Aminomethyl-4,5',8-trimethylpsoralen Research Articles

Spectroscopic view on the interaction between the psoralen derivative amotosalen and DNA

Psoralens are eponymous for PUVA (psoralen plus UV-A radiation) therapy, which inter alia can be used to treat various skin diseases. Based on the same underlying mechanism of action, the synthetic psoralen amotosalen (AMO) is utilized in the pathogen reduction technology of the INTERCEPT® Blood System to inactivate pathogens in plasma and platelet components. The photophysical behavior of AMO in the absence of DNA is remarkably similar to that of the recently studied psoralen 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT). By means of steady-state and time-resolved spectroscopy, intercalation and photochemistry of AMO and synthetic DNA were studied. AMO intercalates with a higher affinity into A,T-only DNA (KD = 8.9 × 10−5 M) than into G,C-only DNA (KD = 6.9 × 10−4 M). AMO covalently photobinds to A,T-only DNA with a reaction quantum yield of ΦR = 0.11. Like AMT, it does not photoreact following intercalation into G,C-only DNA. Femto- and nanosecond transient absorption spectroscopy reveals the characteristic pattern of photobinding to A,T-only DNA. For AMO and G,C-only DNA, signatures of a photoinduced electron transfer are recorded.Graphical abstract

Psoralen Derivatives with Enhanced Potency.

Psoralen is a furocoumarin natural product that intercalates within DNA and forms covalent adducts when activated by ultraviolet radiation. It is well known that this property contributes to psoralen's clinical efficacy in several disease contexts, which include vitiligo, psoriasis, graft-versus-host disease and cutaneous T-cell lymphoma. Given the therapeutic relevance of psoralen and its derivatives, we attempted to synthesize psoralens with even greater potency. In this study, we report a library of 73 novel psoralens, the largest collection of its kind. When screened for the ability to reduce cell proliferation, we identified two derivatives even more cytotoxic than 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), one of the most potent psoralens identified to date. Using MALDI-TOF MS, we studied the DNA adduct formation for a subset of novel psoralens and found that in most cases enhanced DNA binding correlated well with cytotoxicity. Generally, our most potent derivatives contain positively charged substituents, which we believe increase DNA affinity and enhance psoralen intercalation. Thus, we provide a rational approach to guide efforts toward further optimizing psoralens to fully capitalize on this drug class' therapeutic potential. Finally, the structure-activity insights we have gained shed light on several opportunities to study currently underappreciated aspects of psoralen's mechanism.

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.

Ground- and Excited-State Interactions of a Psoralen Derivative with Human Telomeric G-Quadruplex DNA.

G-quadruplex DNA has been a recent target for anticancer agents, and its binding interactions with small molecules, often used as anticancer drugs, have become an important area of research. Considering that psoralens have long been studied in the context of duplex DNA but that very little is known about their potential as G-quadruplex binders and their excited-state interaction with the latter has not been explored, we have studied herein the binding of a planar water-soluble psoralen derivative, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), with the 22-mer human telomeric G-quadruplex-forming sequence, AGGG(TTAGGG)3, labeled here as (hTel22), and investigated the consequences of photoexcitation of AMT by calorimetric and spectroscopic techniques. The results show an enthalpy-driven 1:1 binding of AMT with hTel22 via end-stacking mode. Fluorescence quenching experiments on 6-fluorescein amidite-labeled oligomers indicate that the binding site is nearer to the 3' end of hTel22 in the diagonal loop region. Femtosecond time-resolved transient absorption measurements indicate electron transfer from the guanine moiety of hTel22 to photoexcited AMT, leading to the formation of a radical pair species (AMT•-G•+), which survives for 30 ps and is favored by a parallel/quasi-parallel orientation between the two. The findings reveal psoralens as a prospective class of compounds for the development of anticancer therapeutics by targeting the G-quadruplex DNA.

Rapid characterization of cross-links, mono-adducts, and non-covalent binding of psoralens to deoxyoligonucleotides by LC-UV/ESI-MS and IRMPD mass spectrometry.

Upon UV photoactivation, psoralen analogs form covalent mono-adducts and cross-links with DNA at thymine residues. Electrospray ionization mass spectrometric analysis allowed rapid and efficient determination of the reaction percentages of each psoralen analog with DNA duplexes containing different binding sites after exposure to UV irradiation. The distribution of cross-linked products and mono-adducts was monitored by both LC-UV and IRMPD-MS methods with the highest ratio of cross-linked products to mono-adducts obtained for 8-methoxypsoralen (8-MOP), psoralen (P), and 5-methoxypsoralen (5-MOP). Reactions at 5'-TA sites were favored over 5'-AT sites, and duplexes containing two and three binding sites showed extensive binding by the psoralens. 4'-Aminomethyl-4,5',8-trimethylpsoralen (AMP) bound non-selectively via non-covalent interactions and was the only psoralen analog to show significant binding in the absence of UV irradiation. 8-MOP binding displayed the greatest sequence selectivity among the psoralen analogs. The sites of interstrand cross-linking were determined by fragmentation of the duplex/psoralen complexes by infrared multiphoton dissociation (IRMPD), which produced cross-linked product ions containing an intact single strand, the psoralen analog, and either a w(n) or a(n)-B portion of the complementary strand. IRMPD of DNA/AMP complexes after UV irradiation also produced high abundances of the intact single strands with the AMP ligand attached, products indicative of a significant population of mono-adducts.

Inactivation of Leukocytes in Platelet Concentrates by Photochemical Treatment With Psoralen Plus UVA

A photochemical treatment (PCT) process using a novel psoralen and long wavelength ultraviolet light (UVA, 320-400 nm) has been developed to inactivate bacteria and viruses in platelet concentrates. This study evaluated the efficacy of PCT for inactivation of leukocytes that contaminate platelet preparations. Three psoralens, 8-methoxypsoralen (8-MOP), 4′-aminomethyl 4,5′,8-trimethylpsoralen (AMT), and the novel psoralen S-59, were compared using the following four independent but complementary biological and molecular assays. (1) T-cell viability: Treatment with 150 μmol/L S-59 and 1.0 to 3.0 Joules/cm2 UVA inactivated >5.4 ± 0.3 log10 of T cells in full-sized single-donor plateletpheresis units. Using 1.0 Joule/cm2 UVA, the lowest dose of S-59, AMT and 8-MOP required to reduce the number of T cells to the limit of detection was 0.05 μmol/L, 1.0 μmol/L, and 10.0 μmol/L, respectively. (2) Cytokine synthesis: Treatment with 1.9 Joules/cm2 UVA and 150 μmol/L S-59 or AMT completely inhibited synthesis of the cytokine IL-8 by contaminating leukocytes during 5 days of platelet storage. After treatment with 75 μmol/L 8-MOP and 1.9 Joules/cm2 UVA, only low levels of IL-8 were detected. (3) Psoralen-DNA adduct formation: The combination of 1.9 Joules/cm2 UVA and 150 μmol/L S-59, AMT, or 8-MOP induced 12.0 ± 3.0, 6.0 ± 0.9, and 0.7 psoralen adducts per 1,000 bp DNA, respectively. (4) Replication competence: Polymerase chain reaction (PCR) amplification of small genomic DNA sequences (242-439 bp) after PCT was inhibited. The degree of PCR amplification inhibition correlated with the level of adduct formation (S-59 > AMT > 8-MOP). In contrast, 2,500 cGy gamma radiation, a dose that inactivates >5 log10 of T cells in blood products, had minimal effect on cytokine synthesis and did not induce sufficient DNA strand breaks to inhibit PCR amplification of the same small DNA sequences. These results demonstrate that leukocytes are sensitive to PCT with psoralens and among the psoralens tested S-59 is the most effective. Therefore, PCT has the potential to reduce the incidence of leukocyte-mediated adverse immune reactions associated with platelet transfusion.

Inactivation of Leukocytes in Platelet Concentrates by Photochemical Treatment With Psoralen Plus UVA

AbstractA photochemical treatment (PCT) process using a novel psoralen and long wavelength ultraviolet light (UVA, 320-400 nm) has been developed to inactivate bacteria and viruses in platelet concentrates. This study evaluated the efficacy of PCT for inactivation of leukocytes that contaminate platelet preparations. Three psoralens, 8-methoxypsoralen (8-MOP), 4′-aminomethyl 4,5′,8-trimethylpsoralen (AMT), and the novel psoralen S-59, were compared using the following four independent but complementary biological and molecular assays. (1) T-cell viability: Treatment with 150 μmol/L S-59 and 1.0 to 3.0 Joules/cm2 UVA inactivated >5.4 ± 0.3 log10 of T cells in full-sized single-donor plateletpheresis units. Using 1.0 Joule/cm2 UVA, the lowest dose of S-59, AMT and 8-MOP required to reduce the number of T cells to the limit of detection was 0.05 μmol/L, 1.0 μmol/L, and 10.0 μmol/L, respectively. (2) Cytokine synthesis: Treatment with 1.9 Joules/cm2 UVA and 150 μmol/L S-59 or AMT completely inhibited synthesis of the cytokine IL-8 by contaminating leukocytes during 5 days of platelet storage. After treatment with 75 μmol/L 8-MOP and 1.9 Joules/cm2 UVA, only low levels of IL-8 were detected. (3) Psoralen-DNA adduct formation: The combination of 1.9 Joules/cm2 UVA and 150 μmol/L S-59, AMT, or 8-MOP induced 12.0 ± 3.0, 6.0 ± 0.9, and 0.7 psoralen adducts per 1,000 bp DNA, respectively. (4) Replication competence: Polymerase chain reaction (PCR) amplification of small genomic DNA sequences (242-439 bp) after PCT was inhibited. The degree of PCR amplification inhibition correlated with the level of adduct formation (S-59 > AMT > 8-MOP). In contrast, 2,500 cGy gamma radiation, a dose that inactivates >5 log10 of T cells in blood products, had minimal effect on cytokine synthesis and did not induce sufficient DNA strand breaks to inhibit PCR amplification of the same small DNA sequences. These results demonstrate that leukocytes are sensitive to PCT with psoralens and among the psoralens tested S-59 is the most effective. Therefore, PCT has the potential to reduce the incidence of leukocyte-mediated adverse immune reactions associated with platelet transfusion.

Psoralen-mediated photodecontamination of platelet concentrates: inactivation of cell-free and cell-associated forms of human immunodeficiency virus and assessment of platelet function in vivo.

Treatment of platelet concentrates (PCs) with psoralens and broad-band ultraviolet A (UVA) radiation is being examined for the elimination of pathogens that might be present in donated blood. Previous studies have demonstrated the inactivation of cell-free viruses and the maintenance of platelet integrity with common in vitro assays. Human immunodeficiency virus (HIV) in three forms-cell-free, activity replicating, and latently infected cell lines-was added to PCs and treated with 50-microgram per mL of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), 0.35 mM rutin, and broad- and narrow-band UVA light (320-400 nm and 360-370 nm [UVA1], respectively). The inactivation of added HIV was assessed in tissue culture; platelet hemostatic activity was assessed in thrombocytopenic rabbits. Each form of HIV was inactivated completely (> or = 10(5) infectious units) on treatment with 30 J per cm2 of UVA1 light. Similar results were obtained on treatment of 2.5 mL of PCs in test tubes or intact PC units (50 mL) in blood bags. Latently infected cell lines were substantially more sensitive than cell-free HIV or HIV that was actively replicating. Human platelets treated with 40 J per cm2 of UVA1 light had a fully corrected bleeding time shortly after treatment or after 5 days' storage, as assessed in thrombocytopenic rabbits. Platelet hemostatic function began to decrease with 81 J per cm2 of UVA1 light and was abolished with 113 J per cm2. At similar fluences, broad-band UVA light was more injurious to platelets than was UVA1 light. HIV transmission might be eliminated by PCs after treatment with AMT and UVA1 light and without a reduction in platelet hemostatic function.

Inactivation of Trypanosoma cruzi Trypomastigote Forms in Blood Components with a Psoralen and Ultraviolet A Light

Inactivation of the blood-borne parasite Trypanosoma cruzi by UVA and 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 micrograms/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type photo-reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form of T. cruzi, the trypomastigote, was achieved at a UVA (320-400 nm radiation) fluence of 4.2 J/cm2. We note that while the infectivity of the parasite is eliminated at 4.2 J/cm2 the trypomastigote motility continues for at least 16 h-post-treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of 3H-AMT indicated that at the lethal UVA influence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborne T. cruzi, the causative agent of Chagas disease.

The Solution Structure of a Psoralen Cross-Linked DNA Duplex by NMR and Relaxation Matrix Refinement

The three dimensional structure of the DNA oligomer d(5′-GGGTACCC-3′)2cross-linked with 4′-aminomethyl-4,5′,8-trimethylpsoralen(AMT) has been determined by two-dimensional NMR and a relaxation matrix refinement method. NMR data and structural calculation establish that the cross-linking of the psoralen in the B-DNA duplex retains Watson-Crick type hydrogen bonding throughout the duplex, although the thymine residue which is cross-linked with psoralen forms a weaker hydrogen bond. The psoralen cross-linking in DNA duplex induces significant change of the local DNA structure, but has no important effect on overall DNA helix. Our observation places an upper limit of 10° on overall DNA bending by psoralen cross-linking, which is consistent with the result of HMT-octamer cross link [Spielmann, H. P., Dwyer, T. J., Sastry, S. S., Hearst, J. E., and Wemmer, D. E., (1995)Proc. Natl. Acad. Sci. USA92,2345–2349] and gel electrophoretic study [Haran, T. E., and Crothers, D. E. (1988)Biochemistry27,6967–6971].

PSORALEN‐MEDIATED VIRUS PHOTOINACTIVATION IN PLATELET CONCENTRATES: ENHANCED SPECIFICITY OF VIRUS KILL IN THE ABSENCE OF SHORTER UVA WAVELENGTHS

Treatments with psoralens and long-wavelength ultraviolet radiation (UVA, 320-400 nm; PUVA) have shown efficacy for virus sterilization of platelet concentrates (PC). Our laboratory has employed the psoralen derivative 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), and we have found that platelet integrity is best preserved when rutin, a flavonoid that quenches multiple reactive oxygen species, is present during AMT/UVA treatment of PC. In this report, we examine the effects of different UVA spectra under our standard PC treatment conditions (i.e. 50 micrograms/mL AMT, 0.35 mM rutin and 38 J/cm2 UVA). Added vesicular stomatitis virus (VSV; > or = 5.5 log10) was completely inactivated with the simultaneous maintenance of the platelet aggregation response (> 90% of control) when a UVA light source with transmission mainly between 360 and 370 nm (narrow UVA1) was used. In contrast, with a broad-band UVA (320-400 nm; broad UVA) light source, the aggregation response was greatly compromised (< 50% of control) with only a minor increase in the rate of VSV kill. With this lamp, platelet function could be improved to about 75% of the control by adding a long-pass filter, which reduced the transmission of shorter (< or = 345 nm) UVA wavelengths (340-400 nm; UVA1). At equivalent levels of virus kill, aggregation function was always best preserved when narrow UVA1 was used for PUVA treatment. Even in the absence of AMT, and with or without rutin present, narrow UVA1 irradiation was better tolerated by platelets than was broad UVA.(ABSTRACT TRUNCATED AT 250 WORDS)

4′‐AMINOMETHYL‐4,5′,8‐TRIMETHYLPSORALEN PHOTOCHEMISTRY THE EFFECT OF CONCENTRATION AND UVA FLUENCE ON PHOTOADDUCT FORMATION IN POLY(dAdT) AND CALF THYMUS DNA

The photochemistry of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with poly(dA-dT) and calf thymus DNA was studied. The extent of photoadduct formation and the distribution of photoadducts (3,4- and 4',5'-monoadducts and crosslinks) were determined by liquid scintillation analysis and HPLC, respectively. The adducts were characterized on the basis of their UV absorption spectra and mass spectral analysis. The high DNA binding constant for AMT (1.5 x 10(5) M-1) led to a high fraction of intercalated molecules, which contributed to the high level of AMT photoadduct formation, as many as 102 adducts per kilobase pair. In addition, there is a distinct difference in the adduct distribution compared to the previously studied 8-methoxypsoralen (8-MOP). Under the conditions employed for the photochemical studies, virtually all of the AMT molecules in solution are intercalated, occupying 25% of the base pair sites. Under similar conditions, 8-MOP molecules occupied 10 times fewer sites. Thus, for AMT, DNA base pair sites other than 5'TA, the well-characterized strong binding for psoralens in general, are an additional target for photomodification, which results in the formation of a higher percentage of monoadducts. The proportion of photoadducts formed was virtually independent of AMT concentration and UVA (320-400 nm radiation) fluence.

Determination of residual 4'-aminomethyl-4,5',8-trimethylpsoralen and mutagenicity testing following psoralen plus UVA treatment of platelet suspensions.

Psoralens and UVA light have been used in the laboratory to study the inactivation of viruses that may be infrequently present in platelet concentrates that are prepared for transfusion. In order to evaluate safety aspects of the treatment of platelet suspensions with 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), we have investigated the residual levels and mutagenic potential of AMT after UVA phototreatment. 4'-aminomethyl-4,5',8-trimethylpsoralen, at a final concentration of 40 micrograms/mL, was added to platelet suspensions which contained 16% plasma and a synthetic medium. Platelet suspensions containing AMT were irradiated with up to 7.2 J/cm2 UVA light under normal oxygen levels. Residual levels of AMT were determined by HPLC and a bioassay based on bacteriophage phi 6 inactivation. The photodestruction of AMT or its activity by UVA was characterized by a D37 value of 0.6 and 0.3 J/cm2 with HPLC or bioassay, respectively. At 2.4 J/cm2 UVA, which results in approximately 5 log10 inactivation of vesicular stomatitis virus (VSV) and retention of platelet in vitro properties, 12% (HPLC) to 9% (bioassay) AMT remained. Like other psoralens, AMT was found to bind to serum proteins as shown by ultrafiltration. Results are consistent with approximately 36% of the initial drug load binding primarily to serum albumin. It was determined using 3H-AMT that 9 to 18% of radioactivity was bound to platelets in the absence of irradiation. Similar fractions (13 to 18%) of AMT were bound to platelets after 3.6 J/cm2 UVA irradiation, and 8 to 10% of total AMT was associated with saline-washed irradiated platelets and is presumably tightly bound.(ABSTRACT TRUNCATED AT 250 WORDS)

RELAXATION OF SUPERCOILED DNA BY AMINOMETHYL TRIMETHYLPSORALEN AND UV PHOTONS: ACTION SPECTRUM

An action spectrum for the relaxation of supercoiled plasmid DNA (induction of the first single-strand break) by photoactivated 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) has been determined using monochromatic UV photons from 254 to 405 nm. The spectrum of AMT-induced plasmid DNA relaxation fits closely with the absorbance spectrum of AMT in the spectral region between 313 nm and 405 nm but deviates at wavelengths shorter than 313 nm. This assay also reveals that the psoralen photosensitization reaction with DNA also produces piperidine-labile sites. Addition of mannitol and azide partially quenches the supercoil relaxation reaction, evidence for a role of Type II photosensitization pathway.

Virus sterilization in platelet concentrates with psoralen and ultraviolet A light in the presence of quenchers.

The virucidal and functional effect of the treatment of platelet concentrates (PCs) with long-wave ultraviolet light (UVA) and the psoralen derivative 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) was studied. Cell-free vesicular stomatitis virus (VSV) was completely inactivated (greater than or equal to 6.5 log10) on treatment of PCs with 25 micrograms per mL (85 microM) of AMT and with 20.7 J per cm2 (30 min) of UVA in the presence of air, or with 82.8 J per cm2 (2 hours) of UVA under conditions of reduced oxygen tension. When treatment was in air, the extent and rate of platelet aggregation in response to collagen measured after overnight storage were reduced to about 70 and 50 percent of control values, respectively; however, aggregation responses were similar to those of controls when PCs were treated under reduced oxygen tension. As a means of eliminating the necessity of oxygen depletion during AMT and UVA treatment, we examined the effects of the addition of quenchers of reactive oxygen species. The presence of 2 mM (2 mmol/L) mannitol during treatment of PCs with 25 micrograms per mL of AMT and 20.7 J per cm2 of UVA in air significantly improved the aggregation response and other in vitro indicators of platelet function and had little or no effect on VSV inactivation. Less benefit was observed with the other quenchers examined. Thus, the nucleic acid specificity of psoralen photoinactivation under reduced oxygen conditions may also be attainable when selected free radical scavengers such as mannitol are present during treatment in air.

Dark and photoreactivity of 4′-aminomethyl-4,5′,8-trimethylpsoralen with T7 phage

The dark and photoreactions of 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) with T7 phage were investigated from biological and structural points of view. The dark reaction leads to the structural destabilization of the double helix of the DNA as is shown by optical melting measurements. The genotoxicity of AMT in the dark is comparable with that of known genotoxic drugs as determined by phage inactivation. The photoreaction with UVA light leads to the formation of mono- and di-adducts depending on the wavelength and dose used. Mono- and di-adducts influence DNA stability differently; biologically both types of adducts are genotoxic as measured by action spectra.

Characterization of new furocoumarin derivatives by their dark and light-mediated action on RNA bacteriophage MS2

The monofunctional and bifunctional furocoumarin derivatives 8-methyl-3-carbethoxypsoralen (8Me3CPs) and 8-methoxypsoralen (8MOP) as well as their thiosubstituted derivatives (2-thio-8Me3CPs and 2-thio-8MOP) were compared in terms of their reactivities towards a ribonucleoprotein, the bacteriophage MS2. The order of their photoreactivities, differed from that measured with nuclear DNA and mitochondrial DNA. Besides their widely investigated photoreactivity, their biological activity in the dark and after pre-irradiation was quantified. A parameter was defined which compares the number of molecules acting in the dark for 1 h with the number of absorbed photons which lead to the same degree of inactivation. The parameter for the furocoumarin derivatives examined, including 3-carbethoxypsoralen (3CPs) and 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT), was in the following order: AMT > 3CPs > 8MOP > 8Me3CPs > 8Me3CPsS ≈ 8MOPS. A similar parameter was also determined for the dark effect of pre-irradiated compounds.

Characterization of the effects on ovalbumin mRNA of aminomethyl-trimethylpsoralen photoreaction with hen oviduct mRNA.

We have described the reaction of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with hen oviduct mRNA and have investigated the specific effects of AMT photoaddition on ovalbumin mRNA which constitutes 60-70% of oviduct mRNA. The photoreaction of AMT with hen oviduct mRNA appeared to occur in two phases - a rapid monoaddition followed by a slower conversion of monoadducts to diadducts (i.e. crosslinking). Both nondenaturing and denaturing gel electrophoresis revealed a photoreaction time dependent increase in ovalbumin mRNA electrophoretic mobility indicating the formation of a progressively more compact molecular structure. Identical analysis of photoreacted rabbit globin mRNA revealed no change in electrophoretic mobility suggesting that AMT was stabilizing the AU rich secondary structure of ovalbumin mRNA but having no similar effect on the relatively GC rich secondary structure of globin mRNA. Ovalbumin-specific DNA primer extension was used to demonstrate the selective and secondary structure specific photoaddition of AMT to uracil bases at the 5' end of ovalbumin mRNA.

Cross-linking of tobacco mosaic virus RNA and capped polyribonucleotides to 18S rRNA in wheat germ ribosome-mRNA complexes.

Tobacco mosaic virus RNA, forming 40S or 80S initiation complexes with wheat germ ribosomes, was covalently bound to 18S ribosomal RNA by the photoreaction with an RNA cross-linking agent, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). Synthetic polyribonucleotide, poly(A, U), with the cap structure m7GpppGmC at the 5'-terminal was also cross-linked to 18S ribosomal RNA in 40S or 80S complexes with ribosomes by the AMT photoreaction. Polyuridylic acid with the same 5'-cap structure, forming 40S complexes but not 80S complexes with ribosomes, was most efficiently cross-linked to 18S ribosomal RNA by the psoralen photoreaction. These results suggest that the interactions between mRNA and 18S rRNA are not necessarily of strict complementarity but occur during formation of the complexes in eukaryotes. The 40S complexes would be then converted to 80S complexes in the presence of the AUG initiation codon or AUG-like triplets containing A and U on the polyribonucleotide chains which interact with 18S ribosomal RNA.