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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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

It has been described that treatment of cells with high dose psoralen and UVA induce the production of reactive oxygen species (ROS) leading to DNA damage. Transcription factor nuclear factor kappa B (NFkappaB) plays a crucial role in regulating not only cell growth but also cell differentiation, and ROS seem to be partly involved in these mechanisms. The aim of this research was to find out the effect of a combined treatment with trioxsalen (TMP)/UVA on NFkappaB binding activity in HaCaT keratinocytes. HaCaT keratinocytes were treated with 27 microg/l TMP. This concentration did not affect the proliferation rates, nor was it toxic, as shown by cytotoxicity assays. After treatment with TMP with or without UVA (1 J/cm(2)), NFkappaB binding activity in nuclear protein extracts was measured by electrophoretic mobility shift assays. The effect on cytokines and cytokine receptor genes was investigated using cDNA expression arrays. An inhibitory effect on NFkappaB binding activity was found between 30 and 60 min after TMP supplementation of the culture media. UVA irradiation induced a 2-fold increase in NFkappaB binding activity in TMP supplemented HaCaT keratinocytes compared with the non-irradiated control. In addition, NFkappaB binding activity was higher after UVA irradiation with TMP than in UVA irradiated cells in the absence of TMP. TGF-alpha, IL-1R, IL-2Ralpha, IL-12beta and PDGF expression was induced by UVA. However, all of them except PDGF were inhibited by combined TMP/UVA treatment. Using an inhibitor of NFkappaB activation, we found out that under these conditions, these cytokines or cytokine receptor genes are apparently not regulated by NFkappaB. Our results indicate that a combined TMP/UVA treatment of HaCaT keratinocytes induces NFkappaB binding activity, and that this is a synergistic effect. The investigated cytokines, and cytokine receptor genes do not seem to be NFkappaB regulated; however, TMP shows anti-inflammatory capacities in vitro., (Copyright 2002 S. Karger AG, Basel)

Irradiation of thin films consisting of 4,5',8-trimethylpsoralen (TMP), adenosine and small amounts of alcohols led to TMP-alcohol photoadducts in addition to TMP-adenosine photoadducts. Four TMP-ethanol and two TMP-methanol adducts have been separated and characterized. Covalent bonds were formed between the 4-carbon of TMP and the alpha-carbon to the hydroxy group in the alcohols. The TMP-alcohol photoadducts were formed only in the TMP film containing small amounts of alcohol and adenosine. Furthermore, no photoadduct of TMP and ribose was detected upon photolysis of a TMP-ribose film, suggesting that the adenine moiety plays a specific role in the reaction. The interaction of adenosine with psoralens in a dry film may be related to the DNA sequence selectivity observed for the photoreaction of psoralens with DNA.

A novel photoadduct of 4,5',8-trimethylpsoralen (TMP) and adenosine was isolated and purified by reverse-phase liquid chromatography. The structure of the photoproduct was determined by various spectral methods and found to be a TMP-adenosine 1:1 adduct resulting from the covalent bond formation between the carbon C(4) of TMP and ribose 4'-carbon of adenosine.

A wide variety of viruses are inactivated by psoralen compounds in the presence of ultraviolet A light (UVA). Use of aminomethyltrimethylpsoralen (AMT) and UVA is being evaluated as a method to inactivate viruses that may be present in platelet suspensions prepared for transfusion. Studies have been conducted to assess how variation in various environmental parameters influences the extent of viral inactivation and the retention of platelet properties. Most notably, it was determined that increasing levels of plasma progressively inhibited the inactivation of model viruses. As a result, experiments were routinely conducted at a plasma level of approximately 14.5%, using 40 micrograms/ml AMT, which was determined to be optimal when using this reduced plasma level. The reduced plasma level was achieved by dilution with a nonplasma medium that has been shown to be satisfactory for storage of platelets. Under these conditions, about 5 logs of vesicular stomatitis virus (VSV), pseudorabies, and phi 6 inactivation were achieved. Variation of platelet and leukocyte counts, within normal levels, had a minimal effect on extent of viral inactivation. Although oxygen level (mean levels, 97.9 mm Hg versus 19.2 mm Hg) had only a small influence on viral inactivation with 2.4, 4.8, and 7.2 J/cm2 of UVA (equivalent to 1-3 minutes of exposure), in vitro platelet properties, such as medium pH, morphology characteristics, and aggregation response, were better retained with a longer exposure time at the reduced oxygen level. With normal oxygen (97.9 mm Hg), platelet properties declined substantially relative to untreated controls (no UVA, no AMT) on exposure to 4.8 J/cm2. Our studies have identified two sets of conditions that provide about 5 logs of virus inactivation without extensively altering platelet in vitro properties.

UV irradiation of 4'-hydroxymethyl-4,5',8-trimethyl psoralen (HMT) in aqueous solution yields three major photoproducts. We have isolated and characterized (1) a cyclobutane-bridged dimer in which a cis-syn linkage occurs between the furan end of one HMT moiety and the pyrone end of the other; (2) a cyclobutane-bridged dimer wherein both HMT moieties are linked at their pyrone ends with probable cis-syn configuration; and (3) an isomer of HMT for which we have proposed a structure in which the furan and pyrone ring oxygens assume a para orientation via photoisomerization.

The DNA of mammalian cells treated with 4,5',8-trimethylpsoralen (Trioxsalen) is crosslinked (preventing transcription) only after photoactivation with near ultraviolet light. Suppression of gene action in cocultivated or fused cultures, including both induction and antiviral action of fibroblast interferon, can consequently be limited to pretreated cells, an advantage over actinomycin treatment.

Aminomethyltrioxsalen (AMT), a psoralen, is known to cause interstrand crosslinks in double stranded nucleic acids. We have demonstrated the photochemical reversal of this reaction, and have used this result to develop a method for identification of specific sequences which are adjacent because of RNA secondary structure formation. E. coli 5S rRNA is used as a model system. We isolated and characterized a product that is derived from the stem region of 5S RNA.

The photomodification of 4,5',8-trimethylpsoralen (TMP) and related compounds was studied in aqueous solution in the presence of oxygen. The capacity of these compounds to generate singlet oxygen was also determined and an evident correlation between the capacity to generate singlet oxygen and the rate constant of the photomodification was observed. Moreover the photomodification was increased in the presence of D2O (which increases the lifetime of singlet oxygen) and quenched in the presence of sodium azide, a well known singlet oxygen quencher. These data support the hypothesis that the photomodification of the examined compounds occurs mainly via singlet oxygen. The role of the photomodification of TMP in relation of therapeutical efficacy is discussed.

The structure of herpes simplex virus type 1 (HSV-1) DNA in the nuclei of living infected cells was studied with the DNA photoaffinity probe 4,5',8-trimethylpsoralen. The rate of photobinding to HSV-1 DNA was compared to that of a suitable internal control at different times during infection. The rates of photobinding to DNA packaged in virions, capsids, and prereplicative and postreplicative DNA were characteristically different. By 4 h after infection, after the initiation of DNA replication, the rate of photobinding to HSV-1 DNA increased 4 times relative to the rate of binding to the host DNA. The enhanced rate of photobinding to HSV-1 DNA was maintained at all later times during infection and was not affected when frequent single-strand breaks were introduced in HSV-1 DNA by gamma irradiation of infected cells. The results suggest that the bulk of the replicating herpes DNA is free of torsional tension and that the differing rates of photobinding are attributable to changes in accessibility of the HSV-1 DNA. The results are compatible with previous proposals, based on in vitro studies, that intranuclear HSV-1 DNA is primarily free of nucleosomal organization and suggest that there are few, if any, unrestrained DNA supercoils averaged over the entire HSV-1 genome.

Experimental survival curves for Escherichia coli K 12 (CR 34) were determined after exposure to 4,5',8-trimethylpsoralen and near ultraviolet light. The lethal action was shown to arise exclusively from interstrand crosslinks, cell vulnerability increasing markedly with the doubling time of the culture. To account for these results, two quite different models are considered. The first assumes that a cell survives as long as at least one copy of its genome remains undamaged; a variant of this permits repair by DNA strand exchange. The second model allows for a limited period of time during which DNA repair can take place. A crosslink in a stretch of DNA due to be replicated within this interval constitutes a fatal lesion. Theoretical survival curves are computed for bacterial populations with defined age distributions and chromosome configurations. While the first model completely fails to provide a satisfactory description of the experimental results, the second model does predict the presence of a shoulder in the survival curves and, in one of its forms, it seems to agree rather well with the measured data over a wide range of crosslink concentrations and doubling times.

Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E. coli DNA polymerase I large fragment. By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT. These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues. In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5'exonuclease proof-reading activity of the DNA polymerase.