Studies of the optical properties of the proflavine–DNA complex

We report studies of the optical properties of the proflavine–DNA complex, using absorbance and circular dichroism spectroscopy. From comparison of the absorption spectra of proflavine complexed with calf thymus and T2 DNA, we conclude that stacking of the dyes external to the double helix is comparatively much weaker with T2 DXA, probably because of its glucosylation. Several sources are found for the circular dichroism induced in proflavine when it is complexed with DNA. There is a relatively weak circular dichroism induced when the dye is infinitely dilute on the DNA lattice; this presumably arises from the environmental asymmetry of the binding site. Stronger circular dichroism effects are induced by interaction of intercalated and stacked dyes; studies with T2 DNA, for which stacking seems to be blocked, permit a tentative resolution of effects due to the two modes of binding. One recurring theme of these studies is the observation that the optical properties are quite dependent on environment. The most dramatic example is a strong variation with salt concentration of the amplitude of the circular dichroism induced in the isolated (intercalated) monomer by the surrounding DNA. This suggests that the structure of the intercalated complex is quite sensitive to external conditions.     

Dichroism of TMV in pulsed electric fields

The linear dichroism induced in a solution of electrically anisotropic molecules by a pulsed electric field has been studied. Equations have been obtained which express the dichroism as a function of dipole moment, excess polarizability, field strength, and the angle α between the dipole moment and the transition moment for the absorption band. These expressions have been related to the experimentally observed difference signal in such a way that when the dichroism is measured as a function of field strength the permanent moment, excess polarizability and angle a can be determined. Experiments have been carried out on tobacco mosaic virus (TMV), which is similar in its properties to the theoretical model. The polarizability anisotropy and rotary diffusion constant for the monomer and dimer of TMV have been obtained from these experiments. In addition to the molecular parameters mentioned above, the saturated electric dichroism of the virus was measured as a function of wave length and the presence of an n–π* transition in the tryptophan spectrum was indicated. Further experiments measuring dichroism as a function of pH demonstrated the general denaturation of the virus at high pH (10–11) but also the existence of a stable fraction which is not fragmented even at the high pH involved.     

An unusual electrooptical effect observed for DNA fragments and its apparent relation to a permanent electric moment associated with bent DNA

Dichroism decay curves of DNA fragments with chain lengths in the range of 179-256 bp show an amplitude inversion suggesting the existence of a positive dichroism component, when these fragments are dissolved at monovalent salt concentrations above approx. 5 mM and are exposed to field pulses with amplitudes and/or lengths above critical values. At the critical values, the unusual dichroism is reflected by an apparent acceleration of the decay curves, which can be fitted by single exponentials with time constants much below the values expected from the DNA contour lengths. The critical pulse amplitudes and lengths decrease with increasing DNA chain length and increasing salt concentration. The experimental data are consistent with results obtained by hydrodynamic and electric model calculations on smoothly bent DNA double helices. The DNA is represented by a string of overlapping beads, which is used to calculate the rotational diffusion tensor and the center of diffusion. The distribution of phosphate charges is asymmetric with respect to this center and thus gives rise to a substantial permanent dipole moment. The magnitude of this dipole moment is calculated as a function of DNA curvature and is used together with experimental values of polarizabilities for simulations of dichroism decay curves. The curves simulated for bent DNA show the same phenomenon as observed experimentally. The ionic strength dependence of the unusual dichroism is explained by an independently observed strong decrease of the polarizability with increasing salt concentration. The field strength dependence is probably due to field-induced bending of double helices driven by the change of the dipole moment. Although our calculations are on rigid models of DNA and thus any flexibility of the double helix has not been considered, we conclude that the essential part of our experimental results can be explained by our model.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field induced conformational changes of bacteriorhodopsin were studied in six types of dried film (randomly and electrically oriented membranes of purple as well as cation-depleted blue bacteriorhodopsin) by measuring the frequency dependence of the optical absorbance change and the dielectric dispersion and absorption. For the purple bacteriorhodopsin the optical absorbance change induced by alternating rectangular electric fields of ±300 kV/cm altered the sign twice in the frequency range from 0.001 Hz to 100 kHz (around 0.03 Hz and 100 kHz), indicating that the electric field induced conformational change in these samples consists of, at least, three steps. Similarly, it was found for the blue bacteriorhodopsin that at least two steps are involved. In accord with optical measurements, the dielectric behaviour due to alternating sinusoidal electric fields of±6kV/cm in the frequency range from 10 Hz to 10 MHz showed two broad dispersion/absorption regions, one below 1 kHz and the other around 10–100 kHz. This suggests that the conformational change of bacteriorhodopsin is also reflected by its dielectrical properties and that it is partially induced at 6 kV/cm. Including previous results obtained by analysis of the action of DC fields on purple membrane films, a model for a field-induced cyclic reaction for purple as well as blue bacteriorhodopsin is proposed. In addition it was found that there are electrical interactions among purple membrane fragments in dried films.     

Unusual DNA Binding Exhibited by Synthetic Distamycin Analogues Lacking the N-terminal Amide Unit under High Salt Conditions

Abstract

The binding of three analogues of the minor-groove binding antiviral antibiotic distamycin (Dst) with double-stranded (ds)-DNA were monitored using ds-DNA melting temperature (T_m) measurements, ethidium bromide (EtBr) displacement assay, footprinting analysis and induced circular dichroism (ICD). These compounds contained 3–5 N-methyl-pyrrole-car- boxamide units and lacked the N-terminal formamide unit present in Dst. These experiments suggested that the present analogues did not compromise their AT-specificity despite the deletion of the N-terminal formamide unit. The binding affinities, however, were significantly affected. Interestingly, the analogue with three N-methyl-pyrrole-carboxamide units exhibited an initial decrease in ICD at >40 mM salt concentrations. This was followed by a pronounced recovery of ICD at > 1.6 M salt concentrations, a phenomenon hitherto not observed with any other DNA binding molecules. The pentapyrrole analogue exhibited the highest binding affinity with CT-DNA under normal (40 mM) salt conditions. However, it suffered maximum relative dissociation under high salt conditions and did not exhibit any recovery in ICD at higher NaCl concentrations. The analogues possessing four and five pyrrole rings exhibited intense ICD signals with poly d(GC) in the ligand absorption region in the presence of 40 mM NaCl, unlike the one with three pyrrole rings. These ICD signals were however, highly susceptible to changes in ionic strength. Thus subtle modifications in the ligand molecular structure can have dramatic effect on their DNA binding properties.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

Optical and Nonlinear Optical Limiting Properties of AgNi Alloy Nanostructures

Silver-nickel alloy nanoparticles with varying size were synthesized by reducing the metal precursors chemically using a single-step solution-based synthesis route. The structural, optical, and nonlinear optical properties of the prepared samples were investigated. The synthesized samples having highly agglomerated, interconnected nature and found to exhibit dipole and multipole surface plasmon resonance related optical absorption bands. Nonlinear optical and optical limiting properties were investigated using a single beam open aperture z-scan technique with the use of 532 nm, 5-ns laser pulses. The nonlinearity observed was found to have contributions from saturable absorption (SA) and excited state absorption (ESA) related to free carriers. The effective nonlinear optical absorption was enhanced in AgNi alloy compared to pure Ag nanostructures.     

Base tilt of DNA in various conformations from flow linear dichroism

We have measured the isotropic absorption (Aiso) and linear dichroism (LD) of Escherichia coli DNA in 0.01 M Na+ (10.4 base pairs per turn of B form), 5.5 M NH4F (10.2 base pairs per turn of B form), and 80% trifluoroethanol (A form) into the vacuum UV spectral region. The reduced dichroism spectrum (LD divided by Aiso) of DNA in the A conformation differed from those of the B conformations, demonstrating that LD is a sensitive method for distinguishing DNA conformation. The reduced dichroism spectra of the B conformations were similar, indicating little change in the orientation of the bases for DNA in high salt. The wavelength dependence of the reduced dichroism indicates that the angle between the base planes and the helix axis is less than 76 degrees for all three conformations of DNA.     

Interaction of ethidium bromide with DNA. Optical and electrooptical study

The binding of ethidium bromide to DNA has been studied by various optical methods. From fluorescence polarization studies, and film, electric linear dichroism, and circular dichroism spectra, we propose assignments of the absorption bands of the dye, which are discussed in connection with wave-mechanical calculations recently reported. The optical activity induced in the dye absorption bands upon binding to DNA was attributed to various origins depending on the electronic transition considered. The visible absorption band displayed a circular dichroism due to the asymmetry of the binding site and independent of the amount of binding. The transition identified at 378 nm from the circular dichroism and electric dichroism observations was thought to be due to a magnetic-dipole transition. It remained constant with increasing amounts of dye bound. The main ultraviolet band showed circular dichroism characteristics corresponding to exciton interactions between dye molecules bound to neighboring sites. The electric dichroism observed for the strongly bound dye molecules indicated that the phenanthridinium ring of ethidium bromide was probably not perfectly parallel to the DNA base planes. When the amount of dye bound to DNA exceeded the maximum amount compatible with the exclusion of adjacent binding sites, the electric dichroism decreased owing to the appearance of externally bound dye molecules with no contribution to the dichroism. Sonicated DNA was used to study the lengthening of the DNA molecule upon complexation. Although the viscosity of the complexes increased with the amount of binding, the rotational diffusion coefficient measured by the electric birefringence relaxation was not detectably affected. The absence of variation in the electric birefringence with the binding indicated that the DNA base stacking remained unaltered.     

Matrix-method calculation of linear and circular dichroism spectra of nucleic acids and polynucleotides

Calculations of the optical properties (absorption, linear dichroism, circular dichroism, and anisotropic components of the CD) are presented for polynucleotides of random or regular sequence within the formalism of the matrix method using a set of parameters that includes only the ππ* transitions of the aromatic bases. Experimental solution spectra agree favorably with calculated CD spectra for A-RNA, A-DNA, and B-DNA, when coordinates derived from x-ray studies on fibers are used. Excessive hypochromicity is predicted when parameters intended to reproduce the vacuum-uv absorption of the chromophores are included in the calculations, but total elimination of these parameters leads to an insufficient hypochromicity for the long-wavelength absorption band. Using alternative conformations for DNA in low-salt aqueous solution did not improve the agreement between experimental and calculated spectra, but some features of the optical properties predicted for these variant structures suggest that the tilt of the bases with respect to the helical axis may be larger than that of the fiber B-form. In the case of polynucleotides with regular structure, which have been traditionally less easy to understand in terms of the standard nucleic acid conformations, a series of alternative structures has been examined. Unexpectedly, the calculated spectrum for the Z-DNA structure compares almost quantitatively with the experimental spectrum of poly(dGC·dGC) in low salt. This result, which confirms a recent report [Vasmel, H. & Greve, J. (1981) Biopolymers 20 , 1329–1332], is in contrast with the current identification of Z-DNA with the high-salt form of poly(dGC·dGC). Finally, the optical properties of single-stranded polyribonucleotides appear to be better explained when alternative structures [9₁-helix for poly(rA) and 6₁-helix for poly(rC)] are introduced instead of the A-RNA form.     

Reinterpretation of Linear Dichroism of Chromatin Supports a Perpendicular Linker Orientation in the Folded State

Abstract

We present a reinterpretation of linear dichroism data for the salt induced condensation of chromatin. A conflict between electric and flow linear dichroism data for identical chromatin samples, studied at varying degrees of Mg²⁺ induced folding, can be solved if the orientation in electric fields is mainly determined through the polarization of counter ions along the linker parts, whereas the orientation in flow is governed by the hydrodynamical response of the entire chromatin fiber. The orientation of a chromatin fiber in an electric field would then depend on the linker tilt angle so that at an angle larger than 55° the fiber would tend to orient perpendicular to the applied field. The different orientation distributions obtained with the two methods of alignment may in this way provide extra information about the structure and folding of chromatin.     

Orientation of DNA in agarose gels.

An orientation of the lambda DNA during the electrophoresis in agarose gels was measured by a microscopic linear dichroism technique. The method involved staining the DNA with the dye ethidium bromide and measuring under the microscope the polarization properties of the fluorescence field around the electrophoretic band containing the nucleic acid. It was first established that the fluorescence properties of the ethidium bromide-DNA complex were the same in agarose gel and in a solution. Then the linear dichroism method was used to measure the dichroism of the absorption dipole of EB dye bound to lambda DNA. In a typical experiment the orientation of two-tenth of a picogram (2 x 10(-13)g) of DNA was measured. When the electric field was turned on, the dichroism developed rapidly and assumed a steady state value which increased with the strength of the field and with the size of DNA. A linear dichroism equation related the measured dichroism of fluorescence to the mean orientation of the absorption dipole of ethidium bromide and to an extent to which the orientation of this dipole deviated from the mean. The observed development of dichroism in the presence of an electric field was interpreted as an alignment of DNA along the direction of the field. The increase in the steady state value of dichroism with the rise in the strength of the field and with the increase of the size of DNA was interpreted as a better alignment of DNA along the direction of the field and as a smaller deviation from its mean orientation.     

Poly(L–lysine)–DNA interactions in NaCl solutions: B → C and B → ψ Transitions

Thermal denaturation and circular dichroism (CD) properties of poly(L -lysine)–DNA complexes vary greatly when these complexes are prepared differently, that is, whether by NaCl-gradient dialysis starting from 2.0 M NaCl or by direct mixing at low salt. These differing properties were investigated in more detail by examining complexes, made by direct mixing in the presence of various concentrations of NaCl, both before and after the NaCl was dialyzed out of the complex solution. The precipitation curves of DNA due to polylysine binding indicate that such binding is noncooperative at zero salt; from 0.1 up to 1.0 M NaCl they exhibit varying degrees of cooperatively. Starting from zero salt, as the NaCl concentration used for complex formation is increased, both the CD and the melting properties of the complexes are shifted from those of directly mixed at zero salt to those of reconstitution: in the CD spectra there is a gradual shift from a B → C transition to a B → ψ transition; thermal denaturation results show a gradual increase in the melting temperatures of both free DNA (t_m) and polylysine-bound DNA (t′_m). The progressive shift from B → C to B → ψ suggests a close relationship between these two transitions. Large aggregates of the complexes do not warrant the appearance of ψ-type CD spectra: ψ-spectra have been obtained in the supernatants of polylysine–DNA complexes made and measured at 1.0 M NaCl while slightly perturbed CD spectra in B → C transition have been observed in turbid solutions of fully covered complexes made at very low salt. If the complexes are made at intermediate salts and dialyzed to a very low salt, although up to 60% of the DNA is still bound by polylysine, the CD spectra of the complexes are shifted back to the B-type CD characteristic of pure DNA.     

Electric properties and structure of DNA-restriction fragments from measurements of the electric dichroism

The electric dichroism of 17 homogeneous DNA fragments, ranging in size from 43 to 4362 base-pairs, has been analyzed in high electric fields. The orientation of the small fragments can be described in terms of an induced dipole moment, whereas the large fragments are oriented according to a constant dipole mechanism. In the intermediate size range, DNA orients according to an induced dipole mechanism at low field strengths and according to a constant dipole mechanism at high field strengths. From these observations we propose an orientation mechanism with a saturating induced dipole. The induced dipole observed at low field strengths is saturated at a field strength Eo within a transition range Em to give a constant dipole moment at high field strengths. These parameters together with the polarizability and the limit reduced dichroism are evaluated by a least-squares analysis of the experimental data. Eo and Em are found to decrease with increasing chain length from Eo approximately 40 kV/cm (Em approximately 14 kV/cm) at 65 base-pairs to 10 kV/cm (6 kV/cm) at 194 base-pairs. The polarizability is found to increase with the square of the chain length, whereas the saturated dipole increases with chain length N at low N and goes to a limit value at high N. The temperature dependence of the orientation parameters is found to be very small. The values obtained for the limit dichroism are between -1.0 and -1.3 for chain lengths between 60 and 1000 base-pairs, whereas values around -1.4 are observed at chain lengths greater than 1000 base-pairs. These data indicate that electric fields extend the contour of DNA strands at high chain lengths from a weakly bent to a more linear form. The variations of the limit dichroism observed for short fragments suggest sequence-dependent differences in the secondary structure of the helix. The experimental results are compared with numerical calculations based on simple polyelectrolyte models. For short fragments the magnitude of several electrochemical parameters can be adequately explained by a polarization of the ion cloud around the DNA molecules. However, these polyelectrolyte models do not adequately describe the observed chain length dependence of the orientation phenomena.     

Optical Kerr effect in biopolymer solutions

The electric field in a single mode, YAG laser beam has been used to induce orientational birefringence in solutions of tobacco rattle virus, DNA, heparin, and hyaluronic acid. Using this laser in its “fixed-Q” mode, laser pulses were generated which persisted for up to 200 μsec in which the effective electric field vector rose to 5 kV cm^?1. The birefringence amplitudes so produced had a quadratic dependence on the effective field strength and thus obeyed Kerr's law. From the birefringence decay rates, relaxation times were determined which, by comparison with the birefringence induced by pulsed static electric fields revealed the biopolymer orientational origins of the effects. This indicated how these experiments can lead to the evaluation of particle geometry, the electronic contribution to electrical polarizabilities, and the optical polarizability of biopolymers in solution.     

Unusual DNA binding exhibited by synthetic distamycin analogues lacking the N-terminal amide unit under high salt conditions.

The binding of three analogues of the minor-groove binding antiviral antibiotic distamycin (Dst) with double-stranded (ds)-DNA were monitored using ds-DNA melting temperature (Tm) measurements, ethidium bromide (EtBr) displacement assay, footprinting analysis and induced circular dichroism (ICD). These compounds contained 3-5 N-methyl-pyrrole-carboxamide units and lacked the N-terminal formamide unit present in Dst. These experiments suggested that the present analogues did not compromise their AT-specificity despite the deletion of the N-terminal formamide unit. The binding affinities, however, were significantly affected. Interestingly, the analogue with three N-methyl-pyrrole-carboxamide units exhibited an initial decrease in ICD at > 40 mM salt concentrations. This was followed by a pronounced recovery of ICD at > 1.6 M salt concentrations, a phenomenon hitherto not observed with any other DNA binding molecules. The pentapyrrole analogue exhibited the highest binding affinity with CT-DNA under normal (40 mM) salt conditions. However, it suffered maximum relative dissociation under high salt conditions and did not exhibit any recovery in ICD at higher NaCl concentrations. The analogues possessing four and five pyrrole rings exhibited intense ICD signals with poly d(GC) in the ligand absorption region in the presence of 40 mM NaCl, unlike the one with three pyrrole rings. These ICD signals were however, highly susceptible to changes in ionic strength. Thus subtle modifications in the ligand molecular structure can have dramatic effect on their DNA binding properties.     

Reinterpretation of linear dichroism of chromatin supports a perpendicular linker orientation in the folded state

We present a reinterpretation of linear dichroism data for the salt induced condensation of chromatin. A conflict between electric and flow linear dichroism data for identical chromatin samples, studied at varying degrees of Mg2+ induced folding, can be solved if the orientation in electric fields is mainly determined through the polarization of counter ions along the linker parts, whereas the orientation in flow is governed by the hydrodynamical response of the entire chromatin fiber. The orientation of a chromatin fiber in an electric field would then depend on the linker tilt angle so that at an angle larger than 55 degrees the fiber would tend to orient perpendicular to the applied field. The different orientation distributions obtained with the two methods of alignment may in this way provide extra information about the structure and folding of chromatin.     

Retained binding mode of various DNA-binding molecules under molecular crowding condition

Meso-tetrakis(N-methyl pyridinium-4-yl)porphyrin (TMPyP) intercalates between the base-pairs of DNA at a low [TMPyP]/[DNA base] ratio in aqueous solutions and molecular crowding conditions, which is induced by the addition of Poly(ethylene glycol) (PEG). Studied DNA-binding drugs, including TMPyP, 9-aminoacridine, ethidium bromide, and DAPI (4′,6-diamidino-2-phenylindole) showed similar binding properties in the presence or absence of PEG molecules which is examined by circular and linear dichroism. According to the LD^r (reduced linear dichroism) results of the binding drugs examined in this work, PEG molecules induced no significant change compared to their binding properties in aqueous buffering systems. These results suggest that the transition moments are not expected to be perturbed significantly by PEG molecules. In this study, the experimental conditions of PEG 8000 were maintained at 35% (v/v) of total reaction volume, which is equal to the optimal molar concentration (0.0536 M as final concentration for PEG 8000) to maintain suitable cell-like conditions. Therefore, there was no need to focus on the conformational changes of the DNA helical structure, such as forming irregular aggregate structures, induced by large quantities of molecular crowding media itself at this stage.     

Simulation of electrostatic and hydrodynamic properties of Serratia endonuclease

We analyze the electrostatic and hydrodynamic properties of a nuclease from the pathogenic gram-negative bacterium Serratia marcescens using finite-difference Poisson-Boltzmann methods for electrostatic calculations and a bead-model approach for diffusion coefficient calculations. Electrostatic properties are analyzed for the enzyme in monomeric and dimeric forms and also in the context of DNA binding by the nuclease. Our preliminary results show that binding of a double-stranded DNA dodecamer by nuclease causes an overall shift in the charge of the protein by approximately three units of elementary charge per monomer, resulting in a positively charged protein at physiologic pH. In these calculations, the free enzyme was found to have a negative (−1 e) charge per monomer at pH 7. The most dramatic shift in pK_a involves His 89 whose pK_a increases by three pH units upon DNA binding. This shift leads to a protonated residue at pH 7, in contrast to the unprotonated form in the free enzyme. DNA binding also leads to a decrease in the energetic distances between the most stable protonation states of the enzyme. Dimerization has no significant effect on the electrostatic properties of each of the monomers for both free enzyme and that bound to DNA. Results of hydrodynamic calculations are consistent with the dimeric form of the enzyme in solution. The computed translational diffusion coefficient for the dimer model of the enzyme is in very good agreement with measurements from light scattering experiments. Preliminary electrooptical calculations indicate that the dimer should possess a large dipole moment (approximately 600 Debye units) as well as substantial optical anisotropy (limiting reduced linear electric dichroism of about 0.3). Therefore, this system may serve as a good model for investigation of electric and hydrodynamic properties by relaxation electrooptical experiments. © 1997 John Wiley & Sons, Inc.     

Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores.

It has been shown recently that electrically induced DNA transfer into cells is a fast vectorial process with the same direction as DNA electrophoresis in an external electric field (Klenchin, V. A., S. I. Sukharev, S. M. Serov, L. V. Chernomordik, and Y. A. Chizmadzhev. 1991. Biophys. J. 60:804-811). Here we describe the effect of DNA interaction with membrane electropores and provide additional evidences for the key role of DNA electrophoresis in cell electrotransfection. The assay of electrically induced uptake of fluorescent dextrans (FDs) by cells shows that the presence of DNA in the medium during electroporation leads to a sharp increase in membrane permeability to FDs of M(r) < 20,000. The permeability increases with DNA concentration and the effect is seen even if FD is added to the cell suspension a few minutes after pulse application. The longer the DNA fragment, the greater the increase in permeability. The use of a two-pulse technique allows us to separate two effects provided by a pulsed electric field: membrane electroporation and DNA electrophoresis. The first pulse (6 kV/cm, 10 microseconds) creates pores efficiently, whereas transfection efficiency (TE) is low. The second pulse of much lower amplitude, but substantially longer (0.2 kV/cm, 10 ms), does not cause poration and transfection by itself but enhances TE by about one order of magnitude. In two-pulse experiments, TE rises monotonously with the increase of the second pulse duration. By varying the delay duration between the two pulses, we estimate the lifetime of electropores (which are DNA-permeable in conditions of low electric field) as tens of seconds.(ABSTRACT TRUNCATED AT 250 WORDS)