Role of structure-proteins in the porphyrin–DNA interaction

We studied the complexation of meso-tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) with HeLa nucleosomes and compared it to our earlier results on T7 phage nucleoprotein complex (NP) and isolated DNA. To identify binding modes and relative concentrations of the bound TMPyP forms, the porphyrin absorption spectra were analyzed at various base pair/porphyrin ratios. Spectral decomposition and circular dichroism measurements proved that the two main binding modes of TMPyP, i.e., external binding and intercalation occur also in the nucleosomes. The DNA superstructure maintained by the proteins decreases its accessibility for TMPyP similarly in both nucleoproteins. A difference is observed between the partitioning of the two binding modes: in the case of nucleosome the ratio of intercalation to groove-binding is changed from 60/40 to 40/60 as determined for T7 NP and for isolated DNA-s. Using UV and CD melting studies, we revealed that TMPyP destabilizes the DNA–protein interaction in the nucleosomes but not in the T7 phage. Lastly, photoinduced reaction of bound TMPyP caused alterations in DNA structures and DNA–protein interactions within both nucleoprotein complexes; the nucleosomes were found to be more sensitive to the photoreaction.     

A COMPARISON OF THREE PHOTOSENSITIZERS WITH RESPECT TO EFFICIENCY OF CELL IN ACTIVATION, FLUORESCENCE QUANTUM YIELD AND DNA STRAND BREAKS

Intracellular properties of three photosensitizers relevant to photodynamic cancer therapy were compared using cultured human NHIK 3025 cells. When taken up in the cells, the hydrophilic photosensitizer aluminum phthalocyanine tetra sulfonate required about 10 times more quanta of light absorbed per cell to kill 90% of the cells than did the hydrophobic dyes Photofrin II and tetra(3-hydroxyphenyl)porphyrin. In spite of this, the phthalocyanine molecule was the most efficient dye per quantum of excitation light, since the extinction coefficient of the phthalocyanine is more than 10 times higher than that of the two hydrophobic photosensitizers at therapeutic wavelengths. The two hydrophobic dyes had significantly higher fluorescence quantum yields when taken up by cells than when bound to human plasma or human serum albumin, whereas the opposite was true for the hydrophilic phthalocyanine dye--suggesting intracellular aggregation. Finally, the potential genetic toxicities of the drugs in the form of DNA strand breaks were compared. The aluminum phthalocyanine tetra sulfonate photosensitized more DNA strand breaks than did Photofrin II and tetra(3-hydroxyphenyl)porphyrin when compared at the same level of cell survival.     

PRODUCTION OF BREAKS IN SINGLE- AND DOUBLE-STRANDED FORMS OF BACTERIOPHAGE øx174 DNA BY PROFLAVINE AND LIGHT TREATMENT

Abstract— Irradiation at 440 + 360 nm and a fluence rate of 3.8 kJm^-2 min^-1, of both complexes previously formed between proflavine and either øX circular single-stranded (ss) DNA or øX supercoiled duplex (RFI)DNA, induces single-strand scissions in the two DNAs under consideration. Linear øXSS DNA molecules are detected by sedimentation through alkaline sucrose gradients. After treatment of the øXRFI DNA, however, the degree of degradation is the same whether it is measured under neutral or alkaline conditions, indicating that alkaline-labile bonds are not created; moreover, double-strand breaks can only be detected after accumulation of single-strand breaks. In addition to the amount of proflavine bound to the DNA and the duration of irradiation, the following factors are shown to influence the nicking activity of the treatment: (1) the DNA structure (the øXRFI DNA is much more sensitive than the øXss DNA); (2) the ionic strength of the medium during irradiation (a high value of 0.5 leads to a markedly increased efficiency); (3) the addition of cysteamine (this latter compound decreases the reaction rate) and (4) the irradiation wavelength (after irradiation at 440 nm alone, the reaction occurs at a reduced rate and is sensitive to NaN₃). The kinetics of the nicking reaction does not follow a single-hit curve showing that at least one primary lesion occurs prior to strand breakage. On the other hand, strand scission cannot be detected after irradiation of the proflavine-DNA complexes at the low fluence rate causing a decrease in the infectivity of both øXSS and øXRFI DNAs. Similarly. the sedimentation pattern of the DNA extracted from treated øx174 phages 99.9% inactivated, is identical to that of the control ss DNA, although more drastic treatments are susceptible to induce single strand breaks inside the phage head. Finally, the unknown lesion (s) that is biologically important does not prevent the treated DNAs from penetrating into the hostcells.     

DNA REPAIR IN ULTRAVIOLET LIGHT IRRADIATED HeLa CELLS AND ITS REVERSIBLE INHIBITION BY HYDROXYUREA

Abstract— –The repair of u.v. damaged DNA in HeLa cells can be detected using the alkaline sucrose gradient technique. As a result of pyrimidine dimer excision single strand breaks are produced in DNA of irradiated cells. Rejoining of these breaks occurs during an 8 hr post-irradiation incubation period and is prevented by hydroxyurea and acriflavine. The inhibition of repair by hydroxyurea can be reversed by a mixture of all 4 deoxyribonucleosides at a concentration that does not reverse the inhibition of total DNA synthesis.     

DNA LESIONS AND DNA DEGRADATION IN MOUSE LYMPHOMA L5178Y CELLS AFTER PHOTODYNAMIC TREATMENT SENSITIZED BY CHLOROALUMINUM PHTHALOCYANINE

Two closely related strains of mouse lymphoma L5178Y cells, LY-R and LY-S, have been found to differ in their sensitivity to the cytotoxic effects of photodynamic treatment (PDT) with chloroaluminum phthalocyanine (CAPC) and red light. Strain LY-R is more sensitive to photodynamic cell killing than strain LY-S. Differences in uptake of CAPC could not account for the differences in cytotoxic effects. There was no marked difference between the two strains in the induction of single-strand breaks (which includes frank single-strand breaks and alkali-labile lesions), but substantially more DNA-protein cross-links were formed in strain LY-R by CAPC and light. Repair of single-strand breaks proceeded with similar kinetics in both strains for the first 30 min post-irradiation, suggesting that these lesions are not responsible for the differential sensitivity of the two strains to the lethal effects of photodynamic treatment. Thereafter, alkaline elution revealed the presence of increasing DNA strand breakage in strain LY-R. DNA degradation, as measured by the conversion of prelabeled [14C] DNA to acid-soluble radioactivity, was more rapid and extensive in strain LY-R.     

Cell-cycle delay is induced in cells of a U937 promonocytic cell line by low-intensity light irradiation at 660 nm

Visible-light irradiation (VLI) at 660 nm and 11.5 J/cm2 inhibits proliferation of cells of the U937 promonocytic cell line, as monitored by autoradiographical analysis. The S-phase cell population is reduced at 6 h post-radiation treatment. Flow cytometric analysis confirms this, and also shows that light irradiation of cells induces a statistically significant increase in G2/M cells at 6 h post-radiation treatment. It has been postulated that VLI at 660 nm can alter cell-cycle progression by affecting intracellular concentrations of ions, in particular pH and calcium. However, no significant effects of light irradiation on these intracellular ions have been observed. These effects of VLI are not a consequence of radiation-induced DNA strand breaks, therefore events other than direct DNA damage are involved. These findings demonstrate a direct photobiological effect of VLI at 660 nm on the cell cycle, and indicate a previously unsuspected mechanism for the induction of cell-cycle delay that is neither a result of changes in the concentration of intracellular ions nor initiated by DNA strand breaks.     

The use of silver-stained "comets" to visualize DNA damage and repair in normal and Xeroderma pigmentosum fibroblasts after exposure to simulated solar radiation

The alkaline and neutral comet assays have been widely used to assess DNA damage and repair in individual cells after in vivo or in vitro exposure to chemical or physical genotoxins. Cells processed under neutral conditions generate comets primarily from DNA double strand breaks, whereas under alkaline conditions, comets arise from DNA single and double strand breaks and alkali-labile lesions. A modified version of the alkaline comet assay, as described here, used silver stain to visualize the comets and a Gelbond base to facilitate the manipulation and processing of samples. To demonstrate how these modifications improve the assay, fibroblasts derived from both normal and Xeroderma pigmentosum (Xp) individuals were exposed to simulated solar radiation and the resulting DNA damage and repair evaluated and compared with results from the relevant literature. Comets from normal fibroblasts reached their maximum length at about an hour after irradiation. Dose-dependent increases in comet length were observed up to at least 360 mJ/cm2. In contrast, comet lengths from repair deficient Xp fibroblasts were shorter than normal cells reflecting their reduced capacity to generate single strand breaks by the excision of DNA dimers. For incubation times of more than 1 h, comet lengths from normal fibroblasts underwent a time-dependent decrease, supporting the contention that this change was related to the ligation step in the DNA repair process. These changes were compatible with the model of DNA damage and repair established by others for ultraviolet radiation.     

ALKALINE ELUTION STUDIES OF HEMATOPORPHYRIN-DERIVATIVE PHOTOSENSITIZED DNA DAMAGE AND REPAIR IN CHINESE HAMSTER OVARY CELLS

Abstract We have used alkaline elution to study DNA damage produced by the photosensitizer hematoporphyrin derivative (HPD) in cultured Chinese hamster cells. Dosimetry was performed by measuring fluence and calculating photon absorption by intracellular HPD. HPD photosensitization causes DNA strand breakage. These breaks are repaired by the cell, although their fractional rate of repair is smaller than that for X-ray induced strand breaks at equivalent levels of strand breakage. The combined DNA polymerase inhibitors cytosine arabinoside and hydroxyurea suppress the repair of HPD-photosensitized breaks more strongly than they suppress repair of X-ray induced breaks. Addition of novobiocin to the aforementioned inhibitors causes almost total suppression of photosensitized break repair. A nucleotide excision repair system with inhibitor susceptibility similar to that of the system which removes pyrimidine dimers thus does not act upon HPD-photosensitized damage. The repair rate and inhibitor sensitivity findings together suggest biologically important differences in the chemical nature of X-ray induced and HPD-photosensitized strand breaks. In addition to strand breaks, HPD photosensitization produces covalent DNA-protein crosslinks, some of which persist through at least 90 min incubation, but which are repaired within 180 min.     

Vacuum UV laser induced scission of simian virus 40 DNA

Abstract— The effect of vacuum UV-laser irradiation on Simian virus 40 (SV40) DNA I was studied in vitro. Following exposure of SV40 DNA I to an argon fluoride (ArF) laser (LD = 193 nm), single-strand breaks occur in either strand of the double-stranded superhelical molecule (DNA I) and convert it to the DNA II conformation. Upon increased irradiation times, additional breaks occur in already scissioned strands resulting in the production of smaller fragments as analyzed by alkaline sucrose centrifugation. These results suggest that short periods of irradiation lead to conformational alterations of SV40 DNA I.     

Synthesis, structure and thermal stability of fully hydrophobic porphyrin-DNA conjugates

The DNAs modified with tetraphenyl porphyrin at the center of 13mer oligonucleotide were synthesized using phosphoramidite chemistry and automated DNA synthesis. When the porphyrin modified oligonucleotide was annealed with its complementary strand, they formed a standard B-form duplex. The porphyrin moiety intercalated in the duplex, and moderately lowered the thermal stability.     

DNA analysis of a radiotolerant bacterium Pantoea agglomerans by FT-IR spectroscopy

A radiation tolerance strain, Pantoea agglomerans was isolated from γ-irradiated carrot samples (Daucus carota). D₁₀ determination showed that the radioresistance of this bacterium is five-fold higher than Escherichia coli, both belonging to the family of Enterobacteriaceae. DNA isolated from untreated and irradiated bacterial cells was analyzed by FT-IR spectroscopy to investigate the radiotolerance of this bacterium. At doses <5 kGy, an alteration of the interbase hydrogen networks was observed and characterized mainly by an increase of bands assigned to the carbonyl non-pairing and the free amine groups. Moderate breakage of the DNA backbone and damage of the osidic structure were also observed. Similar spectral profiles were noticed at doses ≥5 kGy, but additional increase of the band intensity of CC and CN suggests damages of nucleobases. High number of asymmetric PO₂⁻ and upper shift of symmetric PO₂⁻ are indicative of DNA strand breaks. Osidic damages were evidenced by decrease of the absorption bands ascribed to deoxyribosyl moieties and by appearance of C–OH band. DNA degradation at high irradiation doses was also noticed by electrophoresis using agarose gel. It appeared that DNA underwent covalent cross-linking, as revealed by agglomeration of DNA in the wells of agarose gel.     

The effect of Ge, Si and Sn phthalocyanine photosensitizers on cell proliferation and viability of human oesophageal carcinoma cells

The photodynamic activity of water soluble mixed sulfonated metallophthalocyanines complexes: GePcSmix, SnPcSmix and SiPcSmix on human oesophageal carcinoma (SNO) cells are reported, and compared with the activity of the unmetallated H2PcSmix and of the newly synthesized water soluble adjacently substituted binaphthalo phthalocyanine (complex 3). The alkaline phosphate (ALP) showed damage to the cell membrane in the presence of complex 3 without irradiation. The GePcSmix complex caused a relatively large increase in inflammation and a high intracellular ATP.     

Fluorometric analysis of DNA unwinding (FADU) as a method for detecting repair-induced DNA strand breaks in UV-irradiated mammalian cells

Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray-induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20 degrees C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).     

BIOLOGICAL PROPERTIES OF SOME BENZOPSORALEN DERIVATIVES

Abstract— The biological activity of some benzopsoralen derivatives, prepared with the aim of obtaining new drugs for photochemotherapy, has been studied. The more interesting compounds are 4-hydroxy-methyl-4',5'-benzopsoralen and 4-hydroxymethyl-4',5'-tetrahydro-benzopsoralen, which were found to be active in the dark also: DNA and RNA synthesis were both inhibited in Ehrlich cells, even if in a partially reversible fashion, while protein synthesis remained unaffected. In Chinese hamster ovary cells cultured in vitro , the clonal growth was strongly inhibited by incubation in the dark with both drugs, while a number of chromosomal aberrations was observed in the fraction of growing cells. Using alkaline elution, DNA strand breaks were detected. In addition, in the presence of aphidicolin, a specific inhibitor of DNA polymerase, the clonal growing capacity was completely restored; in contrast, the number of DNA strand breaks remained unchanged. All these results suggest that DNA topoisomerases are probably the target of these two benzopsoralens. These compounds are also good sensitizers; by UV-A irradiation they have a good capacity to produce singlet oxygen, but they appeared to be unable to induce erythemas on guinea-pig skin. Under UV-A light, they induced a strong inhibition of DNA synthesis in Ehrlich cells. Thus, benzopsoralens appear to be capable of inducing strong antiproliferative effects by two different mechanisms, by UV-A irradiation and in the dark.     

THE REPAIR OF DNA STRAND BREAKS IN HUMAN LYMPHOCYTES EXPOSED TO NEAR UV-RADIATION (UVA) AND FAR UV-RADIATION (UVC)

Abstract— UVA irradiation of human lymphocytes induces DNA strand breaks and a portion of these breaks are closed at a slower rate than X-ray induced DNA strand breaks and the strand breaks generated during repair of UVC induced DNA lesions. In addition, the yield of DNA strand breaks in lymphocytes pretreated with UVA radiation and given a subsequent exposure with UVC radiation is higher and shows a slower decrease with increasing repair time in comparison with the expected yield based on additivity between UVA and UVC induced DNA strand breaks. This indicates that UVA delays the closure of the intermediate strand breaks formed in the repair process of UVC induced DNA lesions.     

Lack of correlation between DNA strand breakage and p53 protein levels in human fibroblast strains exposed to ultraviolet lights

The contribution of DNA strand breaks accumulating in the course of nucleotide excision repair to upregulation of the p53 tumor suppressor protein was investigated in human dermal fibroblast strains after treatment with 254 nm ultraviolet (UV) light. For this purpose, fibroblast cultures were exposed to UV and incubated for 3 h in the presence or absence of l-beta-D-arabinofuranosylcytosine (araC) and/or hydroxyurea (HU), and then assayed for DNA strand breakage and p53 protein levels. As expected from previous studies, incubation of normal and ataxia telangiectasia (AT) fibroblasts with araC and HU after UV irradiation resulted in an accumulation of DNA strand breaks. Such araC/HU-accumulated strand breaks (reflecting nonligated repair-incision events) following UV irradiation were not detected in xeroderma pigmentosum (XP) fibroblast strains belonging to complementation groups A and G. Western blot analysis revealed that normal fibroblasts exhibited little upregulation of p53 (approximately 1.2-fold) when incubated without araC after 5 J/m2 irradiation, but showed significant (three-fold) upregulation of p53 when incubated with araC after irradiation. AraC is known to inhibit nucleotide excision repair at both the damage removal and repair resynthesis steps. Therefore, the potentiation of UV-induced upregulation of p53 evoked by araC in normal cells may be a consequence of either persistent bulky DNA lesions or persistent incision-associated DNA strand breaks. To distinguish between these two possibilities, we determined p53 induction in AT fibroblasts (which do not upregulate p53 in response to DNA strand breakage) and in XP fibroblasts (which do not exhibit incision-associated breaks after UV irradiation). The p53 response after treatment with 5 J/m2 UV and incubation with araC was similar in AT, XPA, XPG and normal fibroblasts. In addition, exposure of XPA and XPG fibroblasts to UV (5, 10 or 20 J/m2) followed by incubation without araC resulted in a strong upregulation of p53. We further demonstrated that HU, an inhibitor of replicative DNA synthesis (but not of nucleotide excision repair), had no significant impact on p53 protein levels in UV irradiated and unirradiated human fibroblasts. We conclude that upregulation of p53 at early times after exposure of diploid human fibroblasts to UV light is triggered by persistent bulky DNA lesions, and that incision-associated DNA strand breaks accumulating in the course of nucleotide excision repair and breaks arising as a result of inhibition of DNA replication contribute little (if anything) to upregulation of p53.     

UPTAKE and PHOTODYNAMIC EFFICIENCY OF HEM ATOPORPH YRIN, HYDROXYETHYLVINYLDEUTEROPORPHYRIN and HEMATOPORPHYRIN DERIVATIVE (PHOTOFRIN II®): A STUDY WITH ISOLATED MITOCHONDRIA

The uptake of Photofrin II (PFII), hematoporphyrin (Hp) and hydroxyethylvinyldeuteroporphyrin (HVD) by isolated mitochondria was studied using the high performance liquid chromatography (HPLC) technique. The various PFII components show a high affinity for mitochondria. At 5.75 micrograms/ml PFII, their ratio of incorporation was found to be very similar, except for Hp which is about two times less incorporated. These results were reproduced with pure Hp and pure HVD. The uptake of Hp and HVD increases with concentration but, while that of Hp reaches a plateau, the uptake of HVD continues to increase. At a high porphyrin concentration (approximately 10(-5) M), the loss of respiratory control is obtained with the same light dose for Hp and PFII. Taking into account the uptake and the known photophysical parameters of the various porphyrins, the photodynamic efficiency of HVD seems equivalent to that of Hp. The present results and known data on cell photoinactivation suggest that the activity of these porphyrins is mainly dependent on their incorporation.     

Biological responses to the simulated Martian UV radiation of bacteriophages and isolated DNA

Mars is considered as a main target for astrobiologically relevant exploration programmes. In this work the effect of simulated Martian solar UV radiation was examined on bacteriophage T7 and on isolated T7 DNA. A decrease of the biological activity of phages, characteristic changes in the absorption spectrum and in the electrophoretic pattern of isolated DNA/phage and the decrease of the amount of PCR products were detected indicating damage of isolated and intraphage T7 DNA by UV radiation. Further mechanistic insights into the UV-induced formation of intraphage/isolated T7 DNA photoproducts were gained from the application of appropriate enzymatic digestion and neutral/alkaline agarose gel electrophoresis. Our results showed that intraphage DNA was about ten times more sensitive to simulated Martian UV radiation than isolated T7 DNA indicating the role of phage proteins in the DNA damage. Compared to solar UV radiation the total amount of DNA damage determined by QPCR was about ten times larger in isolated DNA and phage T7 as well, and the types of the DNA photoproducts were different, besides cyclobutane pyrimidine dimers (CPD), double-strand breaks (dsb), and single-strand breaks (ssb), DNA-protein cross-links were produced as well. Surprisingly, energy deposition as low as 4-6eV corresponding to 200-400nm range could induce significant amount of ssb and dsb in phage/isolated DNA (in phage the ratio of ssb/dsb was approximately 23%/12% and approximately 32%/19% in isolated DNA). 5-8% of the CPD, 3-5% of the AP (apurinic/apyrimidinic) sites were located in clusters in DNA/phage, suggesting that clustering of damage occur in the form of multiple damaged sites and these can have a high probability to produce strand breaks. The amount of total DNA damage in samples which were irradiated in Tris buffer was reduced by a factor approximately 2, compared to samples in phosphate buffer, suggesting that some of the photoproducts were produced via radicals.     

EFFECT OF POLYAMINE DEPLETION ON DNA DAMAGE and REPAIR FOLLOWING UV IRRADIATION OF HeLa CELLS

Treatment of HeLa cells with the polyamine biosynthesis inhibitors, methylglyoxal bis(guanylhydrazone) (MGBG), difluoromethylornithine (DFMO) or a combination of the two, resulted in reduction in cellular polyamine levels. Analysis of UV light-induced DNA damage and repair in these polyamine depleted cells revealed distinct differences in the repair process relative to that seen in cells possessing a normal polyamine complement. Initial yield of thymine dimers and rate of removal of these lesions from cellular DNA appeared normal in polyamine-depleted cells. However, depleted cells exhibited retarded sealing of DNA strand breaks resulting from cellular repair processes, reduced repair synthesis and an increased sensitivity to UV killing. Incision at damaged sites was not affected since ara-C repair-dependent breaks accumulated in a normal fashion. Molecular analysis of inhibited repair sites by exonuclease III and T4 DNA ligase probes suggest that the strand interruptions consist of gaps rather than ligatable nicks, consistent with an interpretation of the repair defect being at the gap-filling stage rather than the ligation step. Observed patterns of differential polyamine depletion by DFMO and MGBG, and partial reversal of repair inhibition by polyamine supplementation, suggests that polyamine depletion per se, rather than some secondary effect of inhibitor treatment, is responsible for the inhibition of repair.     

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.