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.

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.

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.

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.

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.

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.

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.

Stabilisation of self-assembled DNA crystals by triplex-directed photo-cross-linking.

The tensegrity triangle is a robust DNA motif that can self-assemble to generate macroscopic three-dimensional crystals. However, the stability of these crystals is dependent on the high ionic conditions used for crystal growth. Here we demonstrate that a triplex-forming oligonucleotide can be used to direct the specific intercalation, and subsequent photo-cross-linking, of 4,5',8-trimethylpsoralen to single or multiple loci within or between the tiles of the crystal. Cross-linking between the tiles of the crystal improves their thermal stability. Such an approach is likely to facilitate the removal of crystals from their mother liquor and may prove useful for applications that require greater crystal stability.

Detecting RNA-RNA interactions using psoralen derivatives.

Psoralens are tricyclic compounds that intercalate into double-stranded DNA or RNA and, on irradiation with long-wavelength (365-nm) UV light, covalently link pyrimidines on adjacent strands. More rarely, psoralen cross-links can be observed at the ends of helices (i.e., double-stranded-single-stranded boundaries). Although psoralens can, in some instances, cross-link protein to RNA, their primary application is to detect RNA-RNA base-pairing interactions. The most useful psoralen derivative is 4'-aminomethyl trioxsalen (AMT), which is soluble in H2O. This protocol describes the use of AMT to detect RNA-RNA interactions in tissue culture cells or in extracts. Cross-linked RNAs are detectable by their reduced mobility in polyacrylamide gels. Cross-links can be reversed by exposure to short-wavelength (254 nm) UV light.

Synthesis of Oligonucleotides Containing 4,5',8-Trimethylpsoralen at the 2'-O Position and Their Cross-Linking Properties with RNAs.

4,5',8-Trimethylpsoralen-conjugated oligonucleotides have been used in the study of photo-cross-linking with target oligonucleotides and in the field of the photodynamic therapy. This unit describes synthetic procedures for oligonucleotides using 2'-O-methylphosphoramidite units and an adenosine phosphoramidite unit containing a 4,5',8-trimethylpsoralen derivative attached at the 2' position of an adenosine sugar moiety via an ethoxymethylene linkage. Procedures for obtaining the photo-cross-linking efficiency of 2'-O-methyloligonucleotides containing a 4,5',8-trimethylpsoralen derivative with a target oligonucleotide under UV irradiation conditions are also described, together with the procedure for preparation of (32)P-radiolabeled RNA.

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.

Development and characterization of nanocarriers for topical treatment of psoriasis by using combination therapy.

Psoriasis is an autoimmune, chronic, inflammatory skin disease characterized by epidermal hyperplasia, proliferation of blood vessels, and infiltration of leukocytes in dermis and epidermis. Several immunosuppressants such as methotrexate (MXT) and cyclosporine are used but they are associated with adverse effects due to down regulation of immune system. Numerous approaches have been explored to overcome the problems of conventional topical system such as high frequency of application, impermeability to skin barrier, and limited efficacy. Photodynamic therapy is another non-invasive technique currently used for skin diseases. The combination of two drugs is also commonly observed to achieve more effective therapy. In the present study, antipsoriatic activity of niosomal formulations for the treatment of psoriasis in combination with narrow and broad band UV radiation had been explored in experimental animal model.

Modulation of the UVB-induced lethality by furocoumarins in Staphylococcus aureus.

Furocumarins (FCs) are photoactive compounds capable of binding to DNA, and once excited by UVA light (∼365nm), they form photoadducts which can lead to mutagenicity and lethality. However, the biological effects of FCs combined with UVB light (312nm) is still little investigated. In the present study, the lethal effect of UVB light alone and combined with different concentrations of 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP) and 3-carbethoxypsoralen (3-CPs) was evaluated in a strain of Staphylococcus aureus. 8-MOP-UVB and TMP-UVB were more effective in inducing lethality compared to UVB alone, indicating that these FCs act as photosensitizing agents for UVB. The increase in concentration of 8-MOP resulted in a greater mortality. On the contrary, a decrease in mortality was found with an increase in TMP concentration. 3-CPs protected bacteria against damage induced by UVB, which can be attributed to the inhibition of cyclobutyl pyrimidine dimer formation. The different modulatory effects on lethality induced by UVB shown by the FCs tested could be related to differences in the specificity of each compound for particular nucleotide sequences, as well as other chemical characteristics of each molecule could influence the number and types of adducts formed, contributing to the photosensitizing or photoprotective effects observed.

[Simultaneous determination of eight furocoumarines in cosmetics by high performance liquid chromatography and verification by liquid chromatography-tandem mass spectrometry].

A method using high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of eight furocoumarines (8-hydroxypsoralen, psoralen, isopsoralen, 8-methoxypsoralen, 5-methoxypsoralen, trioxsalen, imperatorin and isoimperatorin) in cosmetics. The cosmetic samples, including cream, lotion, shampoo, powder and lipstick, were supersonically extracted with appropriate solvents. The extract was centrifuged, and the supernatant was filtered through a membrane, and then separated on an Agilent Zorbax SB-Phenyl chromatographic column (250 mm x 4.6 mm, 5 microm) by gradient elution at a flow rate of 1.0 mL/min with methanol-acetonitrile-water as mobile phases. The column temperature was set at 30 degrees C. The wavelength of detection was 250 nm. The limits of quantification (LOQs) were 0.25 mg/kg for 8-hydroxypsoralen and 0.5 mg/kg for psoralen, isopsoralen, 8-methoxypsoralen, 5-methoxypsoralen, trioxsalen, imperatorin and isoimperatorin. The recoveries at three spiked levels were in the range of 85.0% - 105.8% with the relative standard deviations (RSDs) of 0.41% - 7.90%. The intra-day precision (n=6) was less than 1%, and the inter-day precision (n = 6) was less than 2% for the peak areas of the eight furocoumarines in a mixed standard solution. The method is accurate, simple, rapid and suitable for the determination of the eight furocoumarines in various cosmetic samples.

Dengue virus photo-inactivated in presence of 1,5-iodonaphthylazide (INA) or AMT, a psoralen compound (4'-aminomethyl-trioxsalen) is highly immunogenic in mice.

Two novel methods of dengue virus inactivation using iodonaphthyl azide (INA) and aminomethyl trioxsalen (AMT) were compared with traditional virus inactivation by formaldehyde. The AMT inactivated dengue-2 virus retained its binding to a panel of 5 monoclonal antibodies specific for dengue-2 envelope protein, whereas inactivation by formaldehyde and INA led to 30-50% decrease in binding. All three inactivated viruses elicited high level virus neutralizing antibodies in vaccinated mice. However, only mice vaccinated with AMT inactivated virus mounted T cell responses similar to live, uninactivated virus.

[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.

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.