Stephen T. Isaacs

Photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light

BACKGROUND: A photochemical treatment process has been developed for the inactivation of viruses and bacteria in platelet concentrates. This process is based on the photochemical reaction of a novel psoralen, S‐ 59, with nucleic acids upon illumination with long‐wavelength ultraviolet light (UVA, 320–400 nm). STUDY DESIGN AND METHODS: High levels of pathogens were added to single‐donor platelet concentrates containing 3 to 5 × 10(11) platelets in 300 mL of 35‐percent autologous plasma and 65‐percent platelet additive solution. After treatment with S‐59 (150 microM) and UVA (0–3 J/cm2), the infectivity of each pathogen was measured with established biologic assays. In vitro platelet function after photochemical treatment was evaluated during 7 days of storage by using a panel of 14 assays. The in vivo recovery and life span of photochemically treated platelets were evaluated after 24 hours of storage in a primate transfusion model. RESULTS: The following levels of pathogen inactivation were achieved:>10(6.7) plaque‐forming units (PFU) per mL of cell‐free human immunodeficiency virus (HIV),>10(6.6) PFU per mL of cell‐associated HIV,>10(6.8) infectious dose (ID50) per mL of duck hepatitis B virus (a model for hepatitis B virus),>10(6.5) PFU per mL of bovine viral diarrhea virus (a model for hepatitis C virus),>10(6.6) colony‐forming units of Staphylococcus epidermidis, and>10(5.6) colony‐forming units of Klebsiella pneumoniae. Expression of integrated HIV was inhibited by 0.1 microM S‐ 59 and 1 J per cm2 of UVA. In vitro and in vivo platelet function were adequately maintained after antiviral and antibacterial treatment. CONCLUSION: Photochemical treatment of platelet concentrates offers the potential for reducing transfusion‐related viral and bacterial diseases.

The reaction of the psoralens with deoxyribonucleic acid

Psoralen photochemistry is specific for nucleic acids and is better understood at the molecular level than are all other methods of chemical modification of nucleic acids. These compounds are used both for in vivo structure analysis and for photochemotherapy since they easily penetrate both cells and virus particles. Apparently, natural selection has selected for membrane and virus penetrability during the evolution of these natural products. Most cells are unaffected by relatively high concentrations of psoralens in the absence of ultraviolet light, and the metabolites of the psoralens have thus far not created a problem. Finally, psoralens form both monoadduct and cross-links in nucleic acid helices, the yield of each being easily controlled by the conditions used during the photochemistry.

Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction.

We describe a photochemical procedure for the sterilization of polynucleotides that are created by the Polymerase Chain Reaction (PCR). The procedure is based upon the blockage of Taq DNA polymerase when it encounters a photochemically modified base in a polynucleotide strand. We have discovered reagents that can be added to a PCR reaction mixture prior to amplification and tolerate the thermal cycles of PCR, are photoactivated after amplification, and damage a PCR strand in a manner that, should the damaged strand be carried over into a new reaction vessel, prevent it from functioning as a template for the PCR. These reagents, which are isopsoralen derivatives that form cyclobutane adducts with pyrimidine bases, are shown to stop Taq polymerase under conditions appropriate for the PCR process. We show that effective sterilization of PCR products requires the use of these reagents at concentrations that are tailored to the length and sequence of the PCR product and the level of amplification of the PCR protocol.

Post-PCR sterilization: development and application to an HIV-1 diagnostic assay

We have developed an effective post-PCR sterilization process and have applied the procedure to a diagnostic assay for HIV-1. The method, which is based on isopsoralen photochemistry, satisfies both the inactivation and hybridization requirements of a practical sterilization process. The key feature of the technique is the use of isopsoralen compounds which form covalent photochemical adducts with DNA. These covalent adducts prevent subsequent extension of previously amplified sequences (amplicons) by Taq polymerase. Isopsoralens have minimal inhibitory effect on the PCR, are activated by long wavelength ultraviolet light, provide sufficient numbers of covalent adducts to impart effective sterilization, modify the amplified sequence such that it remains single-stranded, and have little effect on subsequent hybridization. The sterilization procedure can be applied to a closed system and is suitable for use with commonly used detection formats. The photochemical sterilization protocol we have devised is an effective and pragmatic method for eliminating the amplicon carryover problem associated with the PCR. While the work described here is limited to HIV-1, proper use of the technique will relieve the concern associated with carryover for a wide variety of amplicons, especially in the clinical setting.

A photochemical characterisation of reactions of psoralen derivatives with DNA.

Psoralens are nucleic acid photoreagents which form mono-and diadducts to pyrimidine bases upon irradiation with long wavelength ultraviolet light (Musajo et al., 1967). Diaddition results in the formation of interstrand bridges or crosslinks in nucleic acid dou ble helices (Cole, 1970, 1971). Crosslink formation is a multistep process involving initial dark binding or intercalation of the psoralen, photoaddition of the psoralen to a pyrimidine base forming a monoadduct followed by conversion of the monoadduct to crosslink by a second photoaddition to a second pyrimidine base. Each of these steps may be investigated as a function of the type and position of the substituents of a given psoralen compound.

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.