TMPyP binding evokes a complex, tunable nanomechanical response in DNA.

TMPyP is a porphyrin capable of DNA binding and used in photodynamic therapy and G-quadruplex stabilization. Despite its broad applications, TMPyP's effect on DNA nanomechanics is unknown. Here we investigated, by manipulating λ-phage DNA with optical tweezers combined with microfluidics in equilibrium and perturbation kinetic experiments, how TMPyP influences DNA nanomechanics across wide ranges of TMPyP concentration (5-5120 nM), mechanical force (0-100 pN), NaCl concentration (0.01-1 M) and pulling rate (0.2-20 µm/s). Complex responses were recorded, for the analysis of which we introduced a simple mathematical model. TMPyP binding, which is a highly dynamic process, leads to dsDNA lengthening and softening. dsDNA stability increased at low (<10 nM) TMPyP concentrations, then decreased progressively upon increasing TMPyP concentration. Overstretch cooperativity decreased, due most likely to mechanical roadblocks of ssDNA-bound TMPyP. TMPyP binding increased ssDNA's contour length. The addition of NaCl at high (1 M) concentration competed with the TMPyP-evoked nanomechanical changes. Because the largest amplitude of the changes is induced by the pharmacologically relevant TMPyP concentration range, this porphyrin derivative may be used to tune DNA's structure and properties, hence control the wide array of biomolecular DNA-dependent processes including replication, transcription, condensation and repair.

Imaging the Infection Cycle of T7 at the Single Virion Level.

T7 phages are E. coli-infecting viruses that find and invade their target with high specificity and efficiency. The exact molecular mechanisms of the T7 infection cycle are yet unclear. As the infection involves mechanical events, single-particle methods are to be employed to alleviate the problems of ensemble averaging. Here we used TIRF microscopy to uncover the spatial dynamics of the target recognition and binding by individual T7 phage particles. In the initial phase, T7 virions bound reversibly to the bacterial membrane via two-dimensional diffusive exploration. Stable bacteriophage anchoring was achieved by tail-fiber complex to receptor binding which could be observed in detail by atomic force microscopy (AFM) under aqueous buffer conditions. The six anchored fibers of a given T7 phage-displayed isotropic spatial orientation. The viral infection led to the onset of an irreversible structural program in the host which occurred in three distinct steps. First, bacterial cell surface roughness, as monitored by AFM, increased progressively. Second, membrane blebs formed on the minute time scale (average ~5 min) as observed by phase-contrast microscopy. Finally, the host cell was lysed in a violent and explosive process that was followed by the quick release and dispersion of the phage progeny. DNA ejection from T7 could be evoked in vitro by photothermal excitation, which revealed that genome release is mechanically controlled to prevent premature delivery of host-lysis genes. The single-particle approach employed here thus provided an unprecedented insight into the details of the complete viral cycle.

Comparison of the Efficacy of Two Novel Antitubercular Agents in Free and Liposome-Encapsulated Formulations.

Tuberculosis is one of the top ten causes of death worldwide, and due to the appearance of drug-resistant strains, the development of new antituberculotic agents is a pressing challenge. Employing an in silico docking method, two coumaran (2,3-dihydrobenzofuran) derivatives-TB501 and TB515-were determined, with promising in vitro antimycobacterial activity. To enhance their effectiveness and reduce their cytotoxicity, we used liposomal drug carrier systems. Two types of small unilamellar vesicles (SUV) were prepared: multicomponent pH-sensitive stealth liposome (SUVmixed) and monocomponent conventional liposome. The long-term stability of our vesicles was obtained by the examination of particle size distribution with dynamic light scattering. Encapsulation efficiency (EE) of the two drugs was determined from absorption spectra before and after size exclusion chromatography. Cellular uptake and cytotoxicity were determined on human MonoMac-6 cells by flow cytometry. The antitubercular effect was characterized by the enumeration of colony-forming units on Mycobacterium tuberculosis H37Rv infected MonoMac-6 cultures. We found that SUVmixed + TB515 has the best long-term stability. TB515 has much higher EE in both types of SUVs. Cellular uptake for native TB501 is extremely low, but if it is encapsulated in SUVmixed it appreciably increases; in the case of TB515, quasi total uptake is accessible. It is concluded that SUVmixed + TB501 seems to be the most efficacious antitubercular formulation given the presented experiments; to find the most promising antituberculotic formulation for therapy further in vivo investigations are needed.

Temperature-Dependent Nanomechanics and Topography of Bacteriophage T7.

Viruses are nanoscale infectious agents which may be inactivated by heat treatment. The global molecular mechanisms of virus inactivation and the thermally induced structural changes in viruses are not fully understood. In this study, we measured the heat-induced changes in the properties of T7 bacteriophage particles exposed to a two-stage (65°C and 80°C) thermal effect, by using atomic force microscopy (AFM)-based nanomechanical and topographical measurements. We found that exposure to 65°C led to the release of genomic DNA and to the loss of the capsid tail; hence, the T7 particles became destabilized. Further heating to 80°C surprisingly led to an increase in mechanical stability, due likely to partial denaturation of the capsomeric proteins kept within the global capsid arrangement.IMPORTANCE Even though the loss of DNA, caused by heat treatment, destabilizes the T7 phage, its capsid is remarkably able to withstand high temperatures with a more or less intact global topographical structure. Thus, partial denaturation within the global structural constraints of the viral capsid may have a stabilizing effect. Understanding the structural design of viruses may help in constructing artificial nanocapsules for the packaging and delivery of materials under harsh environmental conditions.

Suitability of GnRH Receptors for Targeted Photodynamic Therapy in Head and Neck Cancers.

Head and neck squamous cell carcinomas (HNSCC) have a high mortality rate, although several potential therapeutic targets have already been identified. Gonadotropin-releasing hormone receptor (GnRH-R) expression is less studied in head and neck cancers, hence, we investigated the therapeutic relevance of GnRH-R targeting in HNSCC patients. Our results indicate that half of the patient-derived samples showed high GnRH-R expression, which was associated with worse prognosis, making this receptor a promising target for GnRH-based drug delivery. Photodynamic therapy is a clinically approved treatment for HNSCC, and the efficacy and selectivity may be enhanced by the covalent conjugation of the photosensitizer to a GnRH-R targeting peptide. Several native ligands, gonadotropin-releasing hormone (GnRH) isoforms, are known to target GnRH-R effectively. Therefore, different 4Lys(Bu) modified GnRH analogs were designed and conjugated to protoporphyrin IX. The receptor binding potency of the novel conjugates was measured on human pituitary and human prostate cancer cells, indicating only slightly lower GnRH-R affinity than the peptides. The in vitro cell viability inhibition was tested on Detroit-562 human pharyngeal carcinoma cells that express GnRH-R in high levels, and the results showed that all conjugates were more effective than the free protoporphyrin IX.

Photochemical and Structural Studies on Cyclic Peptide Models.

Ultra-violet (UV) irradiation has a significant impact on the structure and function of proteins that is supposed to be in relationship with the tryptophan-mediated photolysis of disulfide bonds. To investigate the correlation between the photoexcitation of Trp residues in polypeptides and the associated reduction of disulfide bridges, a series of small, cyclic oligopeptide models were analyzed in this work. Average distances between the aromatic side chains and the disulfide bridge were determined following molecular mechanics (MM) geometry optimizations. In this way, the possibility of cation⁻π interactions was also investigated. Molecular mechanics calculations revealed that the shortest distance between the side chain of the Trp residues and the disulfide bridge is approximately 5 Å in the cyclic pentapeptide models. Based on this, three tryptophan-containing cyclopeptide models were synthesized and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Experimental data and detailed molecular dynamics (MD) simulations were in good agreement with MM geometry calculations. Selected model peptides were subjected to photolytic degradation to study the correlation of structural features and the photolytic cleavage of disulfide bonds in solution. Formation of free sulfhydryl groups upon illumination with near UV light was monitored by fluorescence spectroscopy after chemical derivatization with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) and mass spectrometry. Liquid cromatography-mass spectrometry (LC-MS) measurements indicated the presence of multiple photooxidation products (e.g., dimers, multimers and other oxidated products), suggesting that besides the photolysis of disulfide bonds secondary photolytic processes take place.

Oligo- and polypeptide conjugates of cationic porphyrins: binding, cellular uptake, and cellular localization.

Recently, we have characterized the DNA and nucleoprotein (NP) binding of bis(4-N-methylpyridyl)-15,20-di(4-carboxyphenyl)porphyrin (BMPCP) and meso-tri(4-N-methylpyridyl)-mono(4-carboxyphenyl)porphyrin (TMPCP) and their tetrapeptide conjugates (BMPCP-4P2 and TMPCP-4P, respectively). In this work, we investigated the interaction of TMPCP conjugated to the tetrapeptide branches of branched chain polymeric polypeptide with poly-L-lysine backbone (AK) with DNA or NP using spectroscopic methods. Analysis of absorption spectra revealed the external binding but no intercalation of TMPCP-AK to DNA. There was no evidence for the interaction between TMPCP-AK and encapsidated DNA. Furthermore, we examined the cellular uptake of BMPCP and TMPCP and their tetra- or polypeptide conjugates by flow cytometry and analyzed how charge, size, and structure of the compounds affect their incorporation. In comparison, liposomal association constants of these derivatives were determined. BMPCP-4P2 accumulated the most, and porphyrins with two positive charges (BMPCP and BMPCP-4P2) showed better accumulation than the tri-cationic TMPCP or TMPCP-4P. Cellular uptake of polycationic TMPCP-AK was significantly lower than that of the free or tetrapeptide conjugated derivatives. The subcellular localization of all the five compounds was investigated in co-localization studies by confocal microscopy with special attention to their nuclear localization. Neither free nor conjugated BMPCP or TMPCP was co-localized with nuclear marker. Instead, these derivatives showed co-localization with lysosomal and mitochondrial fluorescent probes. TMPCP-AK conjugate had different localization patterns appearing mainly in mitochondria and cytoplasmic vesicles. Our results may contribute to the further design of DNA-targeting porphyrin-based constructs.

A new daunomycin-peptide conjugate: synthesis, characterization and the effect on the protein expression profile of HL-60 cells in vitro.

Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin ß chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.

In vitro degradation and antitumor activity of oxime bond-linked daunorubicin-GnRH-III bioconjugates and DNA-binding properties of daunorubicin-amino acid metabolites.

Bioconjugates with receptor-mediated tumor-targeting functions and carrying cytotoxic agents should enable the specific delivery of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting the peripheral toxicity. In the present study, gonadotropin-releasing hormone III (GnRH-III; Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was employed as a targeting moiety to which daunorubicin was attached via oxime bond, either directly or by insertion of a GFLG or YRRL tetrapeptide spacer. The in vitro antitumor activity of the bioconjugates was determined on MCF-7 human breast and HT-29 human colon cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their degradation/stability (1) in human serum, (2) in the presence of cathepsin B and (3) in rat liver lysosomal homogenate was analyzed by liquid chromatography in combination with mass spectrometry. The results show that (1) all synthesized bioconjugates have in vitro antitumor effect, (2) they are stable in human serum at least for 24 h, except for the compound containing an YRRL spacer and (3) they are hydrolyzed by cathepsin B and in the lysosomal homogenate. To investigate the relationship between the in vitro antitumor activity and the structure of the bioconjugates, the smallest metabolites produced in the lysosomal homogenate were synthesized and their binding to DNA was assessed by fluorescence spectroscopy. Our data indicate that the incorporation of a peptide spacer in the structure of oxime bond-linked daunorubicin-GnRH-III bioconjugates is not required for their antitumor activity. Moreover, the antitumor activity is influenced by the structure of the metabolites (daunorubicin-amino acid derivatives) and their DNA-binding properties.

Single-particle virology.

The development of advanced experimental methodologies, such as optical tweezers, scanning-probe and super-resolved optical microscopies, has led to the evolution of single-molecule biophysics, a field of science that allows direct access to the mechanistic detail of biomolecular structure and function. The extension of single-molecule methods to the investigation of particles such as viruses permits unprecedented insights into the behavior of supramolecular assemblies. Here we address the scope of viral exploration at the level of individual particles. In an era of increased awareness towards virology, single-particle approaches are expected to facilitate the in-depth understanding, and hence combating, of viral diseases.

Single-Molecule Mechanics in Ligand Concentration Gradient.

Single-molecule experiments provide unique insights into the mechanisms of biomolecular phenomena. However, because varying the concentration of a solute usually requires the exchange of the entire solution around the molecule, ligand-concentration-dependent measurements on the same molecule pose a challenge. In the present work we exploited the fact that a diffusion-dependent concentration gradient arises in a laminar-flow microfluidic device, which may be utilized for controlling the concentration of the ligand that the mechanically manipulated single molecule is exposed to. We tested this experimental approach by exposing a λ-phage dsDNA molecule, held with a double-trap optical tweezers instrument, to diffusionally-controlled concentrations of SYTOX Orange (SxO) and tetrakis(4-N-methyl)pyridyl-porphyrin (TMPYP). We demonstrate that the experimental design allows access to transient-kinetic, equilibrium and ligand-concentration-dependent mechanical experiments on the very same single molecule.

Location of mesoporphyrin in liposomes determined by site-selective fluorescence spectroscopy.

Binding of photosensitizers to target cells is a crucial step during the photodynamic effect. Sensitizer distribution is a good indication of whether the chemical is a good candidate for perturbing cell membrane integrity. Hence, the photophysical properties of porphyrinoid sensitizers in microheterogeneous systems such as liposomes are of outstanding interest. Here we present a site-selective fluorescence study of liposome systems. Monocomponent, small unilamellar vesicles formed of different phosphatidylcholines with incorporated mesoporphyrin were investigated. The size distribution of liposomes was measured by dynamic light scattering after each step of the experiment. On the basis of fluorescence line narrowing spectra of mesoporphyrin, the inhomogeneous distribution function was determined in order to characterize the photosensitizer location. The dual character of the functions revealed two different locations. Decomposition of the inhomogeneous distribution functions into Gaussians and the analysis of the fit results suggest that one of the locations for mesoporphyrin is between the two lipid layers, and the other one is between the hydrocarbon chains of the lipid molecules.

Comparison of light-induced formation of reactive oxygen species and the membrane destruction of two mesoporphyrin derivatives in liposomes.

The photodynamic effect requires the simultaneous presence of light, photosensitizer (PS) and molecular oxygen. In this process, the photoinduced damage of cells is caused by reactive oxygen species (ROS). Besides DNA, the other target of ROS is the membranes, separating internal compartments in living cells. Hence, the ability of ROS formation of porphyrins as PSs, in liposomes as simple models of cellular membranes is of outstanding interest. Earlier we compared the binding parameters and locations of mesoporphyrin IX dihydrochloride (MPCl) and mesoporphyrin IX dimethyl ester (MPE), in small unilamellar vesicles (SUV) made from various saturated phosphatidylcholines. In this study, we used the same kinds of samples for comparing the ROS forming ability. Triiodide production from potassium iodide because of light-induced ROS in the presence of molybdate catalyst was applied, and the amount of product was quantitatively followed by optical spectrometry. Furthermore, we demonstrated and carefully studied SUVs disruption as direct evidence of membrane destruction by the methods of dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS), applying unsaturated phosphatidylcholines as membrane components. Although the ROS forming ability is more pronounced in the case of MPCl, we found that the measured disruption was more effective in the samples containing MPE.

Comparison of the efficiency and the specificity of DNA-bound and free cationic porphyrin in photodynamic virus inactivation.

The risk of transmitting infections by blood transfusion has been substantially reduced. However, alternative methods for inactivation of pathogens in blood and its components are needed. Application of photoactivated cationic porphyrins can offer an approach to remove non-enveloped viruses from aqueous media. Here we tested the virus inactivation capability of meso-Tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) and meso-Tri-(4-N-methylpyridyl)monophenylporphyrin (TMPyMPP) in the dark and upon irradiation. T7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. TMPyP and TMPyMPP reduce the viability of T7 phage already in the dark, which can be explained by their selective binding to nucleic acid. Both compounds proved to be efficient photosensitizers of virus inactivation. The binding of porphyrin to phage DNA was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by DNA bound porphyrin. As optical melting studies and agarose gel electrophoresis of T7 nucleoprotein revealed, photoreactions of TMPyP and TMPyMPP affect the structural integrity of DNA and also of viral proteins, despite their selective DNA binding.

Forced phage uncorking: viral DNA ejection triggered by a mechanically sensitive switch.

The foremost event of bacteriophage infection is the ejection of genomic material into the host bacterium after virus binding to surface receptor sites. How ejection is triggered is yet unknown. Here we show, in single mature T7 phage particles, that tapping the capsid wall with an oscillating atomic-force-microscope cantilever triggers rapid DNA ejection via the tail complex. The triggering rate increases exponentially as a function of force, following transition-state theory, across an activation barrier of 23 kcal mol-1 at 1.2 nm along the reaction coordinate. The conformation of the ejected DNA molecule revealed that it had been exposed to a propulsive force. This force, arising from intra-capsid pressure, assists in initiating the ejection process and the transfer of DNA across spatial dimensions beyond that of the virion. Chemical immobilization of the tail fibers also resulted in enhanced DNA ejection, suggesting that the triggering process might involve a conformational switch that can be mechanically activated either by external forces or via the tail-fiber complex.

The effect of the structure of branched polypeptide carrier on intracellular delivery of daunomycin.

The conjugate of acid labile cis-aconityl-daunomycin (cAD) with branched chain polypeptide, poly[Lys(Glui-DL-Alam)] (EAK) was very effective against L1210 leukemia in mice. However, Dau attached to a polycationic polypeptide, poly[Lys(Seri-DL-Alam)] (SAK) exhibited no in vivo antitumor effect. In order to understand this difference we have performed comparative in vitro studies to dissect properties related to interaction with the whole body (e.g., biodistribution) from those present at cellular or even molecular level. We report here (a) the kinetics of acid-induced Dau liberation, (b) interaction with DPPC phospholipid bilayer, (c) in vitro cytotoxic effect on different tumor cells, and (d) intracellular distribution in HL-60 cells of polycationic (cAD-SAK) and amphoteic (cAD-EAK) conjugates. Fluorescence properties of the two conjugates are also reported. Our findings demonstrate that the kinetics of the drug release, intracellular distribution and in vitro cytotoxic effect are rather similar, while the effect on DPPC phospholipid bilayer and fluorescence properties of the two conjugates are not the same. We also found that the in vitro cytotoxicity is cell line dependent. These observations suggest that the structure of the polypeptide carrier could have marked influence on drug uptake related events.

Interaction of photosensitizers with liposomes containing unsaturated lipid.

Small unilamellar liposomes were made of dipalmitoyl-phosphatidylcholine and dioleoyl-phosphatidylcholine, and photosensitized by a symmetrically or an asymmetrically substituted glycosilated tetraphenyl-porphyrin derivative. As differential scanning calorimetry and electron paramagnetic resonance spectroscopy (EPR) revealed these porphyrin derivatives were localized in different depth within the lipid bilayer. Both porphyrin derivatives were able to induce photoreaction and consequent structural changes in the membrane. 5-, 12-, or 16-doxyl stearic acid labeled lipid bilayers were applied and the efficiency of photoinduced reaction was followed by the decay of their EPR signal amplitude. Light dose-dependent destruction of nitroxide radical proved to be dependent on the position of spin label. In this process the porphyrin localized in closer connection with the double bond of unsaturated fatty acid was more effective. EPR signal decay was also dependent on the unsaturated fatty acid content of the liposome and the oxygen saturation of the solvent.

Stepwise reversible nanomechanical buckling in a viral capsid.

Viruses are nanoscale infectious agents constructed of a proteinaceous capsid that protects the packaged genomic material. Nanoindentation experiments using atomic force microscopy have, in recent years, provided unprecedented insight into the elastic properties, structural stability and maturation-dependent mechanical changes in viruses. However, the dynamics of capsid behavior are still unresolved. Here we used high-resolution nanoindentation experiments on mature, DNA-filled T7 bacteriophage particles. The elastic regime of the nanoindentation force trace contained discrete, stepwise transitions that cause buckling of the T7 capsid with magnitudes that are integer multiples of ∼0.6 nm. Remarkably, the transitions are reversible and contribute to the rapid consolidation of the capsid structure against a force during cantilever retraction. The stepwise transitions were present even following the removal of the genomic DNA by heat treatment, indicating that they are related to the structure and dynamics of the capsomeric proteins. Dynamic force spectroscopy experiments revealed that the thermally activated consolidation step is ∼104 times faster than spontaneous buckling, suggesting that the capsid stability is under strong dynamic control. Capsid structural dynamics may play an important role in protecting the genomic material from harsh environmental impacts. The nanomechanics approach employed here may be used to investigate the structural dynamics of other viruses and nanoscale containers as well.

Interaction of tetraphenyl-porphyrin derivatives with DPPC-liposomes: an EPR study.

The effect of the symmetry and polarity of the porphyrin molecules on their membrane localization and interaction with membrane lipids were investigated by electron paramagnetic resonance (EPR). For this purpose, two glycoconjugated tetraphenyl porphyrin derivatives were selected, respectively, symmetrically and asymmetrically substituted. Small unilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and spin labeled stearic acids were prepared. The spin probe was located at the 5th or 7th or 12th or 16th position of the hydrocarbon chain in order to monitor various regions of the lipid bilayer. EPR spectra of porphyrin-free and porphyrin-bound liposomes were recorded at various temperatures below and above the phase transition temperature of DPPC. The effect on membrane fluidity proved to be stronger with the asymmetrical porphyrin derivative than with the symmetrical one. The rigidity increased when the spin label was near lipid head groups. The difference observed between control and porphyrin-treated samples when measured below the main lipid transition temperature disappeared at higher temperature. When the spin label was near the end of the hydrophobic tails, the symmetrical porphyrin derivative caused increase in fluidity, while the asymmetrical one slightly decreased it. To explain this phenomenon we propose that the asymmetrical derivative exerts a stronger ordering effect caused by its fluorophenyl group located at the level of the lipid heads, which is attenuated to the hydrophobic tails. The perturbing effect of the symmetric derivative could not lead to similar extent of ordering at the head groups and looses the hydrocarbon chains deeper in the membrane.

[Photodynamic inactivation of porphyrin sensitized T7 phages: efficiency and mechanism of action]. / Porfirin származékok alkalmazása T7 fág fotodinamikus inaktivációjában: a reakció hatékonysága és mechanizmusa.

We investigated the efficiency and the mechanism of action of two glycoconjugated tetraphenyl porphyrins in their photoreaction with T7 bacteriophage. Both types of porphyrins sensitized the photoinactivation of T7, but the slopes of inactivation kinetics were markedly different. Our result suggests that both type I and type II reaction play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photo-chemically treated nucleoprotein complex. Polymerase chain reaction (PCR) analysis failed to demonstrate any DNA damage.