Distinguishing Isomeric Cyclobutane Thymidine Dimers by Ion Mobility and Tandem Mass Spectrometry.

Irradiation of the major conformation of duplex DNA found in cells (B form) produces cyclobutane pyrimidine dimers (CPDs) from adjacent pyrimidines in a head-to-head orientation (syn) with the C5 substituents in a cis stereochemistry. These CPDs have crucial implications in skin cancer. Irradiation of G-quadruplexes and other non-B DNA conformations in vitro produces, however, CPDs between nonadjacent pyrimidines in nearby loops with syn and head-to-tail orientations (anti) with both cis and trans stereochemistry to yield a mixture of six possible isomers of the T=T dimer. This outcome is further complicated by formation of mixtures of nonadjacent CPDs of C=T, T=C, and C=C, and successful analysis depends on development of specific and sensitive methods. Toward meeting this need, we investigated whether ion mobility mass spectrometry (IMMS) and MS/MS can distinguish the cis,syn and trans,anti T=T CPDs. Ion mobility can afford baseline separation and give relative mobilities that are in accord with predicted cross sections. Complementing this ability to distinguish isomers is MS/MS collisional activation where fragmentation also distinguishes the two isomers and confirms conclusions drawn from ion mobility analysis. The observations offer early support that ion mobility and MS/MS can enable the distinction of DNA photoproduct isomers.

Post- and Pre-Radiolabeling Assays for anti Thymidine Cyclobutane Dimers as Intrinsic Photoprobes of Various Types of G-Quadruplexes, Reverse Hoogsteen Hairpins, and Other Non-B DNA Structures.

G-quadruplexes are thought to play an important role in gene regulation and telomere maintenance, but developing probes for their presence and location is challenging due to their transitory and highly dynamic nature. The majority of probes for G-quadruplexes have relied on antibody or small-molecule binding agents, many of which can also alter the dynamics and relative populations of G-quadruplexes. Recently, it was discovered that ultraviolet B (UVB) irradiation of human telomeric DNA and various G-quadruplex forming sequences found in human promoters, as well as reverse Hoogsteen hairpins, produces a unique class of non-adjacent anti cyclobutane pyrimidine dimers (CPDs). Therefore, one can envision using a pulse of UVB light to irreversibly trap these non-B DNA structures via anti CPD formation without perturbing their dynamics, after which the anti CPDs can be identified and mapped. As a first step toward this goal, we report radioactive post- and pre-labeling assays for the detection of non-adjacent CPDs and illustrate their use in detecting trans,anti T=(T) CPD formation in a human telomeric DNA sequence. Both assays make use of snake venom phosphodiesterase (SVP) to degrade the trans,anti T=(T) CPD-containing DNA to the tetranucleotide pTT=(pTT) corresponding to CPD formation between the underlined T's of two separate dinucleotides while degrading the adjacent syn TT CPDs to the trinucleotide pGT=T. In the post-labeling assay, calf intestinal phosphodiesterase is used to dephosphorylate the tetranucleotides, which are then rephosphorylated with kinase and [32P]-ATP to produce radiolabeled mono- and diphosphorylated tetranucleotides. The tetranucleotides are confirmed to be non-adjacent CPDs by 254 nm photoreversion to the dinucleotide p*TT. In the pre-labeling assay, radiolabeled phosphates are introduced into non-adjacent CPD-forming sites by ligation prior to irradiation, thereby eliminating the dephosphorylation and rephosphorylation steps. The assays are also demonstrated to detect the stereoisomeric cis,anti T=(T) CPD.

Adjacent and Nonadjacent Dipyrimidine Photoproducts as Intrinsic Probes of DNA Secondary and Tertiary Structure†.

While the photochemistry of duplex DNA has been extensively studied, the photochemistry of nonduplex DNA structures is largely unexplored. Because the structure and stereochemistry of DNA photoproducts depend on the secondary structure and conformation of the DNA precursor, they can serve as intrinsic probes of DNA structure. This review focuses on the structures and stereoisomers of pyrimidine dimer photoproducts arising from adjacent and nonadjacent pyrimidines in A, B and denatured DNA, bulge loops, G-quadruplexes and reverse Hoogsteen hairpins and methods for their detection.

Ability of the Putative Decomposition Products of 2,3-dioxetanes of Indoles to Photosensitize Cyclobutane Pyrimidine Dimer (CPD) Formation and its Implications for the "Dark" (Chemisensitized) Pathway to CPDs in Melanocytes†.

The formation of cyclobutane pyrimidine dimers (CPDs) by a "dark" pathway in melanocytes has been attributed to chemisensitization by dioxetanes produced from peroxynitrite oxidation of melanin or melanin precursors. These dioxetanes are proposed to decompose to triplet state compounds which sensitize CPD formation by triplet-triplet energy transfer. To determine whether such compounds are capable of sensitizing CPD formation, the putative decomposition products of 2,3-dioxetanes of variously substituted indoles were synthesized and their triplet state energies determined at 77 K. Their ability to photosensitize CPD formation was determined by an enzyme-coupled gel electrophoresis assay in comparison with norfloxacin (NFX) which has the lowest triplet energy known to sensitize CPD formation. The decomposition products of 2,3-dioxetanes of 5-hydroxy and 5,6-dimethoxy indoles used as models for melanin precursors had lower triplet energies and were incapable of photosensitizing CPD formation. Theoretical calculations suggest that the decomposition products of the 2,3-dioxetanes of melanin precursors DHI and DHICA will have similarly low triplet energies. Decomposition products of the 2,3-dioxetanes of indoles lacking oxygen substituents had higher triplet energies than NFX and were capable of photosensitizing CPD formation, suggesting that peroxynitrite oxidation of tryptophan could play a hitherto unrecognized role in the dark pathway to CPDs.

The effect of flanking bases on direct and triplet sensitized cyclobutane pyrimidine dimer formation in DNA depends on the dipyrimidine, wavelength and the photosensitizer.

Cyclobutane pyrimidine dimers (CPDs) are the major products of DNA produced by direct absorption of UV light, and result in C to T mutations linked to human skin cancers. Most recently a new pathway to CPDs in melanocytes has been discovered that has been proposed to arise from a chemisensitized pathway involving a triplet sensitizer that increases mutagenesis by increasing the percentage of C-containing CPDs. To investigate how triplet sensitization may differ from direct UV irradiation, CPD formation was quantified in a 129-mer DNA designed to contain all 64 possible NYYN sequences. CPD formation with UVB light varied about 2-fold between dipyrimidines and 12-fold with flanking sequence and was most frequent at YYYR and least frequent for GYYN sites in accord with a charge transfer quenching mechanism. In contrast, photosensitized CPD formation greatly favored TT over C-containing sites, more so for norfloxacin (NFX) than acetone, in accord with their differing triplet energies. While the sequence dependence for photosensitized TT CPD formation was similar to UVB light, there were significant differences, especially between NFX and acetone that could be largely explained by the ability of NFX to intercalate into DNA.

Photocrosslinking of G-Quadruplex-Forming Sequences found in Human Promoters.

While is it well known that human telomeric DNA sequences can adopt G-quadruplex structures, some promoters sequences have also been found to form G-quadruplexes, and over 40% of promoters contain putative G-quadruplex-forming sequences. Because UV light has been shown to crosslink human telomeric G-quadruplexes by cyclobutane pyrimidine dimer (CPD) formation between T's on adjacent loops, UV light might also be able to photocrosslink G-quadruplexes in promoters. To investigate this possibility, 15 potentially UV-crosslinkable G-quadruplex-forming sequences found in a search of human DNA promoters were UVB irradiated in vitro, and three were confirmed to have formed nonadjacent CPDs by mass spectrometry. In addition to nonadjacent T=T CPDs found in human telomeric DNA, a nonadjacent T=U CPD was discovered that presumably arose from deamination of a nonadjacent T=C CPD. Analysis of the three sequences by circular dichroism, melting temperature analysis and chemical footprinting confirmed the presence of G-quadruplexes that could explain the formation of the nonadjacent CPDs. The formation of nonadjacent CPDs from the sequences in vitro suggests that they might be useful probes for the presence of non-B DNA structures, such as G-quadruplexes, in vivo, and if they were to form in vivo, might also have significant biological consequences.

Evidence for Reverse Hoogsteen Hairpin Intermediates in the Photocrosslinking of Human Telomeric DNA Sequences.

UVB irradiation of human telomeric d(GGGTTA)3 GGG sequences in potassium ion solution crosslinks the first and third TTA segments through anti cyclobutane pyrimidine dimer (CPD) formation. The photocrosslinking reaction was first proposed to occur through a form 3 two-tetrad G-quadruplex in which the lateral four-nucleotide GTTA loop can interact with an adjacent TTA loop. Curiously, the reaction does not occur with sodium ion, which was explained by the formation of a basket structure which only has three-nucleotide TTA loops that cannot interact. Sequences known or expected to favor the two-tetrad basket did not show enhanced photocrosslinking, suggesting that some other structure was the reactive intermediate. Herein, we report that anti CPDs form in human telomeric DNA sequences with lithium ion that is known to disfavor G-quadruplex formation, as well as with potassium ion when the bases are modified to interfere with G-quartet formation. We also show that anti CPDs form in sequences containing A's in place of G's that cannot form Hoogsteen hairpins, but can form reverse Hoogsteen hairpins. These results suggest that reverse Hoogsteen hairpins may play a hitherto unrecognized role in the biology and photoreactivity of DNA in telomeres, and possibly in other purine-rich sequences found in regulatory regions.

Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles.

Cyclobutane pyrimidine dimers (CPDs) are DNA photoproducts linked to skin cancer, whose mutagenicity depends in part on their frequency of formation and deamination. Nucleosomes modulate CPD formation, favoring outside facing sites and disfavoring inward facing sites. A similar pattern of CPD formation in protein-free DNA loops suggests that DNA bending causes the modulation in nucleosomes. To systematically study the cause and effect of nucleosome structure on CPD formation and deamination, we have developed a circular permutation synthesis strategy for positioning a target sequence at different superhelix locations (SHLs) across a nucleosome in which the DNA has been rotationally phased with respect to the histone octamer by TG motifs. We have used this system to show that the nucleosome dramatically modulates CPD formation in a T11-tract that covers one full turn of the nucleosome helix at seven different SHLs, and that the position of maximum CPD formation at all locations is shifted to the 5΄-side of that found in mixed-sequence nucleosomes. We also show that an 80-mer minicircle DNA using the same TG-motifs faithfully reproduces the CPD pattern in the nucleosome, indicating that it is a good model for protein-free rotationally phased bent DNA of the same curvature as in a nucleosome, and that bending is modulating CPD formation.

Design, synthesis, and characterization of nucleosomes containing site-specific DNA damage.

How DNA damaged is formed, recognized, and repaired in chromatin is an area of intense study. To better understand the structure activity relationships of damaged chromatin, mono and dinucleosomes containing site-specific damage have been prepared and studied. This review will focus on the design, synthesis, and characterization of model systems of damaged chromatin for structural, physical, and enzymatic studies.

Rapid deamination of cyclobutane pyrimidine dimer photoproducts at TCG sites in a translationally and rotationally positioned nucleosome in vivo.

Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T(m)CG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.

Synergistic modulation of cyclobutane pyrimidine dimer photoproduct formation and deamination at a TmCG site over a full helical DNA turn in a nucleosome core particle.

Sunlight-induced C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C or 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by DNA polymerase η and defines a probable mechanism for the origin of UV-induced C to T mutations. We have now determined the photoproduct formation and deamination rates for 10 consecutive T=(m)CG CPDs over a full helical turn at the dyad axis of a nucleosome and find that whereas photoproduct formation and deamination is greatly inhibited for the CPDs closest to the histone surface, it is greatly enhanced for the outermost CPDs. Replacing the G in a T=(m)CG CPD with A greatly decreased the deamination rate. These results show that rotational position and flanking sequence in a nucleosome can significantly and synergistically modulate CPD formation and deamination that contribute to C to T mutations associated with skin cancer induction and may have influenced the evolution of the human genome.

Effect of sequence and metal ions on UVB-induced anti cyclobutane pyrimidine dimer formation in human telomeric DNA sequences.

Irradiation of G-quadruplex forming human telomeric DNA with ultraviolet B (UVB) light results in the formation of anti cyclobutane pyrimidine dimers (CPDs) between loop 1 and loop 3 in the presence of potassium ions but not sodium ions. This was unexpected because the sequences involved favor the nonphotoreactive hybrid conformations in K(+) solution, whereas a potentially photoreactive basket conformation is favored in Na(+) solution. To account for these contradictory results, it was proposed that the loops are too far apart in the basket conformation in Na(+) solution but close enough in a two G-tetrad basket-like form 3 conformation that can form in K(+) solution. In the current study, Na(+) was still found to inhibit anti CPD formation in sequences designed to stabilize the form 3 conformation. Furthermore, anti CPD formation in K(+) solution was slower for the sequence previously shown to exist primarily in the proposed photoreactive form 3 conformation than the sequence shown to exist primarily in a nonphotoreactive hybrid conformation. These results suggest that the form 3 conformation is not the principal photoreactive conformation, and that G-quadruplexes in K(+) solution are dynamic and able to access photoreactive conformations more easily than in Na(+) solution.

Polyphosphoester-based cationic nanoparticles serendipitously release integral biologically-active components to serve as novel degradable inducible nitric oxide synthase inhibitors.

A degradable polyphosphoester (PPE)-based cationic nanoparticle (cSCK), which is integrated constructed as a novel degradable drug device, demonstrates surprisingly efficient inhibition of inducible nitric oxide synthase (iNOS) transcription, and eventually inhibits nitric oxide (NO) over-production, without loading of any specific therapeutic drugs. This system may serve as a promising anti-inflammatory agent toward the treatment of acute lung injury.

A Simple and Efficient Synthesis of an Acid-labile Polyphosphoramidate by Organobase-catalyzed Ring-Opening Polymerization and Transformation to Polyphosphoester Ionomers by Acid Treatment.

The direct synthesis of an acid-labile polyphosphoramidate by organobase-catalyzed ring-opening polymerization and an overall two-step preparation of polyphosphodiester ionomers (PPEI) by acid-assisted cleavage of the phosphoramidate bonds along the backbone of the polyphosphoramidate were developed in this study. The ultrafast organobase-catalyzed ring-opening polymerization of a cyclic phospholane methoxyethyl amidate monomer initiated by benzyl alcohol allowed for the preparation of well-defined polyphosphoramidates (PPA) with predictable molecular weights, narrow molecular weight distributions (PDI<1.10), and well-defined chain ends. Cleavage of the acid-labile phosphoramidate bonds on the polyphosphoramidate repeat units was evaluated under acidic conditions over a pH range of 1-5, and the complete hydrolysis produced polyphosphodiesters. The thermal properties of the resulting polyphosphoester ionomer acid and polyphosphoester ionomer sodium salt exhibited significant thermal stability. The parent PPA and both forms of the PPEIs showed low cytotoxicities toward HeLa cells and RAW 264.7 mouse macrophage cells. The synthetic methodology developed here has enriched the family of water-soluble polymers prepared by rapid and convenient organobase-catalyzed ring-opening polymerizations and straightforward chemical medication reactions, which are designed to be hydrolytically degradable and have promise for numerous biomedical and other applications.

Nucleic Acid-directed Self-assembly of Multifunctional Gold Nanoparticle Imaging Agents.

Gold nanoparticles have attracted much interest as a platform for development of multifunctional imaging and therapeutic agents. Multifunctionalized gold nanoparticles are generally constructed by covalent assembly of a gold core with thiolated ligands. In this study, we have assembled multifunctionalized gold nanoparticles in one step by nucleic acid hybridization of ODN (oligodeoxynucleotide)-derivatized gold nanoparticles with a library of pre-functionalized complementary PNAs (peptide nucleic acids). The PNAs were functionalized by conjugation with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) for chelating 64Cu for PET imaging, PEG (polyethylene glycol) for conferring stealth properties, and Cy5 for fluorescent imaging. The resulting nanoparticles showed good stability both in vitro and in vivo showing biodistribution behavior in a mouse that would be expected for a PEGylated gold nanoparticle rather than that for the radiolabelled PNA used in its assembly.

Roles of free radicals in type 1 phototherapeutic agents: aromatic amines, sulfenamides, and sulfenates.

Detailed analyses of the electron spin resonance (ESR) spectra, cell viability, and DNA degradation studies are presented for the photolyzed Type I phototherapeutic agents: aromatic amines, sulfenamides, and sulfenates. The ESR studies provided evidence that copious free radicals can be generated from these N-H, N-S, and S-O containing compounds upon photoirradiation with UV/visible light. The analyses of spectral data allowed us to identify the free radical species. The cell viability studies showed that these agents after exposure to light exert cytotoxicity to kill cancer cells (U937 leukemia cell lines HTC11, KB, and HT29 cell lines) in a dosage- and time-dependent manner. We examined a possible pathway of cell death via DNA degradation by a plasmid cleavage assay for several compounds. The effects of photosensitization with benzophenone in the presence of oxygen were examined. The studies indicate that planar tricyclic amines and sulfenamides tend to form π-electron delocalized aminyl radicals, whereas nonplanar ones tend to yield nitroxide radicals resulting from the recombination of aminyl radicals with oxygen. The ESR studies coupled with the results of cell viability measurements and DNA degradation reveal that planar N-centered radicals can provide higher potency in cell death and allow us to provide some insights on the reaction mechanisms. We also found the formation of azatropylium cations possessing high aromaticity derived from azepines can facilitate secondary electron transfer to form toxic O2(•-) radicals, which can further exert oxidative stress and cause cell death.

Efficient protection and transfection of small interfering RNA by cationic shell-crosslinked knedel-like nanoparticles.

Despite the great potential of small interfering RNA (siRNA) as a therapeutic agent, progress in this area has been hampered by a lack of efficient biocompatible transfection agents. Recently, cationic shell-crosslinked knedel-like nanoparticles (cSCKs) were found to possess lower cytotoxicity and better transfection ability for phosphorothioate ODNs and plasmid DNA than the commonly used cationic lipid-based agent Lipofectamine. To determine the usefulness of cSCKs for siRNA transfection, a small library of cSCKs with varying percentage of primary and tertiary amines was assessed for its ability to bind to siRNA, inhibit siRNA degradation in human serum, and to transfect HeLa and mouse macrophage cell lines. The silencing efficiency in HeLa cells was greatest with the cSCK with 100% primary amines (pa100) as determined by their viability following transfection with cytotoxic and non-cytotoxic siRNAs. cSCK-pa100 showed greater silencing efficiency than Lipofectamine 2000 in the HeLa cells, as well in 293T and human bronchial epithelial (HEK) cells, but was comparable in human bronchial epithelial (BEAS-2B) cells and human mammary epithelial (MCF10a) cells. cSCK-pa100 also showed greater silencing of iNOS expression than Lipofectamine 2000 in a mouse macrophage cell line, and provided greater protection from serum degradation, demonstrating its potential usefulness as an siRNA transfection agent. The siRNA silencing of iNOS at lower concentrations of siRNA could be enhanced by complexation with the fusogenic GALA peptide, which was shown to enhance endosomal escape following uptake.

A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis.

Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis-syn thymidine-thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases.

Imaging mRNA expression levels in living cells with PNA·DNA binary FRET probes delivered by cationic shell-crosslinked nanoparticles.

Optical imaging of gene expression through the use of fluorescent antisense probes targeted to the mRNA has been an area of great interest. The main obstacles to developing highly sensitive antisense fluorescent imaging agents have been the inefficient intracellular delivery of the probes and high background signal from unbound probes. Binary antisense probes have shown great promise as mRNA imaging agents because a signal can only occur if both probes are bound simultaneously to the mRNA target site. Selecting an accessible binding site is made difficult by RNA folding and protein binding in vivo and the need to bind two probes. Even more problematic, has been a lack of methods for efficient cytoplasmic delivery of the probes that would be suitable for eventual applications in vivo in animals. Herein we report the imaging of iNOS mRNA expression in live mouse macrophage cells with PNA·DNA binary FRET probes delivered by a cationic shell crosslinked knedel-like nanoparticle (cSCK). We first demonstrate that FRET can be observed on in vitro transcribed mRNA with both the PNA probes and the PNA·DNA hybrid probes. We then demonstrate that the FRET signal can be observed in live cells when the hybrid probes are transfected with the cSCK, and that the strength of the FRET signal is sequence specific and depends on the mRNA expression level.