the Ag(I) ion resides in a [2+2] coordination environment. In addition, the combination with the previously established imidazole nucleoside 1 leads to the formation of Ag(I)-mediated hetero base pairs (2-Ag(I)-1 and 1-Ag(I)-2) with a [2+1] coordination environment. With an increase in the melting temperature of ΔT m=21.8°C upon the formation of a Ag(I)-mediated base pair, the 2-Ag(I)-1 pair with the 13mer duplex is the most stabilizing Ag(I)-mediated base pair so far. Generally, the impact of the formation of a metal-mediated base pair on the properties of the DNA duplex was found to be significantly more prominent for shorter, intrinsically less stable duplexes.

A pyrene dihydrodioxin with pyridinium “arms”: A photochemically active DNA cleaving agent with unusual duplex stabilizing and electron trapping properties

Abstract Pyrene dihydrodioxins (PDHD) comprise effective DNA intercalation agents that are masked ortho-quinones, which can be released by near ultraviolet or visible irradiation. We have studied the binding and photoreactions of chiral dipyridinium PDHDs with herring sperm DNA and an 11-mer duplex DNA containing all 10 basepair steps. Binding affinities to herring sperm DNA were determined for purified enantiomers (Kb = 1.6 ± 0.15 × 105 and 2.3 ± 0.2 × 105 M−1). UV-melting experiments using the 11-mer DNA revealed significant stabilization of duplex DNA, (ΔTm = 11.5° and ΔTm = 15.3° C). Both enantiomers linearized (double-strand cleavage) supercoiled ΦX174 plasmid DNA with high efficiency. PDHDs have specificity to cleave and/or damage DNA duplexes at Gs and have preferable binding to GG DNA sites. A full range of transient absorption spectroscopy from the ultrafast femtosecond to the microsecond domains has been applied in the study of this system. These studies have revealed a novel mechanism for quinone release via the pyrene radical cation and the entrapment of the released electron by the coordinated action of the two pyridinium rings. These same studies have shown that the released pyrenequinone can photochemically initiate the further release of pyrenequinone. Thus, this reaction is autocatalytic and can be initiated with visible light.