Biomedical subjects

M J Waring

Publications and source records attributed to M J Waring.

At least 19 recordsLinked to original sources

PubMed1996

PCR-based development of DNA substrates containing modified bases: an efficient system for investigating the role of the exocyclic groups in chemical and structural recognition by minor groove binding drugs and proteins.

DNA molecules containing inosine in place of guanosine and/or 2,6-diaminopurine in place of adenine have been synthesized and tested as substrates for binding of sequence-selective ligands, both small and large. Footprinting patterns reveal that the binding sites for AT- or GC-specific antibiotics (distamycin or mithramycin, respectively) are completely changed in the modified DNAs, as expected for direct sequence readout involving contact with the purine 2-amino group. However, we also find large changes in the binding of HMG-D, a member of the HMG-1 family of chromosomal proteins, pointing to an indirect influence of the exocyclic amino group on ligand binding via an effect on the deformability of the double helix. This interpretation is confirmed by the finding that deoxyuridine-containing poly- and oligonucleotides, which lack the exocyclic methyl group of thymidine in the major groove, interact 5-10 times more strongly with HMG-D than do their counterparts containing natural nucleotides.

2-Aminopurine↗

PubMed1996

A simple ligation assay to detect effects of drugs on the curvature/flexibility of DNA.

Circular DNA molecules can readily be formed from the 169 bp tyrT fragment in the presence of T4 DNA ligase. We have analyzed the formation of DNA circles in the presence of the clinically important antitumour drugs amsacrine, mitoxantrone and daunomycin. All three are intercalating agents but they affect the closure reaction differently: daunomycin and mitoxantrone progressively inhibit the formation of circles whereas at low concentrations amsacrine strongly enhances the yield of circles suggesting that this drug can increase the flexibility and/or curvature of DNA. The ligation assay described here may prove useful and widely applicable for investigating the effects of small molecules on the secondary structure of DNA.

Amsacrine↗

PubMed1996

Sequence-selective intercalation of antitumour bis-naphthalimides into DNA. Evidence for an approach via the major groove.

LU 79553, a bis-naphthalimide drug highly active against human solid tumour xenografts, has been shown to bis-intercalate into DNA with a helix-unwinding angle of 37 degrees. Footprinting experiments with DNase I reveal that the drug is selective for mixed nucleotide sequences characterised by an alternating purine-pyrimidine motif, particularly those containing GpT (ApC) and TpG (CpA) steps. Derivatives bearing nitro or amino substituents on the naphthalimide chromophores bind at essentially identical sites. The footprinting profiles on tyrT DNA and on two fragments from pBS bear a remarkable resemblance to those determined for nogalamycin, an antibiotic which binds intercalatively leaving bulky carbohydrate substituents blocking both the major and minor grooves of the helix. Several lines of evidence indicate that the bis-naphthalimides recognise their preferred binding sites via the unusual expedient of intercalating from the major groove. Footprints on the complementary DNA strands sometimes appear staggered in the 5'direction. Repositioning the 2-amino group of G.C base pairs, which serves as a critical minor-groove marker, by substitution with inosine and/or 2,6-diaminopurine has little effect on the distribution of binding sites for LU 79553. The bis-naphthalimides affect the guanine-specific reaction with dimethyl sulfate (which reacts with the N7 position of the base located in the major groove) but not reactions with tetrachloropalladinate or methylene blue. Photoactivation of LU 79553-DNA complexes leads to a small amount of strand scission mainly at guanine residues. These observations make a strong case for binding via the major groove of the double helix, in contrast to nearly all common intercalating drugs, which could be important in explaining the unique biological selectivity of bis-naphthalimides.

Amides↗

PubMed1996

Stabilization of triple helical DNA by a benzopyridoquinoxaline intercalator.

Biophysical, footprinting, and chemical probing experiments are described which characterize the triple helix-stabilizing effects of a benzo[f]pyridoquinoxaline derivative BfPQ-4,3 structurally related to the previously reported benzo[f]pyridoindole compound BePI [Mergny et al. (1992) Science 256, 1681-1684]. Two parallel triple helix model systems have been investigated; one in which the third strand matched perfectly a 27 base pair purine-pyrimidine motif in target DNA and another in which the third strand was one nucleotide longer, i.e., a 28-mer. In the latter system, the pairing of the (Y)28 third strand to the (Y.R)27 target induces the formation of a bulge containing at least one unpaired base, which can be evidenced by chemical probing experiments with osmium tetroxide. BPQ, which uinwinds a duplex DNA by 17 degrees as judged by viscometric experiments and otherwise behaves as a typical nonspecific intercalculating drug, promotes the formation of Y.R.Y parallel triple helix containing both T.A.T and C.G.C+ triplets. Both DNase I and MPE.FeII footprinting experiments concur that triplex formation with the target (Y.R)27 sequence can be detected in the presence of BPQ at about 10-fold lower oligonucleotide concentrations than are required to produce an equivalent footprint in the absence of the drug. In addition, BPQ will promote binding to the polypurine-polypyrimidine target sequence by the longer mismatched oligonucleotide, providing significant stabilization of the parallel bulge-containing(Y.R)27,(Y)28 triplex with nearly the same efficiency as the bulge-free (Y.R)27.(Y)28 triplex. Thus in vivo BPQ might enhance the formation of both undesired and desired DNA triplexes. By performing an MPE*FeII probing reaction with a 5'-32 P-labeled oligonucleotide third strand, we have obtained evidence that BPQ is actually bound to the triplex region and may distort in a sequence-specific fashion.

Base Sequence↗

PubMed1996

Interaction of a DNA-threading netropsin-amsacrine combilexin with DNA and chromatin.

Combilexins are a group of DNA ligands having a sequence-specific minor groove binding element combined with an intercalating chromophore which stabilizes the DNA complex and can interfere with topoisomerases. In this study, complementary methods of spectroscopy (absorption, circular dichroism, electric linear dichroism) and biochemistry (viscometry, footprinting) have been applied to explore the nature of the complex formed between a new amsacrine-4-carboxamide-netropsin combilexin and DNA or chromatin. Collectively, the structural and kinetic data concur that the conjugate threads through the DNA double helix so as to intercalate its acridine chromophore, leaving the netropsin moiety and the methanesulfonanilino group positioned within the minor and major grooves of the double helix, respectively. The hybrid retains the AT selectivity conferred by the netropsin moiety. The threading-type intercalation process, evidenced by stopped-flow measurements, is affected when the DNA is wrapped around histones. The composite drug can bind to both the DNA linker segments and the nucleosomal cores in chromatin though, unlike its constituents, it antagonizes the salt-induced condensation of chromatin. As far as its mode of binding to DNA is concerned, the netropsin-amsacrine hybrid molecule exhibits structural features reminiscent of the antitumor antibiotics nogalamycin and pluramycin. The design of DNA-threading combilexins provides an original route for the development of sequence-specific ligands capable of forming stable complexes with DNA.

Amsacrine↗

PubMed1996

DNA recognition by two mitoxantrone analogues: influence of the hydroxyl groups.

The clinically useful anticancer drug mitoxantrone intercalates preferentially into 5'-(A/T)CG and 5'-(A/T)CA sites on DNA. The 5,8 hydroxyl substituents on its anthracenedione chromophore are available to interact with the double helix. Footprinting experiments with two anthraquinone derivatives structurally related to mitoxantrone and ametantrone have been undertaken to assess the influence of the hydroxyl groups on the DNA recognition process. The results confirm that they do play a role in the recognition of preferred nucleotide sequences and suggest that the binding of anthraquinones to a 5'-(A/T)CG site is dependent on the presence of the 5,8 hydroxyl substitutes whereas binding to 5'-(A/T)CA sites appears to proceed just as well without them.

Antineoplastic Agents↗

PubMed1996

Cooperativity in the binding of echinomycin to DNA fragments containing closely spaced CpG sites.

Quantitative footprinting has been used to investigate cooperative binding of the antitumor antibiotic echinomycin to DNA fragments containing closely spaced CpG steps. The sequences of the designed DNA fragments contained two pairs of strong echinomycin binding sites: a pair of ACGT sites together with an ACGT site and a TCGA site, either directly adjacent or separated by two or four A.T base pairs. The results demonstrate that the binding of echinomycin to the sequences ACGTACGT and TCGAACGT is highly cooperative. The extent of cooperativity depends on the nature of the sequences clamped by the antibiotic and diminishes as the distance between the binding sites is increased. Various methods of extracting the information necessary to establish cooperativity have been compared. Beyond the specific interest in echinomycin-DNA interaction, the present quantitative footprinting study provides a model that may be generally applicable for designing investigations into cooperativity in drug-DNA recognition.

Antibiotics, Antineoplastic↗

PubMed1996

Salen-anthraquinone conjugates. Synthesis, DNA-binding and cleaving properties, effects on topoisomerases and cytotoxicity.

A series of amidoethylamino-anthraquinone derivatives bearing either one or two salen (bis(salicylidene)ethylenediamine) moieties complexed with CuII or NiII have been synthesized, and their DNA-binding and cleaving properties examined. The effects of the mono- and di-substituted anthracenedione-salen conjugates on DNA cleavage mediated by topoisomerases I and II have also been determined, as well as their cytotoxicity toward human KB cells. The anthraquinone-salen. NiII conjugates bind to GC-rich sequences in DNA, but do not cleave the macromolecule. By contrast, the anthraquinone-salen. CuII hybrids do not recognize particular nucleotide sequences but efficiently induce single-strand breaks in DNA after activation. The 5,8-dihydroxy-anthraquinone conjugates are more cytotoxic and more potent toward topoisomerase II than the non-hydroxylated analogues, but they are less cytotoxic than the salen-free anthraquinones. The attachment of a salen. CuII complex to the anthraquinone chromophore can confer DNA cleaving properties in vitro, but this is at the expense of cytotoxic activity. Anthraquinone-salen. CuII complexes may find useful employ as footprinting probes for investigating ligand-DNA interactions.

Anthraquinones↗

PubMed1996

Sequence-specific binding of antitumour bisquaternary ammonium heterocycles to DNA and inhibition of polymerase activity in vitro.

Ten bisquaternary ammonium heterocycles (BQA) active against experimental tumours were investigated for possible sequence-selective binding to DNA. Footprinting analyses indicated that several bound preferentially to dAdT runs consisting of at least four base pairs. Shortening of one or two spacer groups between the aromatic rings of the ligands (by replacement of CONH with NH) emerged as a prerequisite for sequence-specific binding. Other relevant factors concerned the overall shape of the ligands and the relative position of their positive charges. Footprinting plots evaluated for the BQA compound SN 6132 on the 167mer EcoRI-RsaI restriction fragment from plasmid pBR322 yielded the highest individual binding constant for the symmetrical base sequence AATTTAA, with approximate K(A) = 2.0 x 10(6)/M. Polymerase-catalysed syntheses of DNA and RNA in vitro were inhibited by all BQA derivatives, but the inhibition was much more pronounced with the sequence-specific binders SN 6999 and SN 6132 than with the non-specific ligand SN 6113.

Antineoplastic Agents↗

PubMed1996

DNA minor groove cleaving agents: synthesis, binding and strand cleaving properties of anthraquinone-oligopyrrolecarboxamide hybrids.

Three 9,10-anthraquinone-oligopyrrolecarboxamide hybrids have been prepared as potential DNA minor groove cleaving agents. Each conjugate was designed to contain a bis- or tris-pyrrolecarboxamide moiety related to netropsin or distamycin covalently linked to a 2-substituted anthraquinone chromophore capable of triggering photocleavage of DNA. AQ(NC)-Dist, having three pyrrole rings, is related to distamycin. AQ(NC)-Net and AQ(CN)-Net are related to netropsin; they differ only by the orientation of the amide bond between the anthraquinone and the netropsin moiety. The binding properties of these compounds to various natural DNAs have been studied by footprinting and circular dichroism. The introduction of the chromophore does not abolish the capacity of the drugs to recognize AT-rich sequences in DNA selectively. There is apparently little correlation between this property and the ability to trigger photo-induced DNA cleavage. AQ(CN)-Net is almost totally inactive in the cleavage assays whereas it manifestly binds selectively to AT-rich tracts. With AQ(NC)-Net and AQ(NC)-Dist, complete conversion of form I to form II of circular DNA is obtained. Moreover, in most cases the cleavage of DNA proved to be non-specific.

Amides↗

PubMed1995

Structures of quinoxaline antibiotics.

The crystal structures of three quinoxaline antibiotics-echinomycin 2QN, triostin C and the C222(1) form of triostin A--have been determined, and the structure of the P2(1)2(1)2(1) form of triostin A has been re-refined against our previously reported data. The molecular conformations are compared with those deduced from NMR data and those reported for two complexes of triostin A with oligonucleotides. Although the depsipeptide ring conformations are basically similar, the effective twofold molecular symmetry is violated by the folding of one of the quinoxaline chromophores in echinomycin 2QN and by a rotation of one of the ester planes with the formation of an intramolecular hydrogen bond in triostin C. In the oligonucleotide complexes of triostin A the chirality of the disulfide bridge is inverted. The alanine NH groups are involved in intermolecular hydrogen bonds in all four structures, and (except in echinomycin 2QN) the stacking of the chromophores in the crystal emulates the intercalation involved in DNA complex formation. In echinomycin 2QN, the antibiotic molecules are hydrogen bonded to form a helix along the crystallographic 6(5) screw axes, with a channel of disordered solvent running through the middle of the helix. Crystal data: (1), echinomycin 2QN, C53H66N10O12S2.2.5(C3H6O).2.5(H2O), M(r) = 1289.5, hexagonal, P6(5), a = b = 22.196(15), c = 24.64 (2) A, V = 10,513 (13) A3, Z = 6, Dx = 1.222 Mg m-3, lambda (Cu K alpha) = 1.5418 A, mu = 1.275 mm-1, T = 193 K, R = 9.0% for 4828 I > 2 sigma (I) and 11.8% for all 7102 unique reflections; (2), triostin C, C54H70N12O12S2.0.67(CHCl3).0.67(H2O), M(r) = 1234.2, orthorhombic, P2(1)2(1)2(1), a = 16.054 (8), b = 17.128 (9), c = 22.706 (12) A, V = 6244 (6) A3, Z = 4, Dx = 1.313 Mg m-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.239 mm-1, T = 188 K, R = 7.7% for 4678 I > 2 sigma (I) and 14.0% for all 7260 unique reflections; (3), triostin A, C50H62N12O12S2.2(C7H14O2), M(r) = 1347.6, orthorhombic, P2(1)2(1)2(1), a = 20.94 (2), b = 18.53 (2), c = 18.80 (2) A, V = 7292 (13) A3, Z = 4, Dx = 1.228 Mg m-3, lambda (Cu K alpha) = 1.5418 A, mu = 1.245 mm-1, T = 293 K, R = 6.8% for 2116 I > 2 sigma (I) and 9.3% for all 2928 unique reflections; (4), triostin A, C50H62N12O12S2.HCl.2(C3H7NO), M(r) = 1269.9, monoclinic, C222(1), a = 10.622 (10), b = 17.035 (17), c = 35.21 (3) A, V = 6371 (10) A3, Z = 4, Dx = 1.324 Mg m-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.199 mm-1, T = 153 K, R = 7.5% for 2164 I > 2 sigma (I) and 13.2% for all 3402 unique reflections. Extensive use was made of restraints on the geometrical and displacement parameters in the successful anisotropic refinement of these structures against weak data.

Anti-Bacterial Agents↗

PubMed1995

Effects of base substitutions on the binding of a DNA-bending protein.

In order to investigate whether the 2-amino group of guanine, which lies in the minor groove of the B-form helix, can directly influence DNA flexibility and major groove recognition by proteins we have examined the properties of DNA molecules containing inosine and/or 2,6-diaminopurine (DAP) residues. Appropriately substituted tyrT(A93) DNA fragments were prepared by the polymerase chain reaction. Their mobility in non-denaturing gels was affected, consistent with changed anisotropic flexibility leading to increased curvature due to G-->I substitution and decreased curvature due to replacement of adenine with DAP. Band-shift assays of FIS protein binding revealed facilitated interaction with inosine-containing DNA and markedly reduced binding to DAP-containing DNA, attributable to altered bendability. DNase footprinting experiments confirmed that fewer sites would bind FIS in DAP-containing DNA at a given protein concentration, whereas higher levels of binding occurred with inosine-containing molecules. Thus base substitutions which affect the placement and presence of the purine 2-amino group in the minor groove can affect both the intrinsic curvature and the bendability of DNA.

2-Aminopurine↗

PubMed1995

Analogue versus digital recognition of DNA by bleomycin: an effect of the carbohydrate moiety.

We have sought to determine the influence of the carbohydrate moiety of the antitumour antibiotic bleomycin on the sequence-specific cleavage of DNA. Both bleomycin A2 and deglycobleomycin A2 produce different cleavage patterns with DNA in which the 2-amino group has been removed from guanine, added to adenine, or both, as well as on a designed DNA fragment containing a few defined cleavage sites. Although each drug cleaves DNA primarily at GpT and GpC sites, the cleavage at these sites is frequently found to be stronger with deglycobleomycin compared with bleomycin A2. Conversely, in most cases the cleavage at secondary sites, in particular at ApT steps, is significantly reduced or even abolished with deglycobleomycin. The results indicate that the gulose-mannose moiety of bleomycin A2 plays a significant role in the recognition of preferred nucleotide sequences and confirm the view that both secondary structure and interaction with guanine are involved in determining sequence-specific cleavage of DNA by bleomycin.

Antibiotics, Antineoplastic↗

PubMed1995

Localized chemical reactivity in double-stranded DNA associated with the intercalative binding of benzo[e]pyridoindole and benzo[g]pyridoindole triple-helix-stabilizing ligands.

Footprinting with methidiumpropyl-EDTA.FeII has been used to map the binding sites on duplex DNA of two closely related benzopyridoindole derivatives which selectively stabilize triple-helical DNA-oligonucleotide complexes. Both ligands bind to many sites, including certain oligopurine.oligopyrimidine tracts, with a weak preference for some (but not all) sequences rich in A.T base pairs. This indifference to primary sequence, with evidence of binding to the commonly disfavoured (A)n.(T)ntracts, may at least partially explain why the ligands stabilize triplex structures composed of T.A.T pairings. Neither 3-methoxy-7H-8-methyl-11- [(3'amino)propylamino]benzo[e]pyrido[4, 3-b]indole (BePI) nor 3- methoxy-7-[3'-diethylamino)propylamino]-10-methyl-11H- benzo[g]pyrido[4,3-b]indole (BgPI) affect the reaction of dimethyl sulphate or potassium tetrachloropalladinate with the N7 of purines in the major groove, but both enhance the reactivity of purines (mostly adenine residues) towards diethylpyrocarbonate, both proximal and distal to their identified binding sites. With potassium permanganate and osmium tetroxide/pyridine, probes for the accessibility of the 5,6 double bond of pyrimidine residues, BgPI has a more potent effect than BePI and, generally, the reaction with KMnO4 is more pronounced than that with OsO4. BgPI conspicuously potentiates the oxidation of pyrimidines in the triplet sequences 3'-ATA, 3'-GTA and 3'-GCA, whereas BePI enhances the reactivity of OsO4 towards thymine in sequences 3'-ATYR, with no effect on cytosine residues. Thus, despite their structural homology and common lack of specific sequence preferences, the two benzopyridoindole derivatives induce distinct conformational changes in duplex DNA, not just within the sites where footprints can be detected.

Animals↗

PubMed1995

The influence of the 2-amino group of guanine on DNA conformation. Uranyl and DNase I probing of inosine/diaminopurine substituted DNA.

The conformation of the DNA helix is supposed to be a critical element in site-specific recognition by ligands both large and small. Groove width is one important measure of the conformation which varies with the local nucleotide composition, perhaps because of the presence of a purine 2-amino group on G.C base pairs. We have probed DNA with G-->inosine (I) and/or A-->diaminopurine (DAP) substitutions to see whether the location of the purine 2-amino group can indeed affect the minor groove width. At acid pH, the reactivity towards uranyl nitrate is modulated in substituted DNA quite differently from natural DNA, consistent with a marked narrowing of the minor groove at sites of G-->I substitution and widening at sites of A-->DAP replacement. The latter exerts the dominant effect. The expected changes in conformation are equally evident in the patterns of susceptibility to DNase I cleavage, but not to hydroxyl radical attack. Nuclease cleavage is maximal in normal and substituted DNA at regions of inferred moderate groove width which are generally little affected by the nucleotide substitutions. Consistent with models of sequence-dependent cutting by DNase I we find that the presence of a purine 2-amino group on the base pair three places upstream of the cutting site has a profound influence on the rate of reaction.

Base Sequence↗

PubMed1995

Transferring the purine 2-amino group from guanines to adenines in DNA changes the sequence-specific binding of antibiotics.

The proposition that the 2-amino group of guanine plays a critical role in determining how antibiotics recognise their binding sites in DNA has been tested by relocating it, using tyrT DNA derivative molecules substituted with inosine plus 2,6-diaminopurine (DAP). Irrespective of their mode of interaction with DNA, such GC-specific antibiotics as actinomycin, echinomycin, mithramycin and chromomycin find new binding sites associated with DAP-containing sequences and are excluded from former canonical sites containing I.C base pairs. The converse is found to be the case for a group of normally AT-selective ligands which bind in the minor groove of the helix, such as netropsin: their preferred sites become shifted to IC-rich clusters. Thus the binding sites of all these antibiotics strictly follow the placement of the purine 2-amino group, which accordingly must serve as both a positive and negative effector. The footprinting profile of the 'threading' intercalator nogalamycin is potentiated in DAP plus inosine-substituted DNA but otherwise remains much the same as seen with natural DNA. The interaction of echinomycin with sites containing the TpDAP step in doubly substituted DNA appears much stronger than its interaction with CpG-containing sites in natural DNA.

2-Aminopurine↗

PubMed1995

Comparison of different footprinting methodologies for detecting binding sites for a small ligand on DNA.

In order to assess the utility of different methods of footprinting applied to the study of sequence-selective small molecule-DNA interaction we have performed a homologous series of experiments on the binding of echinomycin, a bis-intercalator, to a 133 base pair DNA restriction fragment containing a small number of discrete binding sites. Two of those sites each contained a pair of closely clustered CpG steps, the cognate dinucleotide sequence which is the common denominator of sites recognised by echinomycin. DNAse I was found to be much the best enzyme for footprinting in terms of sensitivity, accuracy, and ease of handling. DNAase II and micrococcal nuclease were of limited value. Excellent results were recorded with methidiumpropyl-EDTA.FeII which picked up strong binding sites and yielded sharp footprints from which a parsimonious estimate of site size could be determined. Orthophenanthroline.CuI proved to be a very suitable, sensitive chemical nuclease but hydroxyl radical footprinting with EDTA.FeII was only partially successful. Positive footprinting with conformation-sensitive probes diethylpyrocarbonate, osmium tetroxide and potassium permanganate yielded information to complement that afforded by the enzymic and chemical nucleases. Evidence of binding to both CpG steps in the clustered pair was obtained, with indications of possible cooperativity.

Base Sequence↗

PubMed1995

Synthesis, DNA binding, and sequence specificity of DNA alkylation by some novel cyclic peptide-chlorambucil conjugates.

In an effort to investigate the potential of cyclic peptides as carriers for cytotoxic agents, we synthesized four cyclic peptide-chlorambucil conjugates: cyclo[Lys(CHB)-Lys(CHB)-Gaba-] (peptide Y), cyclo[Lys(CHB)-Gly-Lys(CHB)-Gaba-] (peptide A), cyclo[Lys(CHB)-beta-Ala-Lys(CHB)-Gaba-] (peptide B) and cyclo[Lys(CHB)-Gaba-Lys(CHB)-Gaba-] (peptide C). The cyclic peptides were synthesized by coupling protected amino acid residues in solution and the subsequent cyclization was performed by the pentafluorophenyl ester method as described previously (Sheh et al., 1990, 1993a,b). After deblocking the lysyl-carbobenzyloxy protecting group (Z), the conjugation was achieved by reaction with the pentafluorophenyl ester of chlorambucil (CHB). These cyclic peptides differ from one another in ring size and are disubstituted with CHB via the epsilon-amino group of the lysyl residue. The various conjugates were designed to study the effect of ring size on the mode of DNA binding and alkylation. A DNA-binding assay using lambda-DNA with ethidium bromide showed that whereas peptide Y and CHB have no observable binding affinity, the apparent binding constants for peptide A, peptide B and peptide C on lambda-DNA were determined to be 2.36 x 10(5), 1.27 x 10(5) and 3.50 x 10(5), respectively. Thus, it is suggested that cyclic peptides bearing aliphatic side chains attached to a ring larger in size than 14 members would be more favourable as regards augmenting the binding affinity. DNase I footprinting showed that no footprinting patterns were observed for the 253-mer fragment and 117-mer fragment with peptide A, but two new bands corresponding to G69 and G80 were observed for the 117-mer fragment. DNA alkylation studies using a piperidine cleavage assay on the 117-mer DNA fragment showed that the sequence selectivity, judged by reaction intensity observed with peptide A, peptide B and peptide C, was similar to that seen with CHB alone. The selectivity of alkylation for both CHB and its peptide derivatives appears to be: 3'-Pur-G-Pyr-5' > 3'-Pyr-G-Pyr-5' > 3'-Pyr-G-Pur-5' = 3'Pur-G-Pur-5'. However, there are apparent differences in the intensity of alkylation by peptides A, B, C and CHB at certain guanine residues.

Alkylation↗