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Non-natural peptides with structures and functions similar to natural peptides have emerged lately in biomedical as well as nanotechnological contexts. They are interesting for pharmaceutical applications since they can adopt structures with new targeting potentials and because they are generally not prone to degradation by proteases. We report here a new set of peptidomimetics derived from -peptides, consisting of n units of a 1,5-disubstituted 1,2,3-triazole amino acid (5Tzl). The monomer was prepared using ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) chemistry using [RuCl2Cp*](x) as the catalyst, allowing for simpler purification and resulting in excellent yields. This achiral monomer was used to prepare peptide oligomers that are water soluble independent of peptide chain length. Conformational analysis and structural investigations of the oligomers were performed by 2D NOESY NMR experiments, and by quantum chemical calculations using the B97X-D functional. These data indicate that several conformations may co-exist with slight energetic differences. Together with their increased hydrophilicity, this feature of homo-5Tzl may prove essential for mimicking natural peptides composed of -amino acids, where the various secondary structures are achieved by side chain effects and not by the rigidity of the peptide backbone. The improved synthetic method allows for facile variation of the 5Tzl amino acid side chains, further increasing the versatility of these compounds.

Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F o F 1 ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F 1 performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

Binuclear polypyridine ruthenium compounds have been shown to slowly intercalate into DNA, following a fast initial binding on the DNA surface. For these compounds, intercalation requires threading of a bulky substituent, containing one Ru(II), through the DNA base-pair stack, and the accompanying DNA duplex distortions are much more severe than with intercalation of mononuclear compounds. Structural understanding of the process of intercalation may greatly gain from a characterisation of the initial interactions between binuclear Ru(II) compounds and DNA. We report a structural NMR study on the binuclear Ru(II) intercalator Λ,Λ-B (Λ,Λ-[μ-bidppz(bipy)4Ru2](4+); bidppz = 11,11'-bis(dipyrido[3,2-a:2',3'-c]phenazinyl, bipy = 2,2'-bipyridine) mixed with the palindromic DNA [d(CGCGAATTCGCG)]2. Threading of Λ,Λ-B depends on the presence and length of AT stretches in the DNA. Therefore, the latter was selected to promote initial binding, but due to the short stretch of AT base pairs, final intercalation is prevented. Structural calculations provide a model for the interaction: Λ,Λ-B is trapped in a well-defined surface-bound state consisting of an eccentric minor-groove binding. Most of the interaction enthalpy originates from electrostatic and van der Waals contacts, whereas intermolecular hydrogen bonds may help to define a unique position of Λ,Λ-B. Molecular dynamics simulations show that this minor-groove binding mode is stable on a nanosecond scale. To the best of our knowledge, this is the first structural study by NMR spectroscopy on a binuclear Ru compound bound to DNA. In the calculated structure, one of the positively charged Ru(2+) moieties is near the central AATT region; this is favourable in view of potential intercalation as observed by optical methods for DNA with longer AT stretches. Circular dichroism (CD) spectroscopy suggests that a similar binding geometry is formed in mixtures of Λ,Λ-B with natural calf thymus DNA. The present minor-groove binding mode is proposed to represent the initial surface interactions of binuclear Ru(II) compounds prior to intercalation into AT-rich DNA.

In a majority of living organisms, F o F 1 ATP synthase performs the fundamental process of ATP synthesis. Despite the simple net reaction formula, ADP + P i → ATP + H 2 O, the detailed step-by-step mechanism of the reaction yet remains to be resolved owing to the complexity of this multisubunit enzyme. Based on quantum mechanical computations using recent high resolution X-ray structures, we propose that during ATP synthesis the enzyme first prepares the inorganic phosphate for the γ P-O ADP bond-forming step via a double-proton transfer. At this step, the highly conserved αS344 side chain plays a catalytic role. The reaction thereafter progresses through another transition state (TS) having a planar ion configuration to finally form ATP. These two TSs are concluded crucial for ATP synthesis. Using stepwise scans and several models of the nucleotide-bound active site, some of the most important conformational changes were traced toward direction of synthesis. Interestingly, as the active site geometry progresses toward the ATP-favoring tight binding site, at both of these TSs, a dramatic increase in barrier heights is observed for the reverse direction, i.e., hydrolysis of ATP. This change could indicate a “ratchet” mechanism for the enzyme to ensure efficacy of ATP synthesis by shifting residue conformation and thus locking access to the crucial TSs.

DNA strand exchange is of great importance in vivo for genetic recombination and DNA repair. The detailed mechanism of strand exchange is not understood in full detail despite extensive studies. Simplistic model systems in which molecular parameters can be varied independently are therefore of interest to study. We chose the surface of a positively charged liposome as a scaffold, which we recently demonstrated to be able to catalyze the exchange of fully complementary DNA oligonucleotides. We here study how single base pair mismatches affect the rate of strand exchange on the liposome surface. Interestingly, the rate of the exchange does not simply follow the stability of the duplex in solution, as determined by melting temperatures, but also depends sensitively on the position of the mismatch. For duplexes with similar melting temperatures, the exchange is much faster for a mismatch close to the end than for a mismatch in the middle of the sequence. Our results suggest that the single strands are stabilized by the liposome surface; therefore, the duplex is fraying more and the DNA opens up in a zipperlike fashion on the surface, increasing the probability of strand exchange. We also show that the competition between greater stability (higher T m in solution) and higher concentration is important for the final composition of the duplex when a large excess of single strands is added to a complementary double-stranded DNA. Finally, the similar exchange rate constants for fully base-paired duplexes on the liposome surface when adding fully matched single strands or single strands with a mismatched base indicate that the rate is governed largely by separation of the initial duplex and not by the formation of the product duplex. © 2009 American Chemical Society.

Ruthenium complexes with dipyridophenazine (dppz) type ligands have several characteristics that make them good candidates for use as luminescence probes for hydrophobic environments. Most studies have concerned DNA intercalation, but also lipid membrane fluidity and liposome orientation have been assessed. We report here dipyridophenazine derivatives ([Ru(phen)2dppz]2+) substituted with one or two alkyl ether chains of different lengths aimed at finding the optimum substitution for a high quantum yield when bound to a phospholipid membrane bilayer. The orientation of membrane bound molecules is studied using flow linear dichroism (LD) with phospholipid vesicles as membrane models. LD, excitation anisotropy, steady state luminescence and excited-state lifetime measurements are used to quantitatively investigate the insertion and orientation of the complexes in the vesicles. All complexes are inserted with their long axis of the dppz moiety mainly parallel to the lipid chains, and the degree of orientation is comparable to that of the orientation probe retinoic acid. The ruthenium "head group" with its positive charge functions as a buoy at the water-membrane interface while the hydrophobic chain part embeds the complex down into the bilayer. The complex with two hexyl ether substituents (named D6) has the optimal chain length regarding membrane insertion and orientation, and together with the highest quantum yield, is the best luminescence membrane probe in the two series.

We here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios. Specific as well as nonspecific binding is probed using polarized-light spectroscopy combined with singular value decomposition analysis. Circular and linear dichroism data confirm binding geometries consistent with minor groove binding for both of the ligands. Interestingly, at high and intermediate ligand/DNA ratios the polyamide exhibits no significant sequence discrimination between mixed-sequence (calf thymus) and AT DNA as compared to DST. Each ligand is concluded to exhibit two different binding modes depending upon ligand/DNA ratio and nucleo-base sequence. At high binding ratios, distinct differences between the ligands are observed: circular dichroism spectra exciton effects provide evidence of bimolecular interactions of the polyamide when bound to AT-DNA, whereas no effects are seen with DST or mixed-sequence DNA. Also linear dichroism indicates that a change in binding geometry occurs at high polyamide/AT ratios, and that the effect occurs only with polyamide in contrast to DST. Since the effect is insignificant with DST, or with calf thymus DNA, it is concluded that it relates to the sizes of the ligands and the minor grooves, becoming critical in the limit of crowding.

Photophysical properties of Δ- and Λ-[Ru(phenanthroline)2dipyrido[3,2-a:2‘,3‘-c]phenazine]2+ ([Ru(phen)2DPPZ]2+) bound to poly[d(A−T)2] in the presence and absence of 4‘,6-diamidino-2-phenylindole ...

A conjugate of a triple helix forming oligonucleotide (TFO) and the Lambda and Delta enantiomers of the ruthenium diphenanthroline dipyridophenazine complex [Ru(phen)(2)dppz](2+) was synthesized. The ruthenium complex was attached to the 5'-end of the TFO through the dppz moiety. This conjugate formed a stable triple helix with the polypurine tract (PPT) sequence from HIV proviral DNA. The thermal denaturation temperature of the triplex was increased by 12 degrees C. One remarkable property of the Delta-[Ru(phen)(2)dppz](2+) complex is a strong increase in its fluorescence when it intercalates into DNA. While the fluorescence of the oligonucleotide conjugate was very weak, the formation of a duplex with a complementary sequence or of a triple helix with a target duplex resulted in a large increase in fluorescence of the Delta enantiomer. The increase in fluorescence allowed us to follow the kinetics of duplex and triplex formation by fluorescence spectrometry. In contrast, the Lambda enantiomer gave a much smaller fluorescence change when a triplex was formed, even though the stability of the triplex was comparable to that of the Delta enantiomer. The property was ascribed to intercalation of the dipyridophenazine moiety of the Delta enantiomer into DNA and subsequent threading of the ruthenium complex through the DNA double helix. Salt effects were consistent with the involvement of DNA breathing in the formation of the intercalating complex.

[Fe(1,10-phenanthroline)(3)](2+) is known to racemise in solution and DNA is known to shift the equilibrium point of the Delta/Lambda[Fe(1,10-phenanthroline)(3)](2+) mixture from 50:50 to a position favouring a slight excess of the Delta enantiomer. In this paper it is shown that DNA catalyses the racemisation reaction, presumably by providing vibrational energy to species that bind in a less favoured mode for that enantiomer. The racemisation of [Co(1,10-phenanthroline)(3)](3+), however, is not enhanced by DNA unless [Co(1,10-phenanthroline)(3)](2+) is also added to the solution. In this case, the DNA acts as a template to facilitate the Co-II reversible arrow Co-III electron transfer. Racemisation occurs while the [Co(1,10-phenanthroline)(3)](n+) is in the labile Co-II form.

A library of thrombin inhibitors has been designed using statistical molecular design. An aromatic scaffold was used, with three varied positions corresponding to three pockets at the active site of thrombin (the S-, P-, and D-pockets). The selection was performed in the building block space, and previously acquired data were included in the design procedure. The design resulted in six, four, and six building blocks for the first (S), second (P), and third (D) pockets, respectively. A second round of selection applied to the combined selected building blocks resulted in a subset of 18 compounds. The selected library was synthesized in parallel and biologically evaluated. The compounds were analyzed with respect to their inhibition (pIC(50)) of thrombin; membrane permeability, estimated by migration behavior in micellar media (CE log k') and pK(a); and specificity with respect to inhibition (K(i)) of trypsin. Multivariate QSAR studies of the responses yielded valuable results and information that could only be found using statistical molecular design in combination with multivariate analysis.

The interaction of the cellular delivery vector penetratin with a model system consisting of negatively charged phospholipid vesicles has been studied. Above a certain peptide to lipid molar ratio, the cationic oligopeptide induces vesicle aggregation. Interestingly, the aggregation is followed by spontaneous disaggregation, which may be related to membrane translocation of the peptide. Circular dichroism (CD) measurements indicate a conformational transition, from alpha -helix to antiparallel beta -pleated sheet, which is simultaneous with the aggregation process. The potential influence of spectroscopic artifacts on CD data due to the drastically increased turbidity during aggregation is discussed.

The interactions between the stereoisomers of the chiral bis-intercalator [mu -C4(cpdppz)(2)-(phen)(4)Ru-2](4+) and DNA reveal interesting dynamic discrimination properties. The two enantiomers Delta-Delta and Lambda-Lambda both form very strong complexes with calf thymus DNA with similar thermodynamic affinities. By contrast, they display considerable variations in their binding kinetics. The Delta-Delta enantiomer has higher affinity for calf thymus DNA than for [poly(dA-dT)](2), and the association kinetics of the dimer to DNA, as well as to polynucleotides, requires a multiexponential fitting function. The dissociation reaction, on the other hand, could be described by a single exponential for [poly(dA-dT)](2), whereas two exponentials were required for mixed-sequence DNA. To understand the key mechanistic steps of the reaction, the kinetics was studied at varied salt concentration for different choices of DNA and chirality of the threading complex. The enantiomers were found to have markedly different dissociation rates, the Lambda-Lambda enantiomer dissociating about an order of magnitude faster than the Delta-Delta enantiomer. Also. the salt dependence of the dissociation rate constants differed between the enantiomers, being stronger for the Lambda-Lambda enantiomer than for the Delta-Delta enantiomer. Since the dissociation reaction requires unthreading of bulky parts of the bis-intercalator through the DNA helix, a considerable conformational change of the DNA must be involved, possibly defining the rate-limiting step.

In this paper we report the DNA binding proper ties of piperazinecarbonyloxy-2-propyl derivatives of anthracene (2), pyrene (3), and phenylanthracene (4). An intercalative binding mode is found for 2 and 3, while the phenyl group of 4 prevents intercalation and leads to external binding. Preferential binding of the (S)-enantiomers is found for both anthracene 2 and pyrene 3. However, the enantiomeric preference is small, with K-(R)/K-(S) being around 0.5 for both the anthracene and the pyrene compounds. This is interpreted in terms of orientation polarity in the binding, by which any intrinsic enantioselectivity is canceled by averaging of opposite binding orientations. The affinities for poly(dA-dT)(2) (AT) are 10(4) M-1 for anthracene derivative 2, and 5 x 10(5) M-1 for pyrene derivative 2. The affinities for poly(dG-dC)(2) (GC) are I order of magnitude lower than those for AT. This is explained by steric interference of the piperazinium tail with the exocyclic amino groups of guanine in the minor groove of GC, leading to a more shallow intercalation in GC than in AT, as also indicated by significantly less negative reduced linear dichroism of the intercalator absorption bands in the GC complexes. This behavior is consistent with that observed for the previously studied achiral analogues.(1) Binding thermodynamics support the difference in binding mode between AT and GC. The binding enthalpy of the AT complexes is significantly more negative than that of the corresponding GC complexes. This indicates a larger overlap of intercalating moiety and nucleobases in the AT complexes, consistent with the linear dichroism results.

A combination of measurement of mobility and orientational dynamics of long reptating DNA in agarose gel has been used to reveal how the binding of Delta and Lambda enantiomers of the tris(phenanthroline)ruthenium(II) ion affects the flexibility of the DNA helix. The mobility data, and data over the step length and period time of the reptation cycle, obtained in the presence of the respective enantiomer, are compared with those of free DNA and with data obtained earlier on DNAs with known variations in helix flexibility. The results suggest that the Delta form induces kinks in the DNA helix while the Lambda form gives rise to a local stiffening of the helix.

Two novel substitutionally-inert diastereomeric ruthenium(II) cations of the form Lambda- and Delta-cis-beta-[Ru(RR-picchxn)(phen)](2+), where RR-picchxn is N,N'-dimethyl-N,N'-di(2-picolyl)-IR,2R-diaminocyclohexane and phen is 1,10-phenanthroline, have been studied with respect to their interactions with duplex DNA. NMR investigations show that both diastereomers bind to the oligonucleotide [d(CGCGATCGCG)](2) in the fast exchange regime and that binding predominantly takes place in the minor groove of the oligonucleotide, but that the governing interactions are significantly different for the two Delta and Lambda forms. Linear dichroism data support the latter interpretation, in that the relative orientations of cis-beta-[Ru(RR-picchxn)(phen)](2+) to calf thymus DNA also are observed to differ for the Delta and Lambda diastereomers. Interpretation of these data indicates the Lambda form to be bound with the planar phen ligand closely parallel to the DNA base-pairs, but the average orientation of the phen ligand in the Delta form deviates significantly from a parallel alignment.

Four novel piperazinylcarbonyloxyethyl derivatives of anthracene and pyrene have been prepared and investigated with respect to sequence specificity and synergism between hydrophobic and electrostatic effects upon binding to DNA. Linear and circular dichroism spectroscopy was used to assess the orientation of the aromatic chromophores relative to the nucleobases. Anthracene and pyrene derivatives 2a and 3 are both concluded to bind to homo-polynucleotide poly(dA-dT)(2) by intercalation of their aromatic moieties between base pairs, with a binding constant K-AT of 4 x 10(5) M-1 and 2 x 10(6) M-1, respectively. Significantly reduced affinities (K-GC = 3 x 10(4) M-1 and 10(5) M-1, respectively) are observed with poly(dG-dC)(2), due to less favorable interactions of the piperazinium tail in the minor groove. Base pair specificity is reflected in the binding thermodynamics, with the binding to AT being more enthalpically driven than the binding to GC. Phenyl substitution at the quaternary piperazinium site of the anthracene derivative 2b, does not affect the ratio K-AT/K-GC, but reduces the affinity for both AT End GC slightly. Moreover, the phenyl group in the 10-position of 4 prevents intercalation, and apparently, this compound binds externally to both AT and GC duplex polynucleotides. The results are discussed in terms of general features of the interactions of the intercalating and minor-groove binding molecular moieties, and their interplay with each other, with potentials for tuning specificity.

The binding conformations of single anti- and syn-BPDE-N2-dG adducts in oligonucleotides of varying base composition have been studied by induced circular dichroism (ICD) and light absorption spectroscopy. The sign of the ICD in single-stranded oligonucleotide adducts correlates with the absolute configuration of the cyclohexyl moiety of the BPDE. Adducts in oligonucleotide duplexes with UV lambdamax350 nm exhibiting a significant duplex-induced positive ICD should have a minor groove location as the predominant conformation. Those with UV lambdamax350 nm exhibiting either positive or negative contributions to the ICD should have intercalated binding as the predominant conformation. The magnitude of the ICD is dependent on the sequence context of the adducted strand and the particular BPDE-adduct isomer under study. In some cases, the results suggest structural heterogeneity. For instance, the (+)- and the (-)-trans-anti-BPDE-N2-dG adducts in duplexes where a dT flanks the lesion site exhibit weak positive ICD or negative ICD. These results reflect a bimodal conformational adduct distribution with contributions from both externally and internally located adducts. A key observation for the (+)-cis-syn-BPDE-N2-dG complexes in 5'-d(TGC) and 5'-d(CGC) sequence contexts is that the near-UV absorption spectra showed distinct bands corresponding to minor groove binding (lambdamax congruent with 346 nm) as well as intercalative binding (lambdamax congruent with 354 nm). Evidence for an equilibrium between the different modes of localization is provided by the results from the temperature dependence of the near-UV absorption and ICD characteristics of (+)-cis-syn-BPDE-N2-dG complexes in 5'-d(TGC) and 5'-d(CGC) sequence contexts, respectively.

The binding of 2,7-diazapyrene (DAP), N-methyl-2,7-diazapyrenium monocation (MDAP), and N,N‘-dimethyl-2,7-diazapyrenium dication (DMDAP) to calf thymus DNA has been studied with respect to molecular geometry and thermodynamics. It is concluded from flow linear dichroism (LD) and induced circular dichroism (CD) spectra that the three diazapyrenes bind by intercalation to alternating AT as well as GC polynucleotide duplexes, as indicated by strong interactions with the transitions of the nucleobases in conjunction with approximately perpendicular orientations of the in-plane symmetry axes relative to the DNA helix axis. The reduced LD (LDr = LD/Aiso) of the DNA complexes is characterized by marked fine structure, decreasing in the order DAP > MDAP > DMDAP. This finding is interpreted in terms of a microscopic heterogeneity associated with rotational mobility of the ligand in a tilted intercalation pocket, with the dication DMDAP having less rotational freedom than the neutral DAP has. Other distinct differe...

The binding of a mixed-sequence pentadecamer PNA (peptide nucleic acid) containing all four nucleobases to the fully complementary as well as various singly mismatched RNA and DNA oligonucleotides has been systematically investigated using thermal denaturation and BIAcore surface-interaction techniques. The rate constants for association (k(a)) and dissociation (k(d)) of the duplex formation as well as the thermal stability (melting temperature, T(m)) of the duplexes have been determined. Upon binding to PNA tethered via a biotin-linker to streptavidin at the dextran/gold surface, DNA and RNA sequences containing single mismatches at various positions in the center resulted in increased dissociation and decreased association rate constants. T(m) values for PNA x RNA duplexes are on average 4 degrees C higher than for PNA x DNA duplexes and follow quantitatively the same variation with mismatches as do the PNA x DNA duplexes. Also a faster k(a) and a slower k(d) are found for PNA x RNA duplexes compared to the PNA x DNA duplexes. An overall fair correlation between T(m), k(a), and k(d) is found for a series of PNA x DNA and PNA x RNA duplexes although the determination of k(a) seemed to be prone to artifacts of the method and was not considered capable of providing absolute values representing the association rate constant in bulk solution.

The binding of homochiral [Ru(II)(1,10-phenanthroline)(2)L](2+) complexes {where L = 1,10-phenanthroline (phen), dipyrido[3,2-a:2',3'-c]phenazine (DPPZ) or benzodipyrido[3,2-a:2',3'-c]phenazine (BDPPZ)} to poly(dT*dA-dT) triplex has been investigated by linear and circular dichroism and thermal denaturation. Analysis of the linear dichroism spectra indicates that the extended DPPZ and BDPPZ ligands lie approximately parallel to the base-pair and base-tripler planes consistent with intercalation which is also supported by strong hypochromism in the interligand absorption bands with either duplex or tripler. The spectral properties of any of the metal complex enantiomers were similar for binding to either duplex or tripler DNA, indicating that the third strand, which occupies the major groove of the template duplex, has little effect on the binding geometries and hence supports the hypothesis that the metal complexes all bind from the minor groove with the DPPZ and BDPPZ ligands intercalated but without intercalation in the case of [Ru(phen)(3)](2+). Third-strand stabilization depended on the nature of the third substituted phenanthroline chelate ligand but was not directly related to its size, with stabilizing power increasing in the order phen < BDPPZ < DPPZ. This observation further supports intercalation of the extended ligands from the minor groove of the tripler since the extended BDPPZ ligand that would protrude into the major groove of the template would have greater steric interference than DPPZ with the third DNA strand.

A stretched poly(vinyl alcohol) (PVA) film provides a unique matrix that enables also short DNA oligonucleotide duplex to be oriented and studied by linear dichroism (LD). This matrix further allows controlling DNA secondary structure by proper hydration (A or B form), and such humid films could potentially also mimic the molecular crowding in cellular contexts. However, early attempts to study intercalators and groove binders for probing DNA in PVA failed due to competitive matrix binding. Here we report the successful orientation in PVA of DNA oligonucleotide duplex hairpins with thread-intercalated binuclear complex [μ-(11,11'-bidppz)(phen)4Ru2](4+), and how LD depends on oligonucleotide sequence and metal center chirality. Opposite enantiomers of the ruthenium complex, ΔΔ and ΛΛ, were investigated with respect to enantioselectivity toward GC stretches as long as 22 bp. LD, supported by emission kinetics, reveals that threading intercalation occurs only with ΔΔ whereas ΛΛ remains externally bound, probably in either or both of the grooves of the GC-DNA. Enantioselective binding properties of sterically rigid DNA probes such as the ruthenium complexes could find applications for targeting nucleic acids, e.g., to inhibit transcription in therapeutic context such as treatment of malaria or cancer.

Linear dichroism (LD) has been used to probe the binding geometries of the diastereomeric adducts between DNA and the Delta and Lambda enantiomers of [Ru(phen)(2)L](2+) where the chelate ligand L is either dipyrido[3,2-a:2',3'-c]-phenazine (DPPZ) or its benzologue benzodipyrido[a:3,2-h:2'3'-j]phenazine (BDPPZ). By combined use of LD and emission anisotropy excitation spectra measured in highly viscous solution the absorption envelope of the metal-to-ligand charge transfer (MLCT) region has been fur the first time resolved in a complex of this type bound to DNA. The absorption can be described by four effective polarization directions (three almost orthogonal), along one of which any transition is polarized, assignments which are in good agreement with molecular orbital (INDO/S) calculations. The analysis of the LD spectrum provides the orientation of the complex in terms of several angles. Near perpendicularity relative to the DNA helix axis, found for the in-plane long and short axes of BDPPZ and DPPZ, together with extensive hypochromicity of the corresponding intraligand transitions, supports intercalation of the (B)DPPZ ligands between the DNA bases as earlier studies have indicated. MLCT transition moments that make an oblique angle to the molecular plane of the (B)DPPZ chromophores confirm this orientation and, in addition, provide strong evidence for a rotation (roll) of the complex around the pseudo-dyad axis, The roll is small (varying between 5 and 15 degrees), but significant, and has the same sign (clockwise) for both enantiomeric forms of the two complexes studied and shows only minor variations between calf-thymus DNA and alternating GC or AT homopolymer duplexes, It may reflect a property intrinsic of DNA (tilt of bases) or be a result of steric interference of the two phenanthroline ''propeller blades'' with a groove. The roll provides the first example of an angle in a DNA system that has been determined with its sign from LD spectroscopy.

Peptide nucleic acid (PNA) is a DNA analogue in which the negatively charged sugar phosphate backbone has been substituted by uncharged N-(2-aminoethyl)glycine units. The study of a PNA-DNA duplex and the corresponding DNA-DNA duplex gives a unique opportunity to compare two polyelectrolytes with virtually identical geometry but greatly different linear charge density. The results provide a basis for a study of the applicability of the Poisson-Boltzmann (PB) and counterion condensation (CC) theories. UV and circular dichroism spectroscopy as well as isothermal titration calorimetry (ITC) have been used to study the effect of different ions on the stability and conformation of PNA-DNA, PNA-PNA, and DNA-DNA duplexes having the same base sequences. Cations in general destabilize both antiparallel (N/3') and parallel (N/5') PNA-DNA duplexes whereas they stabilize the DNA-DNA duplex. Studies on the effect of monovalent salt such as NaCl on T-m were carried out over a wide range of salt concentrations (0.01 to 5 M). The decrease in the T-m of the N/3' PNA-DNA duplex with increasing ionic strength in the range of concentrations of 0.01 to 0.5 M, where electrostatic effects predominate, is explained in terms of counterion release upon duplex formation in contrast to the counterion association accompanying the formation of a DNA duplex. The uncharged PNA-PNA duplex shows no significant destabilization in this concentration range. The higher stability of the N/3' PNA-DNA compared to the DNA-DNA duplex (Delta Delta G similar to-7 kcal/mol) is ascribed to more favorable entropic contributions consistent with the counterion release that accompanies the PNA-DNA duplex formation. At high salt concentration (>1 M), where electrostatic contributions saturate, similar trends in the decrease in T-m, were observed for the three types of duplexes irrespective of their backbone charges. The destabilizing effects of a series of Na salts with various monovalent anions on N/3' PNA-DNA and PNA-PNA duplexes were found to follow the Hofmeister series, emphasizing the importance of the hydrophobic interaction between nucleobases for the stability of the PNA complexes in high salt concentration.

Homopyrimidine PNA can recognize a purine target of double-stranded DNA by local displacement of the DNA pyrimidine strand and formation of a PNA-DNA-PNA tripler. In order to elucidate the mechanism of this PNA/DNA recognition reaction we have studied the interaction of PNA-T oligomers with poly(da):poly(dT) as a model system. The PNA binding kinetics can be observed in situ by spectroscopy as a change in the circular dichroism spectrum. The overall reaction rate decreases with increasing ionic strength and proceeds in minutes at 50 mM NaCl. From the temperature dependence of the rate constants an activation energy of about 60 kJ/mol (at 50 mM NaCl) was estimated, consistent with the hypothesis that a rate limiting step could be the DNA opening frequency. As expected from the stability of a DNA double helix toward large-scale openings, also the activation energy of the strand displacement increases somewhat with increasing salt concentration. There is a nonlinear dependence of the overall rate constant of tripler formation on the PNA concentration with an exponent of 2 or somewhat higher. With PNA in excess, the CD signal shows an overshoot suggesting the presence of a third species. The CD spectrum of this intermediate indicates an ordered structure. The presence of intercalators ethidium bromide and 9-aminoacridine is found to increase the overall PNA binding rate, whereas minor groove binder DAPI and major groove binder methyl green both decrease the rate. The mechanism of strand invasion of duplex DNA by PNA is discussed in the light of these results.

Fluorescence and absorption spectroscopy, isothermal titration calorimetry, and viscosity measurements have been used to characterize the interaction of Delta and Lambda [Ru(phen)(2)DPPZ](2+) with calf thymus DNA. The method of continuous variations revealed two distinct binding stoichiometries for both Delta- and Lambda-DPPZ, corresponding to 0.7 and 3 mol of base pair/mol of ligand. Binding isotherms were obtained for the two enantiomers, both of which show strong binding to DNA, with K = 3.2 x 10(6) M(-1) bp and 1.7 x 10(6) M(-1) bp for the Delta and Lambda isomers, respectively, at 25 degrees C in solutions containing 50 mM NaCl. Titration calorimetry gave Delta H values of +0.3 kcal mol(-1) for Delta-DPPZ and +2.9 kcal mol(-1) for Lambda-DPPZ for their interaction with DNA. These small positive enthalpies, which were confirmed using thermal difference spectroscopy, indicated that the binding of these compounds to DNA is entropically driven. An enthalpy of +2.5 kcal mol(-1) was obtained for the binding of the parent compound, tris(phenanthroline)-Ru(II), to DNA. Titration of all three compounds into buffer gave a nonnegligible heat of dilution. The salt dependence of the binding constant was examined for both isomers. The slope SK = (delta logK/delta log[Na+]) was found to be 1.9 and 2.1 for the Delta and Lambda isomers, respectively. By using polyelectrolyte theory to interpret the observed salt dependence of the equilibrium constant, it can be shown that there is a significant nonelectrostatic contribution to the binding constant. Relative viscosity experiments showed that both Delta- and Lambda-DPPZ increase the length of rod-like DNA, in a manner consistent with binding by classical intercalation. Fluorescence energy transfer experiments provided additional evidence for the intercalation of Delta- and Lambda-[Ru(phen)(2)DPPZ](2+) into DNA.

Opposite enantiomers of [Ru(phenanthroline)(3)](2+) affect the persistence length of DNA differently, a long speculated effect of helix kinking. Our molecular dynamics simulations confirm a substantial change of duplex secondary structure produced by wedge-intercalation of one but not the other enantiomer. This effect is exploited by several classes of DNA operative proteins.

Catalysts are agents that by binding reactant molecules lower the energy barriers to chemical reaction. After reaction the catalyst is regenerated, its unbinding energy recruited from the environment, which is associated with an inevitable loss of energy. We show that combining several catalytic sites to become energetically and temporally phase-shifted relative to each other provides a possibility to sustain the overall reaction by internal 'energy recycling', bypassing the need for thermal activation, and in principle allowing the system to work adiabatically. Using an analytical model for superimposed, phase-shifted potentials of F-1-ATP synthase provides a description integrating main characteristics of this rotary enzyme complex.

Peptide nucleic acid (PNA) oligomers where one of the repeating backbone units is extended with a methylene group to either N-(2-aminoethyl)-beta-alanine or N-(3-aminopropyl)glycine were prepared. Alternatively, the linker to the nucleobase was extended from methylenecarbonyl to ethylenecarbonyl. The thermal stability of the hybrids between these PNA oligomers and complementary DNA oligonucleotides was significantly lower than that of the corresponding complexes involving unmodified PNA. However, the sequence selectivity was retained. Thymidyl decamers with all N-(2-aminoethyl)-beta-alanine or N-(3-aminopropyl)glycine backbones were prepared and shown to be unable to hybridize to the complementary (dA)(10) oligonucleotides, whereas a PNA decamer containing only ethylenecarbonyl linkers between the nucleobases and the N-(2-aminoethyl)glycine backbone showed weak but sequence-specific affinity for complementary DNA. All hybrids involving homopyrimidine PNA oligomers exhibited (PNA)(2)/DNA stoichiometry, whereas mixed-sequence PNA oligomers formed PNA/DNA duplexes. The preferred binding direction between the modified PNA and DNA in the duplex motifs was antiparallel, as previously reported for complexes involving unmodified PNA.

The nucleic acid analogues PNA (peptide nucleic acids) hybridize with DNA of complementary sequence. The solution structures of two PNA-DNA duplexes, H-(GCTATGTC)-NH2.d(GACATAGC) and H-(GTAGATCACT)-NH2.d(AGTGATCTAC), have been studied by H-1 NMR. It was found that the PNA-DNA hybrids are base paired by hydrogen bonds, most likely of the Watson-Crick type. From two-dimensional NOESY and COSY results it is concluded that the DNA strand in the PNA-DNA complex adopts a B-like structure with the deoxyribose sugars in the C2'-endo conformation.

The modes of binding of the phenothiazinium dye methylene blue (1) to alternating and nonalternating polynucleotides and to calf thymus (CT) DNA have been characterized using linear dichroism (LD) and circular dichroism (CD) spectroscopy. With the polynucleotide [poly(dG-dC)](2) the interaction at low binding ratios is shown to be purely intercalative and the binding mode is insensitive to changes in ionic strength. The observed CD spectrum is bisignate, which may be due to intercalation at the different base-pair steps (5'G-C3' and 5'C-G3'), giving rise to CD signals of different sign and shape, By contrast, a single intercalative binding mode with the alternating AT polynucleotide [poly(dA-dT)](2) is likely only at very low ionic strength; at high ionic strength (200 mM phosphate, pH 6.9), a second binding mode is also manifest which is attributed to groove binding of the dye. The absorption and linear dichroism spectroscopic features of the methylene blue/CT-DNA (42% GC) complex reflect those of complexes of the dye with both [poly(dA-dT)](2) and [poly(dG-dC)](2); the circular dichroism spectrum of the methylene blue/CT-DNA complex and its variation with ionic strength reflect the complexity of even this simple system where numerous possible binding sites exist. Comparative binding to the nonalternating polynucleotides poly(dA).poly(dT) and poly(da).poly(dC), which each possess only one base-pair step, was also examined. On the basis of the combined LD and CD evidence, it is proposed that the dye is loosely bound with poly(dA).poly(dT), probably in the major groove, and intercalated with poly(dG).poly(dC).

The DNA interaction of derivatives of ellipticine with heterocyclic ring systems with three, four, or five rings and a dimethylaminoethyl side chain was studied. Optical spectroscopy of drug complexes with calf thymus DNA, poly [(dA-dT) · (dA-dT)], or poly [(dG-dC) · (dG-dC)] showed a 10 nm bathochromic shift of the light absorption bands of the pentacyclic and tetracyclic compounds upon binding to the nucleic acids, which indicates binding by intercalation. For the tricyclic compound a smaller shift of 1–3 nm was observed upon binding to the nucleic acids. Flow linear dichroism studies show that the geometry of all complexes is consistent with intercalation of the ring system, except for the DNA and poly [(dG-dC) · (dG-dC)] complexes of the tricyclic compound, where the average angle between the drug molecular plane and the DNA helix axis was found to be 65°. One-dimensional 1H-nmr spectroscopy was used to study complexes between d(CGCGATCGCG)2 and the tricyclic and pentacyclic compounds. The results on the pentacyclic compound show nonselective broadening due to intermediate chemical exchange of most oligonucleotide resonances upon drug binding. The imino proton resonances are in slow chemical exchange, and new resonances with upfield shifts approaching 1 ppm appear upon drug binding, which supports intercalative binding of the pentacyclic compound. The results on the tricyclic compound show more rapid binding kinetics and very selective broadening of resonances. The data suggest that the tricyclic compound is in an equilibrium between intercalation and minor groove binding, with a preference to bind close to the AT base pairs with the side chain residing in the minor groove. © 1994 John Wiley & Sons, Inc.

Utilizing linear dichroism (LD), circular dichroism (CD), and fluorescence energy transfer, the binding geometries of a series of Co(3+)-porphyrins and their free ligands were examined. The compounds studied were Co-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) and its free ligand (H2-TMPyP), Co-meso-tetrakis(N-n-butylpyridinium-4-yl)porphyrin (CoTBPyP) and its free ligand (H2TBPyP), and Co-meso-tetrakis(N-n-octylpyridinium-4-yl)porphyrin (CoTOPyP). The two non-metalloporphyrins exhibit negative LD, having angles of roughly 75 degrees relative to the DNA helix axis. They also display negative CD and a significant contact energy transfer from the DNA bases. On the other hand, the three metalloporphyrins display orientation angles of roughly 45 degrees between the porphyrin plane and the helix axis of DNA. Furthermore, they exhibit positive CD and no contact energy transfer from DNA bases. These observations show that the metalloporphyrins are not intercalated whereas non-metalloporphyrins having four freely rotating meso-aryl groups intercalate between the base pairs of DNA. In the presence of KHSO5, the cobalt porphyrins cleave closed circular PM2 DNA in a single strand manner, i.e., a single activation event on the porphyrin leads to a break in one of the DNA strands. A kinetic analysis of the cleavage data revealed that cleavage rates are in the order CoTMPyP > CoTBPyP > CoTOPyP with the difference being due to different DNA affinities rather than differences in cleavage rate-constants. Based on these and earlier observations, the metalloporphyrins appear bound to a partially melted region of DNA.

Complexes of 4',6-diamidino-2-phenylindole (DAPI) with [poly(dG-dC)]2 and [poly(dI-dC)]2 were studied by optical spectroscopic techniques including linear dichroism (LD), circular dichroism (CD), and fluorescence measurements. The aim was to investigate the importance of the exocyclic amino group of guanine that protrudes into the minor groove of [poly(dG-dC)]2 but is absent in [poly(dI-dC)]2. When bound to [poly(dG-dC)]2, DAPI exhibits a negative, and strongly wavelength-dependent, reduced linear dichroism (LDr) in the DAPI absorption region, a weak positive CD, and a fluorescence behavior that is similar to that of free DAPI with accessibility to quenching by the aqueous solvent. These spectroscopic properties have been interpreted in terms of a major-groove binding geometry by Kim et al. (Kim, S. K.; Eriksson, S.; Kubista, M.; Norden, B. J. Am. Chem. Soc. 1993,115,3441-3447). By contrast, when bound to [poly(dI-dC)]2, DAPI exhibits a strong positive CD in the 300-420-nm region, a positive (wavelength-independent) LD(r), a strong increase of the fluorescence intensity, and shielding to added quencher. These spectroscopic properties closely resemble those of the DAPI-[poly(dA-dT)]2 complex, in which DAPI is situated deep in the minor groove of the polynucleotide. We conclude that the major groove binding geometry of DAPI when complexed with [poly(dG-dC)]2 is a result of a decreased affinity to the minor groove of [poly(dG-dC)]2 due to steric hindrance and decreased electronegative attraction caused by the amino group of guanine.

The Z form of poly [d(G-m5C)2], in presence of Mg2+ ion, is found to be transformed into B form upon interaction with 4',6-diamidino-2-phenylindole (DAPI). The Z --> B transformation is complete at a mixing ratio of about 0.07 DAPI per DNA base pairs, i.e., each DAPI molecule may be related to the conversion of 6-7 base pairs. An interaction between DAPI and poly [d(G-m5C)2] in its Z form at low drug: DNA ratios is suggested from optical dichroism and time-resolved luminescence anisotropy results. The spectroscopic behaviour of DAPI indicates that the Z conformation of DNA does not provide normal binding sites for DAPI, such as groove or intercalation sites, but that the initial association may be of external nature. (C) 1993 John Wiley & Sons, Inc.

We have characterized the interaction of 4',6-diamidino-2-phenylindole (DAPI) with a series of DNA oligonucleotides containing a varying number of contiguous alternating AT base pairs in the center of a GC-stretch. The binding affinity of DAPI to the oligonucleotides increases with the number of contiguous AT base pairs. When entropy effects are taken into account, the microscopic affinity reaches a maximum value when the oligonucleotides contain three AT base pairs. Three is also the minimum number of base pairs that provides a binding site with all the characteristics of binding observed with DNA and [poly(dA-dT)]2 at low binding ratios. With none or only a single AT base pair in the center of a GC dodecamer, the spectral properties of bound DAPI are very similar to those of DAPI bound to [poly(dG-dC)]2. When DAPI is complexed with an oligonucleotide containing two AT base pairs, the absorption and fluorescence properties of DAPI closely resemble those of DAPI complexed with [poly(dG-dC)]2, while the circular dichroism spectrum resembles that of DAPI bound to [poly(dA-dT)]2.

The binding of an eightmer of peptide nucleic acid, H-T8-Lys-NH2 (=PNA-T8), to a polynucleotide, poly(dA), was studied by flow linear dichroism (LD) and circular dichroism (CD) spectroscopy. Whereas the single stranded DNA, due to its high flexibility, does not display any measurable LD signal when subjected to shear flow, the complex with PNA does. A titration shows that saturation occurs at a stoichiometry of two PNA thymine bases per DNA adenine base, indicating the formation of a triplex PNA2-DNA complex. The persistence length of the adduct remains small up to relatively high stoichiometries (above 1:1 T:A) indicating that no significant amounts of PNA:DNA duplex are formed. Instead triplex stretches seem to form surrounded by flexible parts of single stranded poly(dA). Upon approaching the stoichiometry 2:1 of T:A the LD increases dramatically demonstrating that the stiffness of the PNA-DNA triplex arises from base-base contacts preventing bending of the chain. It is also inferred that the main stiffness of duplex DNA very probably has a similar origin and is not primarily a result of the increased phosphate-phosphate repulsion. Circular dichroism spectra support the conclusion that a triplex is formed as the only PNA-DNA complex and that it is a right-handed helix. The wavelength dependence of the reduced linear dichroism shows that the inclination of the bases from perpendicularity relative to the helix axis is small. The base conformation of the poly(dA)[PNA-T8]2 triplex is very similar to that of the conventional poly(dA)[poly(dT)]2 triplex.

Linear dichroism (LD) spectroscopy and steady-state as well as time-resolved luminescence spectroscopy have been used to investigate the interaction of the DELTA and LAMBDA enantiomers of Ru(phen)2DPPZ2+ (phen = 1,10-phenanthroline; DPPZ = dipyrido[3,2-a:2',3'-c]phenazine) with DNA. The pure enantiomers, which were difficult to separate by traditional resolving methods, were synthesized via a chiral precursor. Changes in luminescence, isotropic absorption and excited state lifetimes upon binding, and the LD observed in flow-oriented DNA systems provide detailed information about the DNA binding of the enantiomers. Flow LD shows that both enantiomers bind to DNA in a well-defined manner with an orientation of the dipyridophenazine chromophore consistent with intercalation of this moiety between base-pairs. Both enantiomers are found to show luminescence in the presence of DNA to which they bind very strongly (K almost-equal-to 10(8) M-1); however, the relative luminescence quantum yield of the bound DELTA enantiomer is 6-10 times larger than that of the bound LAMBDA enantiomer. Furthermore, for each enantiomer two distinct excited state lifetimes are found in varying proportions depending on the binding ratio. The large difference in luminescence quantum yield between the enantiomers is interpreted in terms of slightly different intercalation geometries of the dipyridophenazine ligand, resulting in different protections from quenching by solvent water and diastereomeric differences in the interactions between enantiomers bound in contigue on DNA.

Covalent adducts of the carcinogenic polycyclic aromatic hydrocarbon (+)-anti-benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide ((+)-anti-BPDE) in polynucleotides have been studied by fluorescence spectroscopy. The pyrenyl chromophores of the BPDE adducts, linked by the C10 atom to the exocyclic nitrogen of guanine, interact in the photoexcited state, as evidenced by excimer fluorescence. Strong BPDE excimer fluorescence is observed in the alternating poly(dGdC).poly(dGdC) sequence, whereas it is weak in the homopolymeric poly(dG).poly(dC) and in calf thymus DNA. No excimer fluorescence is observed for the BPDE adducts in poly(dAdC).poly(dGdT) or poly(dAdG).poly(dCdT). It is concluded that the formation of BPDE excimers in polynucleotides requires binding to guanines on different strands on consecutive basepairs. The experimental results are supported by graphics modeling and energy minimization of BPDE adducts in various oligonucleotide sequences. The results show that the most favorable arrangement for excimer formation of the BPDE-dG adducts is in a 5'(dCdG-BPDE).5'(dCdG-BPDE) sequence, where the pyrenyl chromophores interact in the minor groove.

The interaction of the uranyl(VI) ion (UO22+) with DNA and its light-induced cleavage of DNA has been studied using flow-linear dichroism and P-32-endlabeled oligonucleotides. It was found that binding of uranyl ion to DNA is a prerequisite for photocleavage; from run-off experiments the binding constant was estimated to be of the order of 10(10) M-1 at pH 4. The angular orientation of the [O=U=O]2+ chromophore is consistent with binding by bridging phosphate groups on opposite strands of the minor groove of DNA; at higher DNA concentration aggregation indicates intermolecular bridging as well. The uranyl-mediated photocleavage of DNA is not influenced by the presence of O2, is more efficient at low pH (

The interaction of tubulin with thiocolchicine and two thiocolchicine analogues, one lacking the B ring and one with a six-membered B ring, has been studied by using near-UV and CD spectroscopies. Rapid, reversible binding of the latter analogue to tubulin demonstrates the ability of the colchicine binding site to accommodate the phenyltropone system with a more coplanar conformation than is present in free colchicine. There is no evidence, however, that bound thiocolchicine should have a much less twisted conformation than free thiocolchicine. Thiocolchicine and the bicyclic analogue appear to have approximately the same conformation of the phenyltropone system, in both the free and the bound states, suggesting that this conformation has an optimal arrangement of the phenyl and tropone rings for binding to tubulin. In contrast to colchicine and related derivatives, the three thiocolchicine analogues show pronounced near-UV CD bands upon association to tubulin. No simple relation could be found between the sign pattern of the CD components in the near-UV band of the thiocolchicinoid chromophore and its axial chirality.

Despite extensive study of DNA interaction with propeller-shaped metal complexes, such as the DELTA and LAMBDA enantiomers of [Ru(1,10-phenanthroline)3]2+, the basis for their enantioselectivity, and even their binding modes, are not yet fully understood. H-1 NMR studies of the interactions with the self-complementary oligonucleotide d(CGCGATCGCG)2 indicate that both enantiomers bind into the minor groove of the central AT-TA region, but with a rapid exchange between the bound and free states. Flow linear dichroism (FLD) and circular dichroism (CD) show different binding geometries for the two enantiomers. These two geometries are found in natural DNA as well as in a number of different B form polynucleotides, virtually independent of base composition and of methylation. The DNA interaction with the [Ru(1,10-phenanthroline)3]2+ complexes will be reconsidered in the light of NMR, FLD, CD and fluorescence results.

High selectivity for long AT sequences can be attained by kinetically controlled DNA threading intercalation by binuclear ruthenium(II) complexes. The rate of intercalation is strongly correlated to the number of consecutive AT basepairs, being up to 2500 times faster with an AT polymer compared to mixed-sequence DNA.

The NaCl-induced transition from B to Z conformation in poly (dG-dC), covalently modified with benzo(a)pyrene diol epoxides (BPDE), has been studied by use of flow linear dichroism (LD), CD, and fluorescence spectroscopy. In contrast to unmodified poly (dG-dC), the BPDE-modified polynucleotide displays a B to Z conversion that is incomplete in 2.7 M NaCl. This and a number of other observations are found consistent with a locally retained BPDE perturbed B-like conformation in the vicinity of the BPDE adduct. In 2.7 M NaCl the polynucleotide orientation in flow is strongly reduced, which could indicate flexibility at the junctions between domains of BPDE perturbed B-like conformation and Z conformation. A virtually stable high-salt B form is found in 2.25 M NaCl at 0°C; its CD spectrum resembles that of the transient so-called B* form, which is observed at an early stage of the B to Z transition. Characteristic B to Z transition times were evaluated by taking this B*-form CD spectrum as the initial state spectrum. The kinetics of the B to Z transition, monitored by the CD signal at 287 nm, shows an increased transition rate with (+)-anti-BPDE modification whereas the (−)-anti- and (±)-syn-BPDE adducts have retarding effects. The difference is discussed against the background of the different binding geometries of the BPDE isomer adducts.

We have used DNase I footprinting and fluorescence melting studies to study the interaction of the hairpin polyamide Im-Py-Py-Py-(R)H2Ngamma-Im-Py-Py-Py-beta-Dp with its preferred binding sites (5'-WGWWCW; W=A or T) and other sequences. DNase I footprinting confirmed that the ligand binds to the sequence AGAACA at nanomolar concentrations and that changing the terminal A to G causes a dramatic decrease in affinity, while there was no interaction with the reverse sequence WCWWGW. Fluorescence melting studies with 11-mer duplexes showed that the polyamide had very different effects on the forward (TGWWCT) and reverse (TCTAGT) sequences. At low concentrations, the polyamide produced biphasic melting curves with TGATCT, TGTACT and TGAACT, suggesting a strong interaction. In contrast, the melting profiles with TCTAGT were always monophasic and showed much smaller concentration dependent changes in Tm. The polyamide also showed weak binding to the sequence TGATCT when one of the central AT pairs was replaced with an AC mismatch. These melting profiles were compared with those produced by the AT-selective minor groove binding agents distamycin and Hoechst 33258 at the same sites and at similar sequences containing A5 and (AT)3, which are expected to bind distamycin in the 1:1 and 2:1 modes, respectively. These ligands produced simple monophasic melting curves in which the Tm steadily increased as the ligand concentration was raised.

Novel platinum(II) square planar coordination complexes, in which two heteroaromatic ligands are held by the metal in an unusual L-shaped geometry orthogonal to each other, have been synthesized, and their interaction with DNA was investigated with absorption and linear dichroism spectroscopy. As a rule, the ligand that is coplanar with the coordination square of Pt is found to be oriented perpendicular relative to the DNA helix axis when bound, suggestive of its intercalation between the base pairs of DNA. However, when this coplanar ligand is replaced by two pyridines, the opposite ligand, orthogonal to the coordination square, is instead preferentially intercalated. This behavior shows that these new complexes do indeed show some properties of true ambintercalators, i.e., compounds that can bind by intercalation of either of two distinct aromatic moieties.

The orientation and conformation of the cell-penetrating peptide "penetratin" associated with phospholipid vesicle membranes has been determined using polarized-light spectroscopy. The magnitude of orientation of penetratin is unprecedented for a solute in our membrane system, which we believe indicates a strong, specific interaction with the membrane. To validate the spectroscopic technique for studying the orientation of the two tryptophan residues of penetratin, we applied tryptophan octyl ester as a model compound. It is found to be incorporated in the membrane and preferentially oriented with its hydrophobic benzene edge of the indole chromophore pointing into the membrane and its hydrophilic groups oriented toward the water. For penetratin, the results indicate that a central alpha-helical part of the peptide is aligned parallel with the membrane surface, while the ends of the peptide adopt a planar structure. The planes of the two tryptophan side chains show a preferred orientation parallel with the membrane surface, indicating that they are not inserted into the membrane.

The association kinetics of RecA protein from Escherichia coli to DNA is strongly enhanced if even a minor fraction of DNA bases has been modified by a carcinogenic (+)-anti metabolite of benzo[a]pyrene (BPDE). The enhancement is much smaller with the less carcinogenic (-)-anti enantiomer of BPDE suggesting the possibility that the RecA protein binds selectively to the proto-oncogenic target. Most importantly, the binding of RecA to DNA modified with the latter enantiomer is found to give rise to a reorganization of this BPDE adduct from an intercalation site into a minor groove site. This indicates that the binding mechanism of RecA is via intercalation of some amino acid moiety, a discovery that could explain the approximately 50% contour length increase of the DNA within its fibrous complex with RecA.

Efficient cellular uptake is crucial for the success of any drug directed towards targets inside cells. Peptide nucleic acid (PNA), a DNA analog with a promising potential as a gene-directed drug, has been shown to display slow membrane penetration in cell cultures. We here used liposomes as an in vitro model of cell membranes to investigate the effect on penetration of a PNA molecule colvalently modified with a lipophilic group, an adamantyl moiety. The adamantyl attachment was found to increase the membrane-penetration rate of PNA three-fold, as compared to corresponding unmodified PNA. From the penetration behaviour of a number of small and large molecules we could conclude that passive diffusion is the mechanism for liposome-membrane passage. Flow linear dichroism (LD) of the modified PNA in presence of rod-shaped micelles, together with octanol-water distribution experiments. showed that the adamantyl-modified PNA is amphiphilic; the driving force behind the observed increased membrane-penetration rate appears to be an accumulation of the PNA in the lipid double layer.