The 1360 bp long basic repeat unit of calf satellite I DNA contains GC rich nucleus of about 140 bp.

Fine melting profiles of calf satellite I DNA and its fragments obtained after digestion with endoR.EcoRI and endoR.AluI nucleases were investigated. It is shown that the 1360 bp basic repeat unit of calf satellite I DNA contains an about 140 bp long GC rich nucleus. It is localized on the 600 bp restriction fragment obtained after digestion of 1360 bp fragment with endoR.AluI nuclease. The main part of satellite I DNA melts as loops between such GC rich nuclei which strongly influence the melting properties of this satellite. There exist significant differences between the thermal stabilities of fragments containing many nuclei, one nucleus and those in which such nucleus is absent.     

Analysis of the Vicia faba genome by use of restriction endonucleases.

DNA of the broad bean, Vicia faba, was cleaved by the restriction endonucleases endoR . EcoRI, endoR . HindIII, endoR . HincII, endoR . BamI, and endoR . BspRI. Separation in agarose gels of the resulting fragments revealed, in addition to the bulk DNA, an enzyme-specific pattern of bands composed of restriction fragments of 300 to more than 30,000 base pairs in length. Bulk DNA was characterized by an unusual size distribution which significantly deviated from that expected according to the random fragmentation theory. It is argued that the observed distribution is due to the high proportion of repetitive DNA within this species (approximately equal to 75%). In all digests, a class of high-molecular-weight restriction fragments of more than 30,000 base pairs in length was observed which comprised 5-8% of the genome. It showed hybridization with highly repetitive DNA (c0t less than or equal to 2 x 10(-2) M . s) and included a fraction (2-3% of the genome) highly resistant to the activity of all the enzymes tested. The buoyant density in CsCl of this resistant DNA was not different from that of the total DNA (36% dG + dC). In endoR . EcoRI digests, the high-molecular-weight fragment class contained, in addition to the resistant DNA, a fraction of relatively high buoyant density (calculated dG + dC content: 61%) containing cleavage sites for the other enzymes used.     

Interaction of synthetic analogs of distamycin with DNA. Role of the conjugated N-methylpyrrole system in specificity of binding

Interaction of two synthetic analogs of distamycin (Dst), PPA and PAP, containing a saturated beta-alanine moiety replacing one N-methylpyrrole ring, with different polynucleotides and natural DNAs were studied using UV and CD spectroscopy. The results indicate that, similar to Dst, these analogs bind to DNA via the minor groove with a specificity towards AT-base pairs. It may be proposed that pyrrole chromophores in Dst probably do not play a role in the AT-base selectivity exhibited by Dst.     

Quantitative structure of a complex between a minor-groove-specific drug and a bent DNA decamer duplex: use of 2D NMR data and NOESY constrained energy minimization

Two-dimensional nuclear magnetic resonance (2D NMR) studies on d(GA4T4C)2 and d(GT4A4C)2 [Sarma, M.H., et al. (1988) Biochemistry 27, 3423-3432; Gupta G., et al. (1988) Biochemistry 27, 7909-7919] showed that A.T pairs are propeller twisted. As a result, A/T tracts form a straight rigid structural block with an array of bifurcated inter base pair H bonds in the major groove. It was demonstrated (previous paper) that replacement of methyl group by hydrogen (changing from T to U) in the major groove does not disrupt the array of bifurcated H bonds in the major groove. In this article, we summarize results of 2D NMR and molecular mechanic studies on the effect of a minor-groove-binding A.T-specific drug on the structure d(GA4T4C)2. A distamycin analogue (Dst2) was used for this study. It is shown that Dst2 binds to the minor groove of d(GA4T4C)2 mainly driven by van der Waals interaction between A.T pairs and the drug; as a consequence, an array of bifurcated H bonds can be formed in the minor groove between amide/amino protons of Dst2 and A.T pairs of DNA. NOESY data suggest that Dst2 predominantly binds at the central 5 A.T pairs. NOESY data also reveal that, upon drug binding, d(GA4T4C)2 does not undergo any significant change in conformation from the free state; i.e., propeller-twisted A.T pairs are still present in DNA and hence the array of bifurcated H bonds must be preserved in the major groove. NOESY data for the A5-T6 sequence also indicate that there is little change in junction stereochemistry upon drug binding.     

DNA curvature in native and modified EcoRI recognition sites and possible influence upon the endonuclease cleavage reaction

The ligation of a decadeoxynucleotide containing the EcoRI recognition site forms a series of multimers which appear to be curved based on observed anomalous gel migration in polyacrylamide gels. The degree of DNA curvature present in the recognition sequence, based upon the observed migration anomaly, can be altered by modifications to the purine functional groups at the 2- and 6-positions. Deletion of the guanine 2-amino group, occurring in the minor groove of the B-DNA helix, is most effective in increasing the observed DNA curvature. Conversely, the displacement of an amino group from the major groove to the minor groove eliminates curvature. DNA curvature is also modulated by the exocyclic group at the purine 6-position with decreasing curvature observed when changing the amino group to a carbonyl or proton substituent. Differences in the kinetic parameters characterizing the cleavage reaction by the endonuclease for many of the modified sequences are the result of modifications of functional groups in the major groove, which are likely to contact the endonuclease during catalysis. However, with two examples, significant decreases in the observed specificity constant (kcat/Km), characterizing the protein-nucleic acid interaction, cannot be easily explained in terms of such functional group contacts. It is more likely in these cases that the functional group modifications affect the efficiency of the endonuclease-DNA interaction by modulation of the structure of the double-stranded DNA helix. With both examples, modifications have been made to minor groove substituents. The extent of DNA curvature is increased significantly for one and decreased for the other, compared with that observed for the native recognition site. The results suggest that curvature of the DNA helix axis is an intrinsic property of the d(GAATTC) sequence which helps to optimize the protein-nucleic acid interactions observed for the EcoRI restriction endonuclease.     

Effects of Polyamines on the Binding of Hoechst 33258 to Calf Thymus DNA

Abstract

The ability of polyamines to displace the minor groove-binding dye Hoechst 33258 from calf thymus DNA was investigated. Polyamines displace non-specific DNA phosphate bound Hoechst in a charge-dependent fashion, but show very little ability to displace the high affinity binding of Hoechst in the minor groove of DNA. This high affinity binding is, however, sensitive to ethidium bromide and the minor groove binding drug berenil. These studies suggest that polyamines probably bind DNA in the minor groove very weakly, if at all, relative to known minor groove binding agents.     

DNA binding properties of novel distamycin analogs that lack the leading amide unit at the N-terminus

First examples of distamycin (Dst) analogs which lack hydrogen bond donor or acceptor groups at the N-terminus have been synthesized. The first molecule of this series, which is a bispyrrole peptide, did not exhibit any detectable binding with double-stranded (ds) DNA. However, all other analogs did bind strongly to AT-rich sequences of ds-DNA, with the binding affinities increasing as a function of the number of repeating pyrrole carboxamide units. These results imply that a hydrogen bond donor or acceptor atom per se at the N-terminus is not a prerequisite for DNA binding in the case of pyrrole carboxamide-based Dst analogs. However, in the absence of H-bond donor or acceptor at the N-terminus, a minimum of three pyrrole carboxamide units is necessary for the onset of DNA binding. Beyond this minimum number, the binding affinity increases as a function of the number of pyrrole units, as a result of the greater availability of hydrogen bonding and van der Waals surface. Experiments with poly[d(G-C)] have shown that the presence of the N-terminus formamide group is not inevitable for GC binding of this class of molecules. The observation that the N-terminus formamide unit can be dispensed with suggests that these molecules, which are much easier to synthesize and functionalize, can be used in place of the conventional analogs of distamycin for the development of novel minor groove binders with extended sequence recognition properties.     

Sequence-specific recognition of cancer drug-DNA adducts by HMGB1a repair protein

The efficacy of cancer drugs such as cisplatin (Cp) and oxaliplatin (Ox), which covalently bind to DNA to form drug-DNA adducts, is linked to their recognition by repair proteins such as HMGB1a. Previous experimental studies showed that HMGB1a's binding affinity for Cp- and Ox-DNA varies with the drug used and the local DNA sequence context of the adduct. We link this differential binding affinity to the free energy of deforming (bending and minor groove opening) the drug-DNA molecule during HMGB1a binding. Specifically, the minimal binding affinity of HMGB1a for Ox-DNA in the TGGA context is explained by its larger deformation free energy compared with Cp-DNA or Ox-DNA in other sequence contexts. Methyl groups on neighboring thymine bases in Ox-TGGA crowd the minor groove and sterically hinder the motion of the diaminocyclohexane ring of Ox, leading to this reduced deformability and resultant decrease in HMGB1a's binding affinity.     

Molecular modelling methods for prediction of sequence-selectivity in DNA recognition

We describe how one can apply molecular modelling methods, based on the molecular mechanics/generalised Born (MM/GB) approach, to the prediction of the relative affinity of DNA minor groove binding ligands for different DNA sequences. We discuss the theoretical background to the technique, some variations in the methodology that can be employed, and illustrate its application through a case study: analysis of the energetics of binding of Hoechst 33258 to the minor groove of various A/T-rich DNA duplexes. We show how the underpinning molecular dynamics (MD) simulations can be set up, how they can be analysed for satisfactory behaviour, and various approaches to extracting thermodynamics of drug binding from them. We find that while certain elaborations to the basic MM/GB method can improve the agreement with experimental data (e.g., calculating the DNA perturbation energy), others have to be analysed with more caution (e.g., calculating configurational entropy changes). Overall, these methodologies can rank the affinity of a ligand for the minor groove of different DNA sequences fairly well, but the calculation of absolute binding affinities is not very reliable.     

Effect of LNA modifications on small molecule binding to nucleic acids

Locked nucleic acid (LNA) is a conformationally constrained DNA analogue that exhibits exceptionally high affinity for complementary DNA and RNA strands. The deoxyribose sugar is modified by a 2'-O, 4'-C oxymethylene bridge, which projects into the minor groove. In addition to changing the distribution of functional groups in the groove and the overall helical geometry relative to unmodified DNA, the bridge likely alters the hydration of the groove. Each of these factors will impact the ability of small molecules, proteins and other nucleic acids to recognize LNA-containing hybrids. This report describes the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes. Using UV-vis, fluorescence and circular dichroism spectroscopies, we find that the minor groove binder as well as the intercalators exhibit significantly lower affinity for LNA-containing duplexes. The lone exception is the alkaloid ellipticine, which intercalates into LNA-DNA and LNA-RNA duplexes with affinities comparable to unmodified DNA-DNA and RNA-DNA duplexes.     

Distamycin-induced inhibition of homeodomain-DNA complexes.

The mobility shift assay was used to study the competition of the minor groove binder distamycin A with either an Antennapedia homeodomain (Antp HD) peptide or derivatives of a fushi tarazu homeodomain (ftz HD) peptide for their AT-rich DNA binding site. The results show that distamycin and the homeodomain peptides compete under the conditions: (i) preincubation of DNA with distamycin and subsequent addition of HD peptide; (ii) simultaneous incubation of DNA with distamycin and HD peptide; and (iii) preincubation of DNA with HD peptide and subsequent addition of distamycin. There is also competition when using a peptide which lacks the N-terminal arm of ftz HD that is involved in contacts in the minor groove. It is proposed that the protein's binding affinity is diminished by distamycin-induced conformational changes of the DNA. The feasibility of the propagation of conformational changes upon binding in the minor groove is also shown for the inhibition of restriction endonucleases differing in the AT content of their recognition site and of their flanking DNA sequences. Thus, it is demonstrated that minor groove binders can compete with the binding of proteins in the major groove, providing an experimental indication for the influence of biological activities exerted by DNA ligands binding in the minor groove.     

Mode of reversible binding of neocarzinostatin chromophore to DNA: evidence for binding via the minor groove

Two general approaches have been taken to understand the mechanism of the reversible binding of the nonprotein chromophore of neocarzinostatin to DNA: (1) measurement of the relative affinity of the chromophore for various DNAs that have one or both grooves blocked by bulky groups and (2) studies on the influence of adenine-thymine residue-specific, minor groove binding agents such as the antibiotics netropsin and distamycin on the chromophore-DNA interaction. Experiments using synthetic DNAs containing halogen group (Br, I) substituents in the major groove or natural DNAs with glucosyl moieties projecting into the major groove show that obstruction of the major groove does not decrease the binding stoichiometry or the binding constant for the DNA-chromophore interaction. Chemical methylation of bases in both grooves of calf thymus DNA, resulting in 13% methylation of N-7 of guanine in the major groove and 7% methylation of N-3 of adenine in the minor groove, decreases the binding affinity and increases the size of the binding site for neocarzinostatin chromophore. Similar results were obtained whether binding parameters were determined directly by spectroscopic measurements or indirectly by measuring the ability of the DNA to protect the chromophore against degradation. On the other hand, netropsin and distamycin compete with neocarzinostatin chromophore for binding to the minor groove of DNA, as shown by their decrease in the ability of poly(dA-dT) to protect the chromophore against degradation and their reduction in chromophore-induced DNA damage as measured by thymine release.(ABSTRACT TRUNCATED AT 250 WORDS)     

A demonstration of the intrinsic importance of stabilizing hydrophobic binding and non-covalent van der Waals contacts dominant in the non-covalent CC-1065/B-DNA binding

The comparative DNA binding properties and cytotoxic activity of CDPIn methyl esters (n = 1-5) vs. PDE-In methyl esters (n = 1-3) are detailed in studies which provide experimental evidence for the intrinsic importance of stabilizing hydrophobic binding and non-covalent van der Waals contacts dominant in the CC-1065/B-DNA minor groove binding. High affinity minor groove binding to DNA was established through: (1) the observation of CDPI3 binding (UV) but not unwinding of supercoiled DNA (phi 174 RFI DNA) thus excluding intercalative binding; (2) the observation of CDPI3 binding to T4 phage DNA (UV, delta Tm) in which the major groove is occluded by glycosylation thus excluding major groove binding; (3) the observation of salt (Na+) concentration independent high affinity CDPI3 binding to poly(dA . poly(dT) thus excluding simple electrostatic binding to the DNA phosphate backbone; and further inferred through (4) the observation of an intense induced dichroism (ICD, poly(dA) . poly(dT) and poly(dG) . poly(dC) [phi]23(358) = 24,000 and 23,500). This high affinity minor groove binding is sufficient to produce a potent cytotoxic effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of distamycin A and netropsin to the 12mer DNA duplexes containing mixed AT.GC sequences with at most five or three successive AT base pairs

Circular dichroism (CD), isothermal calorimetric titrations (ITC), and temperature-dependent UV spectroscopy were used to investigate binding of the minor groove-directed ligands distamycin A (Dst) and netropsin (Net) to the following duplexes: d(GTTAGTATTTGG). d(CCAAATACTAAC), d(GTTAGTATATGG).d(CCATATACTAAC), d(GTTAGTACTTGG). d(CCAAGTACTAAC), and d(GTTAGTAGTTGG).d(CCAACTACTAAC). Our results reveal that Dst binds within the minor grooves of these dodecamers that contain five-AT and/or four-AT.GC binding sites exclusively in a dimeric high-affinity 2:1 binding mode (K approximately 10(16) M(-)(2)). By contrast, Net exhibits high-affinity binding only when it binds in a 1:1 mode (K(1) approximately 10(9) M(-)(1)) to the two duplexes that contain five-AT sites (5'-TATTT-3' and 5'-TATAT-3'). Its further binding to these two duplexes occurs in a low-affinity mode (K(2) approximately 10(6) M(-)(1)) and results in the formation of 2:1 Net-DNA complexes. To the other two duplexes that contain sequences with at most three AT consecutive base pairs Net binds in two distinctive low-affinity 1:1 binding modes (K(1) approximately 10(7) M(-)(1), K(2) approximately 10(6) M(-)(1)). Competition experiments (CD and ITC titrations) reveal that Dst entirely displaces Net from its 1:1 and 2:1 complexes with any of the four duplexes. We discuss and interpret our optical and calorimetric results in the context of the available structural information about the complexes between DNA and the sequence-specific minor groove binders Dst and Net.     

Solution structure of a highly stable DNA duplex conjugated to a minor groove binder.

The tripeptide 1,2-dihydro-(3 H )-pyrrolo[3,2- e ]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder is conjugated to the 5'-end of short oligonucleotides the conjugates form unusually stable hybrids with complementary DNA and thus may have useful diagnostic and/or therapeutic applications. In order to gain an understanding of the structural interactions between the CDPI3minor groove binding moiety and the DNA, we have determined and compared the solution structure of a duplex consisting of oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 and its complementary strand to an unmodified control duplex of the same sequence using nuclear magnetic resonance techniques. Thermal denaturation studies indicated that the hybrid of this conjugate with its complementary strand had a melting temperature that was 30 degrees C higher compared with the unmodified control duplex. Following restrained molecular dynamics and relaxation matrix refinement, the solution structure of the CDPI3-conjugated DNA duplex demonstrated that the overall shape of the duplex was that of a straight B-type helix and that the CDPI3moiety was bound snugly in the minor groove, where it was stabilized by extensive van der Waal's interactions.     

Effect of LNA Modifications on Small Molecule Binding to Nucleic Acids

Abstract

Locked nucleic acid (LNA) is a conformationally constrained DNA analogue that exhibits exceptionally high affinity for complementary DNA and RNA strands. The deoxyribose sugar is modified by a 2′-O, 4′-C oxymethylene bridge, which projects into the minor groove. In addition to changing the distribution of functional groups in the groove and the overall helical geometry relative to unmodified DNA, the bridge likely alters the hydration of the groove. Each of these factors will impact the ability of small molecules, proteins and other nucleic acids to recognize LNA-containing hybrids. This report describes the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes. Using UV-vis, fluorescence and circular dichroism spectroscopies, we find that the minor groove binder as well as the intercalators exhibit significantly lower affinity for LNA-containing duplexes. The lone exception is the alkaloid ellipticine, which intercalates into LNA-DNA and LNA-RNA duplexes with affinities comparable to unmodified DNA-DNA and RNA-DNA duplexes.     

Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder.

The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.     

Molecular dynamics simulations and binding free energy analysis of DNA minor groove complexes of curcumin

Curcumin is a natural phytochemical that exhibits a wide range of pharmacological properties, including antitumor and anticancer activities. The similarity in the shape of curcumin to DNA minor groove binding drugs is the motivation for exploring its binding affinity in the minor grooves of DNA sequences. Interactions of curcumin with DNA have not been extensively examined, while its pharmacological activities have been studied and documented in depth. Curcumin was docked with two DNA duplexes, d(GTATATAC)₂ and d(CGCGATATCGCG)₂, and molecular dynamics simulations of the complexes were performed in explicit solvent to determine the stability of the binding. In all systems, the curcumin is positioned in the minor groove in the A·T region, and was stably bound throughout the simulation, causing only minor modifications to the structural parameters of DNA. Water molecules were found to contribute to the stability of the binding of the ligand. Free energy analyses of the complexes were performed with MM-PBSA, and the binding affinities that were calculated are comparable to the values reported for other similar nucleic acid–ligand systems, indicating that curcumin is a suitable natural molecule for the development of minor groove binding drugs.