Fluorescence study of DNA binding and bending by EcoRI DNA methyltransferase

We have applied fluorescence anisotropy and fluorescence resonance energy transfer (FRET) techniques to study the interaction between EcoRI DNA methyltransferase (M.EcoRI) and its target DNA in solution. Upon binding with M.EcoRI, the dsDNA containing GAATTC bends to flip out the second adenine for methylation. The binding affinity of M.EcoRI to two dsDNA fragments (20 and 38 bp) was studied with fluorescence anisotropy. Their binding constants at different temperatures from 20 to 40 degrees C were obtained, and the thermodynamic parameters of binding were derived. The results showed that M.EcoRI had a higher binding affinity to the short dsDNA strand than to the long one, and its binding to DNA was primarily entropy-driven. By labeling the 5' ends of the 20-bp dsDNA with two fluorescent dyes, fluorescein (FAM) and tetramethylrhodamine (TMR), we were able to monitor the enhanced TMR fluorescence in the presence of M.EcoRI. The end-to-end distance of the dsDNA determined from the FRET efficiency was changed from 72.4 to 63.4 A, and the DNA bending angle was estimated as 57.8 degrees .     

Mobility and activation energy of single-stranded DNA in denaturing cross-linked polyacrylamide slab gels

An original apparatus based on laser-induced fluorescence detection is presented. One lane migration combined to four equidistant detection points allows the study of the dynamics of DNA bands during electrophoresis. We focus this article on the study of the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T, 5% C and 4.3% T, 5% C cross-linked polyacrylamide gels at an electric field of 45 V/cm [T=(g acrylamide+g N,N'-methylenebisacrylamide)/100 ml solution; C=g N,N'-methylenebisacrylamide/% T]. Activation energy has been investigated under these experimental conditions with a temperature varying from 25 to 50 degrees C. The activation energy for migration through the cross-linked polyacrylamide gel decreases with fragment length under our experimental conditions and it varies along the migration.     

Estimation of polyacrylamide gel pore size from Ferguson plots of normal and anomalously migrating DNA fragments. I. Gels containing 3% N,N'-methylenebisacrylamide

The mobilities of normal and anomalously migrating DNA fragments were determined in polyacrylamide gels of different acrylamide concentrations, polymerized with 3% N,N'-methylenebisacrylamide as the crosslinker. The DNA samples were a commercially available 123-bp ladder and two molecular weight ladders containing multiple copies of two 147-base pair (bp) restriction fragments, obtained from the MspI digestion of plasmid pBR322. One of the 147 bp fragments is known to migrate anomalously slowly in polyacrylamide gels. Ferguson plots were constructed for all multimer ladders, using both absolute mobilities and relative mobilities with respect to the smallest DNA molecule in each data set. If the retardation coefficients were calculated from the relative mobilities, and the rms radius of gyration was used as the measure of DNA size, the Ogston equations were obeyed and the gel fiber parameters could be calculated. The effective pore sizes of the gels were estimated from the gel concentration at which the mobility of a given DNA molecule was reduced to one-half its mobility at zero gel concentration. The estimated pore radii ranged from approximately 130 nm for 3.5% gels to approximately 70 nm for 10.5% gels. These values are much larger than the pore sizes previously determined for the polyacrylamide matrix.     

FLUORESCENCE OF COMPLEXES OF QUINACRINE MUSTARD WITH DNA—II. DEPENDENCE ON THE STAINING CONDITIONS

Abstract—Measurements of time-resolved fluorescence of Micrococcus lysodeikticus DNA stained with quinacrine mustard are presented in this paper. In particular, the dependence of the fluorescence decay law both on the degree of DNA saturation by the dye and on the ionic strength of the solutions is investigated. It is shown that the quinacrine mustard molecules intercalating the DNA base pairs exhibit two different fluorescence behaviors which make them either donors or acceptors in a Forster energy transfer mechanism. A mathematical formulation of this mechanism allows us to interpret all the experimental fluorescence decay curves. In addition, some new properties of the external binding mode are obtained.     

Sequence‐specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid‐DNA) in capillary and microfluidic electrophoretic separations

Reversible noncovalent but sequence‐dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite‐modified oligonucleotides has enabled sequence‐specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self‐assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid‐DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence‐dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information‐processing systems.     

Nanometal surface energy transfer in optical rulers, breaking the FRET barrier

Optical-based distance measurements are essential for tracking biomolecular conformational changes, drug discovery, and cell biology. Traditional Forster resonance energy transfer (FRET) is efficient for separation distances up to 100 A. We report the first successful application of a dipole-surface type energy transfer from a molecular dipole to a nanometal surface that more than doubles the traditional Forster range (220 A) and follows a 1/R(4) distance dependence. We appended a 1.4 nm Au cluster to the 5' end of one DNA strand as the energy acceptor and a fluorescein (FAM) to the 5' end of the complementary strand as the energy donor. Analysis of the energy transfer on DNA lengths (15, 20, 30, 60bp), complemented by protein-induced DNA bending, provides the basis for fully mapping the extent of this dipole surface type mechanism over its entire usable range (50-250 A). Further, protein function is fully compatible with these nanometal-DNA constructs. Significantly extending the range of optical based methods in molecular rulers is an important leap forward for biophysics.     

Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration

The electrophoretic mobilities of curved and normal DNA molecules of the same size have been measured in polyacrylamide gels containing various acrylamide concentrations and cross-linker ratios. Ferguson plots were constructed to extrapolate the observed mobilities to zero gel concentration. The DNA samples were two 147-bp restriction fragments, called 12A and 12B, obtained from the MspI digestion of plasmid pBR322, and head-to-tail multimers of each fragment. Fragment 12A is stably curved and migrates anomalously slowly in polyacrylamide gels; fragment 12B has the conformation of normal DNA and migrates with normal electrophoretic mobilities. The extrapolated mobilities of the curved fragment 12A and its multimers at zero gel concentration are lower than the extrapolated mobilities of the normal fragment 12B and its multimers. The free solution mobility of the curved fragment 12A, measured by CE, is also lower than that of the normal fragment 12B. The combined results indicate that the extrapolated mobilities observed for curved DNA molecules at zero polyacrylamide gel concentration reflect the intrinsic differences in their free solution mobilities.     

Enhanced mixing in polyacrylamide gels containing embedded silica nanoparticles as internal electroosmotic pumps

Many biosensors, including those based on sensing agents immobilized inside hydrogels, suffer from slow response dynamics due to mass transfer limitations. Here we present an internal pumping strategy to promote convective mixing inside crosslinked polymer gels. This is envisioned as a potential tool to enhance biosensor response dynamics. The method is based on electroosmotic flows driven by non-uniform, oscillating electric fields applied across a polyacrylamide gel that has been doped with charged colloidal silica inclusions. Evidence for enhanced mixing was obtained from florescence recovery after photobleaching (FRAP) measurements with fluorescein tracer dyes dissolved in the gel. Mixing rates in silica-laden gels under the action of the applied electric fields were more than an order of magnitude faster than either diffusion or electrophoretically driven mixing in gels that did not contain silica. The mixing enhancement was due in comparable parts to the electroosmotic pumping and to the increase in gel swelling caused by the presence of the silica inclusions. The latter had the effect of increasing tracer mobility in the silica-laden gels.     

Stern-Volmer modeling of steady-state Forster energy transfer between dilute, freely diffusing membrane-bound fluorophores

Two different metrics are used to assess Forster resonance energy transfer (FRET) between fluorophores in the steady state: (i) acceptor-quenching of donor fluorescence E (also known as transfer efficiency) and (ii) donor-excited acceptor fluorescence F(A) (Dex). While E is still more widely used, F(A) (Dex) has been gaining in popularity for practical reasons among experimentalists who study biomembranes. Here, for the special case of membrane-bound fluorophores, we present a substantial body of experimental evidence that justifies the use of simple Stern-Volmer expressions when modeling either FRET metric under dilute-probe conditions. We have also discovered a dilute-regime correspondence between our Stern-Volmer expression for E and Wolber and Hudson's series approximation for steady-state Forster quenching in two dimensions (2D). This novel correspondence allows us to interpret each of our 2D quenching constants in terms of both (i) an effective Forster distance and (ii) two maximum acceptor-concentration limits, each of which defines its own useful experimental regime. Taken together, our results suggest a three-step strategy toward designing more effective steady-state FRET experiments for the study of biomembranes.     

Mobility, diffusion and dispersion of single-stranded DNA in sequencing gels

Electrophoresis of single-stranded DNA in denaturing polyacrylamide gels is presently a standard procedure for the sequencing of DNA fragments. A thorough understanding of the factors that determine the resolution of DNA fractionated in polyacrylamide gels is necessary to optimize the performance of DNA sequencers. Significant research on the mobility of double-stranded (ds)DNA molecules in agarose and polyacrylamide gels has been performed, and the phenomenon of band broadening of single-stranded (ss)DNA fragments in DNA sequencing gels has received attention only recently. In this paper, we present a detailed study of mobility, diffusion and dispersion of ssDNA in sequencing gels as a function of molecular size, gel concentration and electric field strength. DNA mobility is shown to be essentially independent of electric field in the range of 0-60 V/cm. The band broadening is greatly enhanced in the presence of an electric field and the dispersion coefficient (DE) can be an order of magnitude higher than the field-free diffusion coefficient. The measured migration parameters approximately follow the predictions of the biased reptation including fluctuations (BRF) theory. However, deviations due to nonidealities of the separation conditions are observed. The measured migration parameters can be used to optimize the performance of separation systems.     

Rhodamine B nanocrystals: elaborations,characterizations and functionalizations for biosensing applications

A biological sensor based on fluorescent organic nanocrystals (NCs) of Rhodamine B grown in sol–gel thin films was developed. The original signalization function is based on fluorescence contrasts of NCs, which exhibit a simple fluorescence signature, good photostability and higher fluorescence intensities compared to dispersed dye molecules. Thanks to a well-controlled dissolution process of the sol–gel surface, accurately followed by atomic force microscopy, the NCs were made emerging just a few nanometers above the silicate thin films to be directly accessible to biological macromolecules. Thus, hairpin-shaped DNA, functionalized by a probe-molecule (DNA probe), has been grafted onto nanocrystal surfaces leading to a fluorescence quenching by Forster resonance energy transfer. After hybridization of these hairpin-shaped DNA probes with their complementary DNA-target, the molecular probes and NCs are pulled apart, stopping thus the quenching. This “turn-on” of nanocrystal fluorescence allows thus a label-free DNA detection. The preparation methodology of the signalization function, its functionalization by hairpin-shaped DNA probes and first DNA-sensor experiments are presented.     

Fluorescence Energy Transfer Study of Interstrand DNA Cross-linking Caused by Rigid Bisintercalator

The intermolecular cross-linking of DNA with a rigid bisintercalator, 1,4-bis(( N -methylquinolinium-4-yl)vinyl)benzene (pMQVB) has been studied using fluorescence resonance energy transfer (FRET), fluorescence anisotropy measurements, and dynamic fluorescence microscopy. Short DNA duplexes, single-labeled with fluorescein (donor) and x-rhodamine (acceptor), were used as energy transfer partners. Due to the quenching effect of pMQVB on the emission of both fluorescein and x-rhodamine, the energy transfer was monitored using the corrected Stern-Volmer plots. The cross-linking ability of pMQVB depended on the ligand structure; the planar E , E isomer cross-linked DNA contrary to the non-planar E , Z isomer. Dynamic fluorescence microscopy observation also demonstrated the ability of pMQVB to cross-link large T4 DNA molecules.     

Effect of linear polymer additives on the electroosmotic characteristics of agarose gels in ultrathin-layer electrophoresis.

Electroosmotic properties of agarose gels with low, medium, high and super high electroendosmosis (EEO) were evaluated based on the apparent electric field mediated mobility of a neutral, fluorescent marker under constant field strength using ultrathin-layer separation configuration. Electroosmotic flow mobility values were measured in different gel concentrations and also in the absence and the presence of various linear polymer additives. Under ultrathin-layer separation conditions, a slight decrease in electroosmotic flow mobility was observed with increasing agarose gel concentration of 1 to 3% for all agarose gels investigated. When linear polymer additives, such as linear polyacrylamide, hydroxyethyl cellulose or polyethylene oxide were added to 1% low electroendosmosis agarose gel, significant reduction of the electroosmotic flow properties were observed with increasing additive concentration. Effect of the intrinsic electroosmotic properties of the various electroendosmosis agaroses on the apparent mobilities and separation performance of double-stranded DNA fragments during automated ultrathin-layer agarose gel electrophoresis was also investigated.     

Estimation of polyacrylamide gel pore size from Ferguson plots of linear DNA fragments. II. Comparison of gels with different crosslinker concentrations, added agarose and added linear polyacrylamide.

The mobilities of various DNA fragments in two normally migrating molecular weight ladders were studied in polyacrylamide gels containing different concentrations of the crosslinker N,N'-methylenebisacrylamide (Bis). The acrylamide concentration ranged from 2.5-10.5%T (w/v); the Bis concentration ranged from 0.5-10%C (w/w), with respect to total acrylamide. Ferguson plots were constructed for each of the DNA fragments in gels of each composition. The Ferguson plots of the different multimers in each molecular weight ladder were nearly parallel in gels containing 0.5-3%C, converged close to a common intercept at zero gel concentration in gels containing 4%C, and crossed at approximately 1.5%T in gels containing 5 and 10%C. If the mobilities observed for the different DNA fragments at zero gel concentration were also extrapolated to zero DNA molecular weight, a common limiting mobility was observed in gels of all crosslinker concentrations. This limiting mobility was approximately equal to the free solution mobility of DNA. The effective pore radius of each gel was estimated from Ferguson plots based on relative mobilities, using the mobility of the smallest DNA fragment in each molecular weight ladder as the reference mobility. The calculated gel pore radii ranged from 142 nm to 19 nm, respectively, for gels containing 4.6%T, 1.5%C, and 10.5%T, 5 or 10%C. These pore radii are an order of magnitude larger than previously accepted values, but are consistent with scanning electron microscope measurements (Rüchel, R., et al., J. Chromatogr. 1978, 42, 77-90).(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence properties of fluorescein, tetramethylrhodamine and Texas Red linked to a DNA aptamer

We report the picosecond time-scale fluorescence dynamics of a dye-labeled DNA oligonucleotide or "aptamer" designed to bind specifically to Immunoglobulin E. Comparison of the photophysics of Texas Red (TR), fluorescein and 5'-carboxytetramethylrhodamine (TAMRA)-labeled aptamers reveals surprising differences with significant implications for measurements of oligonucleotide structure and dynamics. The fluorescence decay of the TR-aptamer is a simple single exponential with a weak temperature dependence. The fluorescence decay of the fluorescein-aptamer (fl-aptamer) is pH dependent and displays a complex temperature dependence with significant changes on melting of the aptamer tertiary structure. Despite its similarities to TR, TAMRA is strongly quenched when conjugated to the aptamer and displays complex fluorescence kinetics best described by a distributed rate model. Using the maximum entropy method, we have discovered two highly temperature-dependent fluorescence lifetimes for the TAMRA-aptamer. One of these lifetimes is similar to that of free TAMRA and displays the same temperature dependence. The other lifetime is quenched and displays a temperature dependence characteristic of a charge transfer reaction. These data set TR apart as an attractive alternative to TAMRA and fluorescein for studies such as fluorescence polarization and fluorescence resonance energy transfer, where environmental sensitivity of the probe is not desired.     

Sieving of ionic constituents across moving boundaries in gel electrophoresis

The representative beta-hydroxyethylmorpholinium-chloride-bicinate moving boundary with a trailing ion net mobility relative to Na+ of 0.41, detected by precipitation of chloride with silver nitrate, exhibits a decreasing chloride mobility at increasing polyacrylamide gel concentrations from 3.5 to 45%T, 5%CBis. This decrease, largely due to an increase of field strength at constant current, is described by a convex* plot of log (mobility) vs. %T (Ferguson plot) and signifies that chloride/bicinate are sieved by the gel. In agarose gels, the same plot of mobility vs. gel concentration is constant below 7% gel concentration, since in those gels field strength and migration rate remain the same within that gel concentration range. Both in polyacrylamide and in agarose gels the displacement rate of the chloride-bicinate boundary as a function of the time of electrophoresis or distance migrated remains invariant within 15%. The plot of log (mobility) vs. gel concentration extrapolated to 0%T is 5.85 and 5.41 (10(-5) cm2s-1V-1) for polyacrylamide and for agarose (SeaKem HGT-P,FMC) gels, respectively. The slightly decreased mobility intercept at 0%T for agarose is presumably due either to the electroendosmotic properties of agarose HGT-P and/or failure to Sufficiently take into account the flattening of the Ferguson plot in the polyacrylamide concentration range below 3% in which a transition from a gel to a fluid (sol) medium takes place.     

Studies on the interaction mechanism between CXS and papain by spectroscopic and molecular modeling methods

We have studied the interaction between cefuroxime sodium (CXS) and papain at different temperatures by a fluorescence method, and confirmed that the mechanism of fluorescence quenching of CXS to papain is mainly static quenching. We also determined the binding constant K. Based on the thermodynamic functions at different temperatures, the results show that the major forces between CXS and papain are van der Waals’ forces and H bond. According to the Forster non-radiation energy transfer mechanism, we determined the binding distance between CXS and papain, and studied the confirmation effect of CXS to papain by synchronous fluorescence and UV–Vis spectroscopy. Molecular simulations show that the binding types of CXS and papain are van der Waals’ forces, hydrophobic interaction, and H-bond.     

Characteristics of migration patterns of DNA oligomers in gels and the relationship to the question of intrinsic DNA bending

We have developed a methodology that is capable of quantitatively describing the electrophoretic mobility patterns of oligomeric B-DNA through polyacrylamide gels (PAG) in the presence of varying concentration of the organic solvent 2-methyl-2,4-pentanediol (MPD), used routinely to induce DNA crystallization. The model includes the ion atmosphere and its polarization, electrostatic excluded volume, hydrodynamic interactions, and fluctuation effects that characterize the overall size of the migrating polyion. Using this model, and by critically examining the mobility patterns of linear random-sequence B-DNA molecules in PAG as a function of MPD, we address the question of the discrepancy between current models used to explain the molecular origins of A-tract-induced DNA bending. Direct analysis of the mobility of B-DNA oligomers on PAG, and comparison to the mobility of A-tract-containing oligomers, shows a significant apparent effect of MPD on the mobility of generic B-DNA sequences, which is larger than the effect on A-tract-containing oligomers. The effect is chain-length dependent, especially at lower MPD concentration. Thus, the apparent reduction in gel mobility, as a function of MPD, is not unique to A-tract regions or A-tract-containing molecules. However, our analysis suggests that MPD molecules are probably excluded from the surface of both B-DNA and A-tract molecules. This is supported by circular dichroism studies on A-tract and B-DNA molecules in solutions containing various MPD concentrations.     

Fluorescence resonance energy transfer from a bio-active imidazole derivative 2-(1-phenyl-1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenol to a bioactive indoloquinolizine system

Novel donor imidazole derivative, 2-(1-phenyl-1H-imidazo [4,5-f][1,10]phenanthrolin-2-yl)-phenol (PIPP) was screened as highly sensitive chemisensor for transition metal ions and it can be used as a "multi-way" optically switchable material. Solvatochromic effects on the fluorescence behaviour of PIPP were studied in different solvents. The fluorescence of PIPP was highly sensitive to both the polarity as well as protic nature of the solvent. Fluorescence (Forster) resonance energy transfer (FRET) process from PIPP to a potent bioactive indoloquinolizine molecule was studied and it is argued that long-range dipole-dipole interaction is operating for the energy transfer mechanism. The energy transfer efficiency (E) and the distance between the acceptor and the donor (r(0)) have been determined.     

丝裂霉素C与牛血清蛋白结合作用的研究

结合光谱法与微量热法研究了水溶液中丝裂霉素C与牛血清白蛋白分子间的结合反应,测定了反应的结合常数K~A,结合位点数n及热力学函数△~rG~m,△~rH~m和△~rS~m,并确定了分子间作用力性质;依据Forster非辐射能量转移机制,确定了授体-受体间的结合距离和能量转移效率;采用同步荧光技术考察了丝裂霉素C对牛血清白蛋白构象的影响。