Unique Crosslinking Properties of Psoralen-Conjugated Oligonucleotides Developed by Novel Psoralen N-Hydroxysuccinimide Esters.

Psoralens and their derivatives, such as trioxsalen, have unique crosslinking features to DNA. However, psoralen monomers do not have sequence-specific crosslinking ability with the target DNA. With the development of psoralen-conjugated oligonucleotides (Ps-Oligos), sequence-specific crosslinking with target DNA has become achievable, thereby expanding the application of psoralen-conjugated molecules in gene transcription inhibition, gene knockout, and targeted recombination by genome editing. In this study, we developed two novel psoralen N-hydroxysuccinimide (NHS) esters that allow the introduction of psoralens into any amino-modified oligonucleotides. Quantitative evaluation of the photo-crosslinking efficiencies of the Ps-Oligos to target single-stranded DNAs revealed that the crosslinking selectivity to 5-mC is the unique feature of trioxsalen. We found that the introduction of an oligonucleotide via a linker at the C-5 position of psoralen can promote favorable crosslinking to target double-stranded DNA. We believe our findings are essential information for the development of Ps-Oligos as novel gene regulation tools.

MiR-CLIP reveals iso-miR selective regulation in the miR-124 targetome.

Many microRNAs regulate gene expression via atypical mechanisms, which are difficult to discern using native cross-linking methods. To ascertain the scope of non-canonical miRNA targeting, methods are needed that identify all targets of a given miRNA. We designed a new class of miR-CLIP probe, whereby psoralen is conjugated to the 3p arm of a pre-microRNA to capture targetomes of miR-124 and miR-132 in HEK293T cells. Processing of pre-miR-124 yields miR-124 and a 5'-extended isoform, iso-miR-124. Using miR-CLIP, we identified overlapping targetomes from both isoforms. From a set of 16 targets, 13 were differently inhibited at mRNA/protein levels by the isoforms. Moreover, delivery of pre-miR-124 into cells repressed these targets more strongly than individual treatments with miR-124 and iso-miR-124, suggesting that isomirs from one pre-miRNA may function synergistically. By mining the miR-CLIP targetome, we identified nine G-bulged target-sites that are regulated at the protein level by miR-124 but not isomiR-124. Using structural data, we propose a model involving AGO2 helix-7 that suggests why only miR-124 can engage these sites. In summary, access to the miR-124 targetome via miR-CLIP revealed for the first time how heterogeneous processing of miRNAs combined with non-canonical targeting mechanisms expand the regulatory range of a miRNA.

Synthesis and Evaluation of Oligonucleotide-Containing 2'-O-{[(4,5',8-Trimethylpsoralen)-4'-ylmethoxy]ethylaminocarb-onyl}adenosine as Photo-crosslinkable Gene Targeting Tools.

Several psoralen-conjugated oligonucleotides (Ps-Oligos) have been developed as photo-crosslinkable oligonucleotides targeting DNA or RNA. To avoid potential off-target effects, it is important to investigate the selective photo-crosslinking reactivity of Ps-Oligos to DNA or RNA. However, the selectivity of these Ps-Oligos has not been reported in detail thus far. In this study, we evaluated the photo-crosslinking properties of two Ps-Oligos, 5'-Ps-Oligo and a novel Ps-Oligo containing 2'-O-{[(4,5',8-trimethylpsoralen)-4'-ylmethoxy]ethylaminocarbonyl}adenosine (APs2-Oligo). Notably, 5'-Ps-Oligo preferentially crosslinked with DNA, whereas APs2-Oligo preferentially crosslinked with RNA. These results demonstrate the interesting crosslinking properties of Ps-Oligos, which will provide useful information for the molecular design of novel Ps-Oligos in future studies.

Nucleotide Excision Repair, XPA-1, and the Translesion Synthesis Complex, POLZ-1 and REV-1, Are Critical for Interstrand Cross-Link Repair in Caenorhabditis elegans Germ Cells.

Interstrand cross-links (ICLs) are adducts of covalently linked nucleotides in opposing DNA strands that obstruct replication and prime cells for malignant transformation or premature cell death. ICLs may be caused by alkylating agents or ultraviolet (UV) irradiation. These toxic lesions are removed by diverse repair mechanisms such as the Fanconi anemia (FA) pathway, nucleotide excision repair (NER), translesion synthesis (TLS), and homologous recombination (HR). In mammals, the xeroderma pigmentosum group F (XP-F) protein participates in both the FA pathway and NER, while DNA polymerase ζ (POLZ-1) and REV-1 mediate TLS. Nevertheless, little is known regarding the genetic determinants of these pathways in ICL repair and damage tolerance in germ cells. In this study, we examined the sensitivity of Caenorhabditis elegans germ cells to ICLs generated by trimethylpsoralen/ultraviolet A (TMP/UV-A) combination, and embryonic mortality was employed as a surrogate for DNA damage in germ cells. Our results show that XPA-1, POLZ-1, and REV-1 were more critical than FA pathway mediators in preserving genomic stability in C. elegans germ cells. Notably, mutant worms lacking both XPA-1 and POLZ-1 (or REV-1) were more sensitive to ICLs compared to either single mutant alone. Moreover, knockdown of XPA-1 and REV-1 leads to the retarded disappearance of RPA-1 and RAD-51 foci upon ICL damage. Since DNA repair mechanisms are broadly conserved, our findings may have ramifications for prospective therapeutic interventions in humans.

Tracing the Photoaddition of Pharmaceutical Psoralens to DNA.

The psoralens 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP) and 5-methoxypsoralen (5-MOP) find clinical application in PUVA (psoralen + UVA) therapy. PUVA treats skin diseases like psoriasis and atopic eczema. Psoralens target the DNA of cells. Upon photo-excitation psoralens bind to the DNA base thymine. This photo-binding was studied using steady-state UV/Vis and IR spectroscopy as well as nanosecond transient UV/Vis absorption. The experiments show that the photo-addition of 8-MOP and TMP involve the psoralen triplet state and a biradical intermediate. 5-MOP forms a structurally different photo-product. Its formation could not be traced by the present spectroscopic technique.

The Photoaddition of a Psoralen to DNA Proceeds via the Triplet State.

Psoralens are natural compounds that serve in the light dependent treatment of certain skin diseases (PUVA therapy). They are DNA intercalators that upon photoexcitation form adducts with thymine bases. For one psoralen derivative, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), the photoreactions are characterized here by nanosecond UV-vis and IR absorption spectroscopy. The triplet state of AMT is identified as the reactive one. On the 1-10 µs time scale this local triplet state transforms into a triplet biradical bearing one single bond between the addends. Within ∼50 µs this biradical forms the final adduct featuring a cyclobutane ring. This kinetic behavior is in stark contrast to the closely related photoaddition of two thymine moieties within the DNA. Origins of the differences are discussed.

Systematic analysis of DNA crosslink repair pathways during development and aging in Caenorhabditis elegans.

DNA interstrand crosslinks (ICLs) are generated by endogenous sources and chemotherapeutics, and pose a threat to genome stability and cell survival. Using Caenorhabditis elegans mutants, we identify DNA repair factors that protect against the genotoxicity of ICLs generated by trioxsalen/ultraviolet A (TMP/UVA) during development and aging. Mutations in nucleotide excision repair (NER) components (e.g. XPA-1 and XPF-1) imparted extreme sensitivity to TMP/UVA relative to wild-type animals, manifested as developmental arrest, defects in adult tissue morphology and functionality, and shortened lifespan. Compensatory roles for global-genome (XPC-1) and transcription-coupled (CSB-1) NER in ICL sensing were exposed. The analysis also revealed contributions of homologous recombination (BRC-1/BRCA1), the MUS-81, EXO-1, SLX-1 and FAN-1 nucleases, and the DOG-1 (FANCJ) helicase in ICL resolution, influenced by the replicative-status of the cell/tissue. No obvious or critical role in ICL repair was seen for non-homologous end-joining (cku-80) or base excision repair (nth-1, exo-3), the Fanconi-related proteins BRC-2 (BRCA2/FANCD1) and FCD-2 (FANCD2), the WRN-1 or HIM-6 (BLM) helicases, or the GEN-1 or MRT-1 (SNM1) nucleases. Our efforts uncover replication-dependent and -independent ICL repair networks, and establish nematodes as a model for investigating the repair and consequences of DNA crosslinks in metazoan development and in adult post-mitotic and proliferative germ cells.

XPF knockout via CRISPR/Cas9 reveals that ERCC1 is retained in the cytoplasm without its heterodimer partner XPF.

The XPF/ERCC1 heterodimeric complex is essentially involved in nucleotide excision repair (NER), interstrand crosslink (ICL), and double-strand break repair. Defects in XPF lead to severe diseases like xeroderma pigmentosum (XP). Up until now, XP-F patient cells have been utilized for functional analyses. Due to the multiple roles of the XPF/ERCC1 complex, these patient cells retain at least one full-length allele and residual repair capabilities. Despite the essential function of the XPF/ERCC1 complex for the human organism, we successfully generated a viable immortalised human XPF knockout cell line with complete loss of XPF using the CRISPR/Cas9 technique in fetal lung fibroblasts (MRC5Vi cells). These cells showed a markedly increased sensitivity to UVC, cisplatin, and psoralen activated by UVA as well as reduced repair capabilities for NER and ICL repair as assessed by reporter gene assays. Using the newly generated knockout cells, we could show that human XPF is markedly involved in homologous recombination repair (HRR) but dispensable for non-homologous end-joining (NHEJ). Notably, ERCC1 was not detectable in the nucleus of the XPF knockout cells indicating the necessity of a functional XPF/ERCC1 heterodimer to allow ERCC1 to enter the nucleus. Overexpression of wild-type XPF could reverse this effect as well as the repair deficiencies.

Methods for single/low-copy integration by ultraviolet and trimethylpsoralen treatment in Caenorhabditis elegans.

Single/low-copy transgene integration is essential for avoiding overexpression, ectopic expression and gene silencing in the germline. Here, we present an overview of a method that uses ultraviolet and trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations in Caenorhabditis elegans. Single/low-copy transgenes from extrachromosomal arrays are integrated into the genome using positive selection based on temperature sensitivity with a vps-45 rescue fragment and negative selection based on benzimidazole sensitivity with a ben-1 rescue fragment. The copy number of the integrated transgenes is determined using quantitative PCR. Our UV/TMP integration method, which is based on familiar extrachromosomal transgenics, provides a simple approach for generating single/low-copy gene integrations.

Reverse genetics by chemical mutagenesis in Caenorhabditis elegans.

Traditional reverse genetics on yeast, mice and other organisms uses homologous recombination with transgenic DNA to interrupt a target gene. Here we report that target-selected gene inactivation can be be achieved in Caenorhabditis elegans with the use of chemical mutagens. We use PCR to selectively visualize deletions in genes of interest; the method is sensitive enough to permit detection of a single mutant among more than 15,000 wild types. A permanent frozen mutant collection of more than a million mutagenized animals has been established, and deletion mutants of several G-protein genes were isolated from it. The approach is suitable to be scaled up for systematic inactivation of all 17,000 C. elegans genes. Because it requires no transgenesis or cell culturing, it may also be applicable to small organisms usually considered to be outside the realm of reverse genetics (for example, other nematodes and insects). Any sequenced gene in any organism that can be handled in very large numbers can possibly be targeted in this way.

Single/low-copy integration of transgenes in Caenorhabditis elegans using an ultraviolet trimethylpsoralen method.

BACKGROUND: Transgenic strains of Caenorhabditis elegans are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are semi-stable and their expression is silenced in the germline. Mos1 transposon or microparticle bombardment methods have been developed to create single- or low-copy chromosomal integrated lines. Here we report an alternative method using ultraviolet trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations. RESULTS: We successfully integrated low-copy transgenes from extrachromosomal arrays using positive selection based on temperature sensitivity with a vps-45 rescue fragment and negative selection based on benzimidazole sensitivity with a ben-1 rescue fragment. We confirmed that the integrants express transgenes in the germline. Quantitative PCR revealed that strains generated by this method contain single- or low-copy transgenes. Moreover, positive selection marker genes flanked by LoxP sites were excised by Cre recombinase mRNA microinjection, demonstrating Cre-mediated chromosomal excision for the first time in C. elegans. CONCLUSION: Our UV/TMP integration method, based on familiar extrachromosomal transgenics, provides a useful approach for generating single/low-copy gene integrations.

'RNA walk' a novel approach to study RNA-RNA interactions between a small RNA and its target.

In this study we describe a novel method to investigate the RNA-RNA interactions between a small RNA and its target that we termed 'RNA walk'. The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT-PCR or real-time PCR. Domains carrying the cross-linked adducts fail to efficiently amplify by PCR compared with non-cross-linked domains. This method was calibrated and used to study the interaction between a special tRNA-like molecule (sRNA-85) that is part of the trypanosome signal recognition particle (SRP) complex and the ribosome. Four contact sites between sRNA-85 and rRNA were identified by 'RNA walk' and were further fine-mapped by primer extension. Two of the contact sites are expected; one contact site mimics the interaction of the mammalian Alu domain of SRP with the ribosome and the other contact sites include a canonical tRNA interaction. The two other cross-linked sites could not be predicted. We propose that 'RNA walk, is a generic method to map target RNA small RNAs interactions in vivo.

A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding.

The helical tension of chromosomal DNA is one of the epigenetic landmarks most difficult to examine experimentally. The occurrence of DNA crosslinks mediated by psoralen photobinding (PB) stands as the only suitable probe for assessing this problem. PB is affected by chromatin structure when is done to saturation; but it is mainly determined by DNA helical tension when it is done to very low hit conditions. Hence, we developed a method for genome-wide analysis of DNA helical tension based on PB. We adjusted in vitro PB conditions that discern DNA helical tension and applied them to Saccharomyces cerevisiae cells. We selected the in vivo cross-linked DNA sequences and identified them on DNA arrays. The entire procedure was robust. Comparison of PB obtained in vivo with that obtained in vitro with naked DNA revealed that numerous chromosomal regions had deviated PB values. Similar analyses in yeast topoisomerase mutants uncovered further PB alterations across specific chromosomal domains. These results suggest that distinct chromosome compartments might confine different levels of DNA helical tension in yeast. Genome-wide analysis of psoralen-DNA PB can be, therefore, a useful approach to uncover a trait of the chromosome architecture not amenable to other techniques.

[Simultaneous determination of four kinds of furocoumarins in cosmetics by high performance liquid chromatography].

OBJECTIVE: A high performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of 4 furocoumarins, including 8-methoxycoumarin, 5-methoxycoumarin, trioxsalen and imperatorin in cosmetics. METHODS: The separation was carried on Diamonsil C18 column (4.6 mm x 250 mm, 5 microm) by gradient elution with methanol-water as mobile phase at a flow rate of 1.0 ml/min and column temperature of 30 degrees C. The analysis of 4 furocoumarins was performed by HPLC with diodearray detector at the wavelength of 248 nm. RESULTS: Identification of 4 furocoumarins was achieved by retention time, and quantification analysis was performed by the external standard method. The limits of quantitation (LOQ) for 4 furocoumarins were 0.54, 0.52, 0.58 and 0.50 microg/g, respectively. The method showed a good linearity in the range of 0.05-10 microg/ml with correlation coefficients more than 0.999. The mean recoveries at spiked levels were in the range of 89.9%-105.2% with RSDs of 0.20%-1.08%. CONCLUSION: The method was accurate and simple, and was suitable for the determination of 4 furocoumarins in cosmetics.

Trimethylangelicin reduces IL-8 transcription and potentiates CFTR function.

Chronic inflammatory response in the airway tract of patients affected by cystic fibrosis is characterized by an excessive recruitment of neutrophils to the bronchial lumina, driven by the chemokine interleukin (IL)-8. We previously found that 5-methoxypsoralen reduces Pseudomonas aeruginosa-dependent IL-8 transcription in bronchial epithelial cell lines, with an IC(50) of 10 µM (Nicolis E, Lampronti I, Dechecchi MC, Borgatti M, Tamanini A, Bezzerri V, Bianchi N, Mazzon M, Mancini I, Giri MG, Rizzotti P, Gambari R, Cabrini G. Int Immunopharmacol 9: 1411-1422, 2009). Here, we extended the investigation to analogs of 5-methoxypsoralen, and we found that the most potent effect is obtained with 4,6,4'-trimethylangelicin (TMA), which inhibits P. aeruginosa-dependent IL-8 transcription at nanomolar concentration in IB3-1, CuFi-1, CFBE41o-, and Calu-3 bronchial epithelial cell lines. Analysis of phosphoproteins involved in proinflammatory transmembrane signaling evidenced that TMA reduces the phosphorylation of ribosomal S6 kinase-1 and AKT2/3, which we found indeed involved in P. aeruginosa-dependent activation of IL-8 gene transcription by testing the effect of pharmacological inhibitors. In addition, we found a docking site of TMA into NF-κB by in silico analysis, whereas inhibition of the NF-κB/DNA interactions in vitro by EMSA was observed at high concentrations (10 mM TMA). To further understand whether NF-κB pathway should be considered a target of TMA, chromatin immunoprecipitation was performed, and we observed that TMA (100 nM) preincubated in whole living cells reduced the interaction of NF-κB with the promoter of IL-8 gene. These results suggest that TMA could inhibit IL-8 gene transcription mainly by intervening on driving the recruitment of activated transcription factors on IL-8 gene promoter, as demonstrated here for NF-κB. Although the complete understanding of the mechanism of action of TMA deserves further investigation, an activity of TMA on phosphorylating pathways was already demonstrated by our study. Finally, since psoralens have been shown to potentiate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport, TMA was tested and found to potentiate CFTR-dependent chloride efflux. In conclusion, TMA is a dual-acting compound reducing excessive IL-8 expression and potentiating CFTR function.

Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease.

Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen-modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex approximately 3 bases from the 3'-side and approximately 9 bases from the 5'-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.

Photoallergic contact dermatitis to 8-methoxypsoralen in Ficus carica.

BACKGROUND: Photocontact dermatitis to Ficus carica is induced by furocoumarins present in sap. These substances are generally considered to cause phototoxic reactions. OBJECTIVES: We conducted a patch test and histopathological study of patients with phytophoto contact dermatitis from the fig tree to evaluate the mechanism underlying the photoreaction. PATIENTS AND METHODS: Patch and photopatch testing with serial dilutions of two natural furocoumarins [5-methoxypsoralen and 8-methoxypsoralen (8-MOP)] contained in plant sap were performed in 47 patients. A synthetic furocoumarin, 4,5',8-trimethylpsoralen, was also tested. Histopathological analyses were made of some positive photoreactions. RESULTS: Positive photopatch tests reactions to 8-MOP were obtained in 12 of 47 patients, in 4 of them down to a concentration of 0.0001%. Patch tests and photopatch tests to the other two furocoumarins were negative. Histopathological findings on biopsies from positive photopatch tests to 8-MOP showed a dermatitis. CONCLUSIONS: Allergic photoreactions induced by contact with plants containing coumarins are generally regarded as chance findings. This study has demonstrated that phytophoto allergic contact dermatitis resulting from furocoumarins is not an exceptional finding, and should be suspected in subjects with diffuse clinical manifestations in photo-exposed but also non-exposed sites. To differentiate allergic from toxic photoreactions, patch tests need to be performed with serial dilutions of furocoumarins. Histological analysis of a biopsy sample from a positive test site will reveal alterations compatible with a photoallergic contact dermatitis.

Design of a Fluorescence Turn-on and Label-free Aptasensor Using the Intrinsic Quenching Power of G-Quadruplex to AMT.

A novel fluorescent aptasensor based on the G-quadruplex induced fluorescent quenching of psoralen and the competitive interactions between 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), adenosine triphosphate (ATP) and G-rich DNA functionalized split ATP aptamer was proposed. The binding of ATP to the G-rich DNA functionalized split aptamer induced a significant enhancement in fluorescence emission intensity while undergoing excitation at 340 nm. Under the optimal conditions, the developed aptasensor showed high selectivity and good accuracy for detecting ATP. The practicality of the proposed aptasensor has been confirmed by successfully analyzing ATP in spiked human blood serum samples with satisfactory results. As far as we know, this is the first time that the intrinsic quenching ability of G-quadruplex was applied to simply construct a fluorescence turn-on and label-free aptasensor. On account of the superiority of the simplicity of the design strategy, more work is expected in the future to develop a variety of novel sensors for other important analytes using the quenching capability of G-quadruplex through reasonable designs.

Photochemotherapy: identification of a metabolite of 4, 5', 8-trimethylpsoralen.

A compound, 4, 8-dimethyl, 5'-carboxypsoralen (DMeCP), has been identified in mouse urine as a major metabolite of the photoactive drug, 4, 5', 8-trimethylpsoralen (TMeP). This drug is widely used in the treatment of vitiligo and psoriasis. DMeCP is fluorescent, and nonphotosensitizing when tested on guinea pig skin. DMeCP also occurs in the urine of human patients receiving TMeP orally.

Small nuclear RNA U2 is base-paired to heterogeneous nuclear RNA.

Eukaryotic cells contain a set of low molecular weight nuclear RNA's. One of the more abundant of these is termed U2 RNA. The possibility that U2 RNA is hydrogen-bonded to complementary sequences in other nuclear RNA's was investigated. Cultured human (HeLa) cells were treated with a psoralen derivative that cross-links RNA chains that are base-paired with one another. High molecular weight heterogeneous nuclear RNA was isolated under denaturing conditions, and the psoralen cross-links were reversed. Electrophoresis of the released RNA and hybridization with a human cloned U2 DNA probe revealed that U2 is hydrogen-bonded to complementary sequences in heterogeneous nuclear RNA in vivo. In contrast, U2 RNA is not base-paired with nucleolar RNA, which contains the precursors of ribosomal RNA. The results suggest that U2 RNA participates in messenger RNA processing in the nucleus.