Recent advances in the synthesis and structure determination of site specifically psoralen-modified DNA oligonucleotides.

We have developed novel methods for the preparation of multimicromole quantities of extremely pure, uniquely photoadducted psoralen-DNA cross-links, furan-side monoadducted DNA and pyrone-side monoadducts. Psoralen cross-linked and furan-side monoadducted DNA were produced by employing high intensity argon ion and krypton ion lasers as light sources. Pyrone-side monoadducts were prepared by base-catalyzed photoreversal of psoralen cross-links. The various psoralen-adducted DNA oligomers were efficiently purified by high performance liquid chromatography. These methods have permitted us to synthesize 4 mumol each of a self-complementary 8-mer d(GCGTACGC) 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) furan-side monoadduct and HMT cross-link. Preliminary nuclear magnetic resonance (NMR) data on the HMT cross-linked 8-mer d(GCGTACGC) have been obtained which confirmed the presence of the diadducted psoralen at the unique 5'TpA3' site. NMR data obtained from the 8-mer furan-side monoadduct revealed that the psoralen molecule is intercalated into the DNA double helix. Preliminary crystals of 8-mer cross-linked DNA molecule have been grown. Conditions for the growth of X-ray diffraction-quality crystals and the further analysis of these crystals are now in progress.     

THE SITE-SPECIFIC INHIBITION OF Bgl I CLEAVAGE BY PSORALEN PHOTOADDUCTS

Abstract— We have investigated the site specificity of furocoumarins by using fluorescent densitometry to examine the frequency of cleavage by the restriction enzyme Bgl I. This enzyme has an 11 base pair (bp) recognition sequence which varies slightly from site to site because it includes a 5 base pair neutral region. Cleavage at all three Bgl I recognition sites in pBR322 was inhibited by the photoaddition of the psoralen derivative 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) which forms both crosslinks and monoad-ducts in a dose-dependent manner. One site, which contains two thymidines in a crosslinkable configuration, was observed to be markedly more sensitive to HMT photoadducts. In contrast Bgl I cleavage at all sites was relatively resistant to the derivative 5-methylisopsoralen (5-MIP), which forms only monoadducts. When HMT-reacted DNA was generated with widely different ratios of monoad-ducts to crosslinks (3% and 40% crosslinks), essentially the same level and pattern of inhibition was observed in both cases. Taken together, the data imply that differences in inhibition seen at the three cutting sites of Bgl I with HMT are attributable to DNA sequence and the role it plays in adduct positioning.     

ACTION SPECTRA AND CHROMOPHORES FOR LETHAL PHOTOSENSITIZATION OF Candida albicans BY DNA MONOADDUCTS FORMED BY 8-METHOXYPSORALEN AND MONOFUNCTIONAL FUROCOUMARINS

The red-shift of furocoumarin action spectra, compared with their absorption spectra, has been investigated. An action spectrum for 8-methoxypsoralen (8-MOP) monoadduct formation in the yeast Candida albicans has been determined. The yeast cells were initially exposed to sublethal doses of monochromatic UVA at different wavelengths. Monoadduct formation was monitored by growth inhibition induced, after washing out any unbound 8-MOP, by re-irradiation with a constant second (non-lethal) dose of 330 nm radiation. A comparison between this action spectrum and the absorption spectrum of the dark complex of 8-MOP and DNA was made. In addition, the action spectra of monoadduct formation of five monofunctional compounds including a coumarin derivative have been determined. These action spectra were compared with their respective DNA dark complex absorption spectra. In general, the peaks of the furocoumarin DNA dark complexes show a red-shift when compared with the free furocoumarin molecule and the action spectra show peaks which correspond with the peaks of the dark complexes. Such data indicate that the DNA dark complex is the chromophore for growth inhibition in yeast rather than the free furocoumarin. The similarity of the 8-MOP monoadduct formation action spectrum and 8-MOP action spectra suggests that spectral dependence for the photobiological effects (including the red-shift) is dependent on monoadduct formation rather than, as previously suggested by several authors, crosslink formation. The action spectrum for the coumarin derivative 4-methyl N-ethylpyrrolo (3,2-g) coumarin (PCNEt) correlated well with the free molecule absorption spectrum rather than DNA dark complex indicating that the free molecule is the chromophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and photoreactivity of psoralen linked to triple helix-forming oligonucleotides

Triple helix-forming oligonucleotides conjugated to a psoralen (psoTFO) have been designed to bind to three distinct purine-rich sequences within the human interstitial collagenase (MMP1) gene. Gel mobility shift assays indicate that these psoTFO bind to and photoreact with model target DNA sequences following ultraviolet A (UVA) irradiation. The dissociation constants for binding of the psoTFO to their targets range from 0.3 to 4 microM. Psoralen monoadducts with the purine-rich target strand and interstrand crosslinks are efficiently formed on targets containing either 5'-ApT-3' or 5'-TpA-3' sequences adjacent to the TFO binding sequence. The dependence of adduct formation on UVA dose has provided quantitative estimates of the overall rate constants for psoralen monoadduct and crosslink formation in the presence of a TFO. When psoralen is tethered to a TFO, the rate of monoadduct formation exceeds that of crosslinking for all sequences studied. This contrasts with the relatively low rate of monoadduct formation that has been reported for free psoralens, suggesting that the bound TFO facilitates the initial photochemistry that generates monoadducts, but does not significantly affect interstrand crosslink formation. psoTFO and UVA treatment inhibit DNA cleavage by a restriction endonuclease when the psoralen covalently reacts directly at the endonuclease site. The particular TFO studied do not completely inhibit endonuclease activity when they are noncovalently bound or when the covalent psoralen adduct does not coincide with the endonuclease site. Our findings confirm that TFO are capable of directing psoralen photoadducts to specific DNA targets and suggest that TFO can significantly modulate psoralen photoreactivity and DNA-protein interactions.     

CHARACTERIZATION OF THE PHOTOREACTION BETWEEN DNA AND AMINOMETHYL-TRIMETHYLPSORALEN USING ABSORPTION AND FLUORESCENCE SPECTROSCOPY

–The use of absorption and fluorescence spectroscopy for following the progress of the photo-reaction between DNA and 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) has been investigated. Absorption at long wavelengths and fluorescence both decline upon intercalation of AMT into the DNA helix. The loss of fluorescence from AMT and the accompanying appearance of monoadduct fluorescence upon irradiation by UV light can be easily followed by using the excitation beam of a spectro-fluorometer as the source of irradiation and monitoring the changing emission spectrum. Where cross-link formation is possible, the subsequent decline of monoadduct fluorescence is seen as well. This suggests that the 4,5-tnonoadduct is a precursor of cross-links. Both monoaddition and cross-linking are more rapid with poly d(A·T) than with calf thymus DNA or poly d(A·T). Excitation spectra can be helpful in resolving the levels of AMT and 4',5'-monoadduct when both are contributing to the emission spectrum. Some changes are observed in the emission spectrum of AMT–poly d(A·T) monoadducts after prolonged irradiation which indicate further photoreaction.     

PHOTOTOXIC EFFECTS OF FOUR PSORALENS ON L1210 CELLS. THE CORRELATION WITH DNA INTERSTRAND CROSS-LINKING

L1210 mouse leukaemia cells were treated with psoralen [S-methoxy-(XMOP), 4,5′,8-trimethyI-(TMP), 4′-hydroxymethyl-4,5′,8-trimethyl-(HMT) or 4′-amino- methyl-4,5′,8-trimethylpsoralen (AMT)] in combination with long wavelength ultraviolct irradiation (Λ～ 365 nm). In order to investigate the relative photobiological activities of the psoralens, cell viability and DNA-synthesis activity as well as psoralen-DNA photoaddition and DNA interstrand cross-linking were measured after the treatment. In all assays the activity ranking order was found to he: TMP > HMT > AM7 > 8MOP. Furthermore, a direct correlation between phototoxicity, psoralen induced DNA interstrand cross-links and inhibition of DNA synthesis was indicated. Finally, psoralen uptake by the cells appears to be an important determinant for phototoxicity, whereas their DNA photoreactivity does not.     

Mutagenesis Mediated by Triple Helix–Forming Oligonucleotides Conjugated to Psoralen: Effects of Linker Arm Length and Sequence Context

Targeted mutagenesis and gene knock-out can be mediated by triple helix-forming oligonucleotides (TFO) linked to mutagenic agents, such as psoralen. However, this strategy is limited by the availability of homopurine/ homopyrimidine stretches at or near the target site because such sequences are required for high-affinity triplex formation. To overcome this limitation, we have tested TFO conjugated to psoralen via linker arms of lengths varying from 2 to 86 bonds, thereby designed to deliver the psoralen at varying distances from the third strand binding site present at the 3'end of the supFG1 mutation reporter gene. Following triplex formation and UVA irradiation, mutations were detected using an SV40-based shuttle vector assay in human cells. The frequency and distribution of mutations depended on the length of the linker arm. Precise targeting was observed only for linker arms of length 2 and 6, which also yielded the highest mutation frequencies (3 and 14%, respectively). Psoralen–TFO with longer tethers yielded mutations at multiple sites, with the maximum distance from the triplex site limited by the linker length but with the distribution within that range influenced by the propensity for psoralen intercalation at A:T base-pair-rich sites. Thus, gene modification can be extended beyond the site of third strand binding but with a decrease in the precision of the targeting.     

Psoralen-Derivatized Oligothymidine Methylphosphonates form Triple Helices with DNA and Crosslink to a Specific Strand

Oligothymidine methylphosphonates derivatized at the 5′-end with 4′-aminoalkyl-4,5′,8-trimethylpsoralen (AMT) were prepared. The interaction of these oligonucleoside methylphosphonates with double-stranded DNA was studied. Oligothymidine tnethylphosphonates, T₇ and T₁₄, were found to form triple helix with an oligodeoxyribonucleotide 45-mer DNA duplex which contains an A₁₅-T₁₅ sequence. Upon irradiation with 365 nm (UV light, AMT crosslinked to accessible thymidine residues in the target DNA. Both AMT-derivatized T₇ and T₁₄ crosslink to the T₁₅ containing strand of the double-stranded DNA target, but they do not crosslink to the A₁₅ containing strand which also contains a potential thymidine crosslinking site. Methylphosphonate oligomer, T₇, was derivatized with AMT using either an ethyl-, butyl- or hexyl-linker. The efficiency of crosslinking is affected by the length of the aminoalkyl linker arm connecting the AMT to the methylphosphonate oligomer. The relative crosslinking efficiencies of the oligomers with these three types of linkers were different. Greatest crosslinking, 45%, was obtained using an oligomer having a butyl-or a hexyl-linker. The interaction of oligothymidine methylphosphonates with DNA can be enhanced by using two shorter AMT-oligomers instead of using one full-length AMT-derivatized oligomer. This strategy was demonstrated by the interaction of AMT-derivatized T₇ with duplex DNA 35-mer and 45-mer target. The extent of crosslinking to the 45-mer target, whose binding site can accommodate two molecules of AMT-derivatized T₇, is 45% whereas that with the 35-mer target, which can accommodate only one T₇ molecule, is only 3%. The results of our experiments suggest that AMT-derivatized oligothymidine methylphosphonates can form triple-stranded complex and psoralen photoadduct with DNA. The formation of such complexes may be useful in probing and controlling gene expression at the DNA level.     

Detection of DNA base variation and cytosine methylation at a single nucleotide site using a highly sensitive fluorescent probe

A fluorescent DNA probe containing an anthracene group attached via an anucleosidic linker can identify all four DNA bases at a single site as well as the epigenetic modification C/5-MeC via a hybridisation sensing assay.     

LONG PERSISTENCE OF MONOFUNCTIONAL 8-METHOXYPSORALEN-DNA ADDUCTS IN HUMAN SKIN in vivo

Human skin can be persistently photosensitized by topical application of aqueous 8-methoxypsoralen plus immediate irradiation with a non-erythemogenic dose of wavelengths above 380 nm. Re-exposure of skin thus sensitized to broadband UV-A produces phototoxic erythema 72-120 h later. The persistence of the photosensitization was demonstrated by phototoxic erythema after re-exposure up to 15 days after the first sensitizing irradiation. According to the concept that the first exposure induces primarily psoralen monoadducts, we consider this an investigation of psoralen monoadduct persistence. In contrast to several earlier studies, this sensitive method indicates that psoralen monoadducts may remain in human skin in vivo for more than 2 weeks after formation.     

Site‐Specifically Arraying Small Molecules or Proteins on DNA Using An Expanded Genetic Alphabet

A class of replicable unnatural DNA base pairs formed between d 5SICS and either d MMO2 , d DMO , or d NaM were developed. To explore the use of these pairs to produce site‐specifically labeled DNA, the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase‐mediated replication, and subsequent site‐specific modification of the amplified DNA by Click chemistry is reported. With the d 5SICS scaffold a propynyl ether linker is accommodated better than its aliphatic analogue, but not as well as the protected propargyl amine linker explored previously. It was also found that with the d MMO2 and d DMO analogues, the d MMO2 position para to the glycosidic linkage is best suited for linker attachment and that although aliphatic and ether‐based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogues, a variety of them were used to site‐selectively attach a biotin tag to the amplified DNA. Finally, we use d 5SICS^CO –d NaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps “evolution” of nanomaterials.     

SENSITIVITY OF DNA REPAIR-DEFICIENT STRAINS OF ESCHERICHIA COLI K-12 TO VARIOUS FUROCOUMARINS AND NEAR-ULTRAVIOLET RADIATION

Abstract— Survival curves were obtained for DNA repair-deficient strains of Escherichia coli K-12 ( polA1, uvrB5 , and recA56 ) exposed to near-ultraviolet radiation [black light (BL)] in the presence of the DNA cross-linking agent 8-methoxypsoralen (8-MOP) or in the presence of photosensitizers forming primarily monoadducts with DNA [angelicin; 3-carbethoxypsoralen (3-CPs); 5,7-dimethoxycoumarin (DMC)], and after exposure to blue light (BluL) in the presence of 8-MOP or 3-CPs. An interpretation of these data suggests that DNA polymerase I is required for the major pathway of monoadduct repair, but appears to play little or no role in the repair of 8-MOP cross-links. The uvrB and recA strains were very sensitive, both to the cross-linking agent and to the monoadduct formers. The markedly different results for BL plus DMC or 3-CPs compared to angelicin suggests that the DMC and 3-CPs monoadducts are repaired by a different mechanism than are the angelicin monoadducts, or else DMC and 3-CPs undergo photochemical side reactions that produce DNA lesions other than the expected monoadducts. From photochemical evidence, we predicted that fewer 8-MOP monoadducts should be converted to cross-links by BluL vs BL; this appears to be the case. 3-CPs showed dramatically different biological results when irradiated with BL vs BluL, suggesting that 3-CPs may form more types of photoproducts than the expected monoadducts; BluL, however, appears to favor monoadduct formation.     

Context-dependent photodimerization in isolated thymine-thymine steps in DNA

The effect of 280 nm irradiation on a family of synthetic DNA hairpins possessing an alkane linker connecting a six-base pair stem having a single T-T step located at different positions within the hairpin has been investigated. A single adduct assigned to the product of 2+2 dimerization is obtained except in the case of a T-T step located adjacent to the linker, in which case both 2+2 and 6-4 adducts are obtained. The efficiency of dimerization is similar for three hairpins having a T-T step located within the duplex interior. Lower efficiency is observed for a T-T step located at the open end of the hairpin and in T overhangs, whereas higher efficiency is observed for the T-T step adjacent to the linker and in a single T bulge. The context-dependence of dimerization efficiency is discussed.     

Preparation and characteristics of photocrosslinkable hydrophilic polymer having cinnamate moiety

The reaction of copolymer of N,N-dimethylacrylamide (DMAA) and bromoethyl methacrylate with potassium cinnamate produced water-soluble photosensitive polymers. Photosensitive polyDMAA films were irradiated with a 400 W high-pressure mercury lamp (λ > 280 nm) to produce crosslinked polymers, which were swollen in water. The degree of swelling was controlled by the irradiation time and content of cinnamate moieties in copolymers. Higher cinnamoylation and longer irradiation time resulted in higher yield of crosslinked polymers and less swellability. Partial degelation upon irradiation at λ ～ 254 nm was observed. The advantage of gelation via photodimerization over conventional chemical crosslinking methods is discussed in conjunction with biomedical applications. © 1992 John Wiley & Sons, Inc.     

Oxygen-independent inactivation of Haemophilus influenzae transforming DNA by monochromatic radiation: action spectrum, effect of histitine and repair.

Abstract— The action spectrum for the oxygen-independent inactivation of native transforming DNA from Haemophilus influenzae with near-UV radiation revealed a shoulder beginning at 334 and extending to 460 nm. The presence of 0.2 M histidine during irradiation produced a small increase in inactivation at 254, 290 and 313 nm, a large increase at 334 nm and a decrease in inactivation at 365, 405 and 460 nm. Photoreactivation did not reverse the DNA damage produced at pH 7.0 at 334, 365, 405 and 460 nm, but did reactivate the DNA after irradiation at 254, 290 and 313 nm. The inactivation of DNA irradiated at 254, 290 and 313 nm was considerably greater when the transforming ability was assayed in an excision-defective mutant compared with the wild type, although DNA irradiated at 334, 365, 405 and 460 nm showed smaller differences. These results suggest that the oxygen-independent inactivation of H. influenzae DNA at pH 7 by irradiation at 334, 365, 405 and 460 nm is caused by lesions other than pyrimidine dimers.     

Synthesis and properties of macrocyclic diazene switch with binaphthalene unit attached via acrylamide linkers

2,2′-Diiodo-1,1′-binaphthalene undergoes a tandem Heck reaction with methyl acrylate to afford methyl 2-(7H-dibenzo[c,g]fluoren-7-ylidene)acetate. As a consequence, the target macrocyclic diazene with binaphthalene unit attached via acrylamide linker was prepared by the stepwise building of acrylamide at a binaphthalene moiety, including the Doebner modification of the Knoevenagel condensation, and completed by oxidative macrocyclisation of aniline end-groups. Despite being an equimolar mixture of monomer and dimer, it exhibited remarkable changes in CD spectra due to reversible (E/Z) isomerisation of N=N diazene bonds upon irradiation at 365/465 nm. Although the dimer isomerises from (E) to (Z) isomer 7.4 times faster than the monomer, the latter’s contribution to the change in ellipticity at 307 nm in the photostationary state is 2.4 times greater.     

Effect of glutaraldehyde on the activity of some DNA restriction endonucleases

The effect of the bifunctional crosslinking reagent glutaraldehyde on the activity of the restriction enzymes Bam HI,Hind III, EcoRI, and Tthlll I was investigated. The four enzymes exhibited differential sensitivity to inactivation. Tthlll I was the most sensitive, with activity losses occurring at levels of 0.0025% and above.Hind III was the most stable of the four and remained fully active at concentrations as high as 0.075%. Addition of BSA to incubation mixtures generally had a stabilizing effect. Implications of these results for the design of glutaraldehyde-based methods for the immobilization of restriction endonucleases are discussed.     

THE EXCITATION OF 8-METHOXYPSORALEN WITH VISIBLE LIGHT: REVERSED PHASE HPLC QUANTITATION OF MONOADDUCTS and CROSS-LINKS

Abstract— The formation of 8-methoxypsoralen-DNA monoadducts and cross-links is presumed to be responsible for the efficacy of photochemotherapies that employ 8-methoxypsoralen activated with long-wavelength ultraviolet radiation (UVA,320–400 nm). In this report it is shown that 8-methoxypsoralen can also be activated with visible light (419 nm). Bovine aorta smooth muscle cells were treated with 8-methoxypsoralen (1000 ng/mL) and 419 nm light (up to 12 J/cm²). Cellular DNA was isolated, hydrolyzed using nucleolytic enzymes and then analyzed by reversed-phase high-performance liquid chromatography. The primary effect of using visible light instead of long-wavelength ultraviolet radiation is a more than 10-fold reduction in the extent of cross-link formation. Because the extent of monoadduct and cross-link formation has not been routinely measured in experiments in which cellular assays have been performed, it is difficult to correlate cell response to the presence of a particular type of 8-methoxypsoralen photoadduct (monoadduct or cross-link). Thus, the use of visible light allows the study of cells containing nearly 100% monoadducts. In addition, the reduction in cross-link formation when visible light is used to activate the compound may also reduce the mutagenicity of 8-methoxypsoralen and hence enhance its therapeutic efficacy.     

PHOTOCHEMISTRY OF HALOGEN PYRIMIDINES: IODINE RELEASE STUDIES

The feasibility of using direct iodide (I-) measurements to monitor the photochemistry of the halogenated pyrimidines 5-iodocytosine and 5-iodouracil and their corresponding deoxynucleosides was examined. Radiation from either a germicidal lamp (lambda = 254 nm) or a sunlamp (lambda greater than 290 nm) was employed to induce homolytic splitting of the carbon-iodine bond and the release of iodine atoms. These atoms combine to form I2 which reacts with water to ultimately form I- and iodate (IO3-). The formation of I- was followed using either high performance liquid chromatography with electrochemical detection or a specific ion electrode. IO3- was assayed spectroscopically following its conversion to triiodide. The yields of I- relative to starting material destroyed were either close to the theoretical limit of 83% or higher depending upon (a) the compound being irradiated, (b) the irradiation wavelength and (c) the extent of exposure. Yields of iodide greater than 83% are generally accounted for by a concomitant reduction in the yield of iodate such that the sum I(-) + IO3- is approx. 100%. Because iodate is photochemically reduced to iodide by 254 nm but not sunlamp irradiation, exhaustive irradiation at 254 nm converts all of the iodate present to iodide. These studies have application to the use of photochemical methods for quantitating the percent substitution of iodinated pyrimidines in DNA, and should be useful in following the photochemistry of IdUrd and IdCyd substituted DNA.     

Fluorescent hydrophobic zippers inside duplex DNA: interstrand stacking of perylene-3,4:9,10-tetracarboxylic acid bisimides as artificial DNA base dyes

Perylene-3,4:9,10-tetracarboxylic acid bisimides (PBs) were incorporated synthetically into oligonucleotides by using automated DNA building-block chemistry. The 2'-deoxyribofuranoside of the natural nucleosides was replaced by (S)-aminopropan-2,3-diol as an acyclic linker between the phosphodiester bridges that is tethered to one of the imide nitrogen atoms of the PB dye. The S configuration of this linker was chosen to mimic the stereochemical situation at the 3'-position of the natural 2'-deoxyribofuranosides. By using this strategy, up to six PB dyes were incorporated in the middle of 18-mer DNA duplexes by using interstrand alternating sequences of PBs with thymines or an abasic site analogue. Both PB dimers and PB hexamers as artificial base substitutions inside the duplexes yield characteristic excimer-type fluorescence. The stacking properties of the PB chromophores are modulated by the presence or absence of thymines opposite the PB modification site in the counterstrand. The interstrand PB dimers can be regarded as hydrophobically interacting base pairs, which display a characteristic fluorescence readout signal. Hence, for the PB hexamers, we proposed a zipperlike recognition motif that is formed inside duplex DNA. The PB zipper shows characteristic excimer-type emission as a fluorescence readout signal for the pairing interaction.