A theoretical study of anthracene and phenanthrene derivatives acting as A-T specific intercalators.

Theoretical computations are performed on the comparative A-T versus G-C binding selectivities of two DNA intercalating molecules recently synthesized by Wilson et al. These are derivatives of phenanthrene and anthracene with side chains containing an hydroxy group bound to its C alpha carbon and a cationic amino group bound to its C beta carbon. We have optimized the binding energies of these phenanthrene and anthracene derivatives (1 and 2, respectively) to the double-stranded tetramers d(ATAT)2 and d(GCGC)2, the intercalation occurring in the central pyrimidine (3'-5') purine sequence. The sum of the intercalator-oligonucleotide intermolecular interaction energy plus the conformational energy variation of the intercalator upon binding were computed by the SIBFA procedures, which use empirical formulas based on ab initio SCF computations. Both compounds are found to bind more favourably to the AT sequence than to the GC one. Moreover, the affinity of 1 for the AT oligomer is computed to be larger than that of 2, whereas conversely that of 2 is larger than that of 1 for the GC oligomer. The AT versus GC binding selectivity of 1 is significantly larger than that of 2. These results are in excellent agreement with the experimental findings of Wilson et al. However, contrary to the suggestion of these authors the alpha-hydroxy group of the side chain of the intercalators does not seem to play a decisive role in determining the A-T specificity.     

A tentative model of the intercalative binding of the neocarzinostatin chromophore to double-stranded tetranucleotides.

Theoretical computations are performed of the intercalative binding of the neocarzinostatin chromophore (NCS) with the double-stranded oligonucleotides d(CGCG)2, d(GCGC)2, d(TATA)2 and d(ATAT)2. Minor groove binding is preferred over major groove binding. It is found that the long axis of the stacked naphtoate ring lies approximately parallel to the long axis of the base pairs of the intercalation site. The galactosamine ammonium group interacts with specific sites of the groove (O2/N3 of bases 2 and O1' of sugar S3), whereas the dodecadyine ring system wraps around the groove towards the backbone. An overall AT versus GC preference is derived. Intercalation in a central purine-(3', 5')-pyrimidine sequence appears to be preferred over that in a central pyrimidine-(3', 5')-purine sequence.     

High field 1H-NMR analysis of the 1:1 intercalation complex of the antitumor agent mitoxantrone and the DNA duplex [d(CpGpCpG)

Complete 1H-nmr assignment has been achieved of the stoichiometric 1:1 complex of the antitumor agent mitoxantrone with the duplex oligomer [d(CpGpCpG)]2. The techniques used included 2D-COSY, 1D-NOE and 2D-HH-INADEQUATE. Comparisons of 1H and 13C chemical shift changes upon addition of drug suggest symmetrical intercalative binding to the center of the tetramer. NOE difference measurements and 31P studies suggest binding of the terminal OH groups of the side chains to the central phosphate groups such that the methylene groups are proximate to C(3)6, C(3)6 and G(4)8 base protons all in the major groove. The data suggest that the side chains bind to the neighboring base pairs from the intercalation site. This is in accord with independent evidence of G,C base preference for binding from spectroscopic and electron microscopy studies.     

Modelling of the binding specificity in the interactions of cationic porphyrins with DNA.

A theoretical investigation is performed of the complexes of a tetracationic porphyrin, tetra-(4-N-methylpyridyl)-porphyrin, (T4MPyP), with the hexanucleotides d(CGCGCG)2 and d(TATATA)2, considering the possibility of both the intercalative and the groove binding interactions. These computations demonstrate that T4MPyP manifests a significant preference for intercalation in its complex with d(CGCGCG)2 but for non intercalative binding in the minor groove in its complex with d(TATATA)2. Such a dual binding behaviour of T4MPyP as a function of the sequence to which it is attached is fully consistent with available experimental data. It demonstrates that intercalation and groove binding may be viewed as two potential wells on a continuous energy surface. In agreement with experiment, the computations indicate that in the here considered case the deepest well is associated with intercalation.     

Single-strand DNA triple-helix formation

Chemical modification studies provide evidence that single-stranded oligodeoxyribonucleotides can form stable intrastrand triple helices. Two oligonucleotides of opposite polarity were synthesized, each composed of a homopurine-homopyrimidine hairpin stem linked to a pyrimidine sequence which is capable of folding back on the hairpin stem and forming specific Hoogsteen hydrogen bonds. Using potassium permanganate as a chemical modification reagent, we have found that two oligodeoxyribonucleotides of sequence composition type 5'-(purine)8(N)4(pyrimidine)8(N)6(pyrimidine)8-3' and 5'-(pyrimidine)8N6(pyrimidine)8N4(purine)8-3' undergo dramatic structural changes consistent with intrastrand DNA triple-helix formation induced by lowering the pH or raising the Mg2+ concentration. The intrastrand DNA triple helix is sensitive to base mismatches.     

The interaction of multiply charged intercalating heterocycles with DNA

The interaction with DNA of two aromatic nitrogen heterocycles, 1 and 2 , which at pH 6 have two positive charges on their ring systems and two cationic side chains, have been determined. A third similar compound, 3 , with a single side chain and reduced ring charge, was analyzed as a control. Viscometric titrations with sonicated DNA indicated that all three compounds bind to DNA by intercalation. Spectrophotometric binding studies as a function of ionic strength indicated that both 1 and 2 bind to DNA as tetracations at pH 6. These are the first examples of intercalators with two charges directly on the intercalating ring system. Dissociation kinetics experiments as a function of ionic strength confirmed that 1 and 2 bind to DNA as tetracations. Compound 1 has a G · C base-pair binding preference, 2 seems to prefer binding to alternating pyrimidine–purine sequences regardless of the composition, and 3 has no significant binding specificity.     

Stopped-flow kinetic analysis of the interaction of anthraquinone anticancer drugs with calf thymus DNA, poly[d(G-C)].poly[d(G-C)], and poly[d(A-T)].poly[d(A-T)]

The sodium dodecyl sulfate driven dissociation reactions of daunorubicin (1), mitoxantrone (2), ametantrone (3), and a related anthraquinone without hydroxyl groups on the ring or side chain (4) from calf thymus DNA, poly[d(G-C)]2, and poly[d(A-T)]2 have been investigated by stopped-flow kinetic methods. All four compounds exhibit biphasic dissociation reactions from their DNA complexes. Daunorubicin and mitoxantrone have similar dissociation rate constants that are lower than those for ametantrone and 4. The effect of temperature and ionic strength on both rate constants for each compound is similar. An analysis of the effects of salt on the two rate constants for daunorubicin and mitoxantrone suggests that both of these compounds bind to DNA through a mechanism that involves formation of an initial outside complex followed by intercalation. The daunorubicin dissociation results from both poly[d(G-C)]2 and poly[d(A-T)]2 can be fitted with a single exponential function, and the rate constants are quite close. The ametantrone and 4 polymer dissociation results can also be fitted with single exponential curves, but with these compounds the dissociation rate constants for the poly[d(G-C)]2 complexes are approximately 10 times lower than for the poly[d(A-T)]2 complexes. Mitoxantrone also has a much slower dissociation rate from poly[d(G-C)]2 than from poly[d(A-T)]2, but its dissociation from both polymers exhibits biphasic kinetics. Possible reasons for the biphasic behavior with the polymers, which is unique to mitoxantrone, are selective binding and dissociation from the alternating polymer intercalation sites and/or dual binding modes of the intercalator with both side chains in the same groove or with one side chain in each groove.     

High Field 1H-NMR Analysis of the 1:1 Intercalation Complex of the Antitumor Agent Mitoxantrone and the DNA Duplex [d(CpGpCpG)]2

Abstract

Complete ¹H-nmr assignment has been achieved of the stoichiometric 1:1 complex of the antitumor agent mitoxantrone with the duplex oligomer [d(CpGpCpG)]₂. The techniques used included 2D-COSY, 1D-NOE and 2D-HH-INADEQUATE. Comparisons of ¹H and ¹³C chemical shift changes upon addition of drug suggest symmetrical intercalative binding to the center of the tetramer. NOE difference measurements and ³¹P studies suggest binding of the terminal OH groups of the side chains to the central phosphate groups such that the methylene groups are proximate to C(3)6, C(3)6 and G(4)8 base protons all in the major groove. The data suggest that the side chains bind to the neighboring base pairs from the intercalation site. This is in accord with independent evidence of G,C base preference for binding from spectroscopic and electron microscopy studies.     

Interaction of the antineoplastic agent mitoxantrone with double-stranded nucleic acids

The binding of mitoxantrone with double-helical nucleic acids was investigated by the methods of isothermal microcalorimetry, circular dichroism and absorption at the ionic strength mu = 0.11 and 0.011 M NaCl at temperature region of 30 divided by 60 degrees C. The investigation shows, that at mu = 0.11 M NaCl mitoxantrone interacts with double-helical nucleic acids in one way only. For such conditions using spectrophotometric titration data Scatchard plots for the binding of mitoxantrone with double-helical nucleic acids were constructed. The calculations show that the saturation stoichiometry is one mitoxantrone molecule per 2 divided by 3 base pairs DNA and 6 divided by 8 base pairs RNA. The dependence of binding constant from GC-content is observed. It is shown that the binding enthalpy of mitoxantrone with DNA and RNA increases linearly and reaches -(3.0 +/- 0.5) kkal per 1 mol mitoxantrone. It is shown that a binding mitoxantrone with double-helical nucleic acids, besides the intercalation of rings, a determinate contribution in the binding is given also by electrostatic interaction of side chains mitoxantrone with nucleic acids.     

Conformational properties of branched RNA fragments in aqueous solution

The conformational properties of branched trinucleoside diphosphates ACC, ACG, AGC, AGG, AUU, AGU, AUG, ATT, GUU, and aAUU [XYZ = X(2'p5'Y)3'p5'Z] have been studied in aqueous solution by nuclear magnetic resonance (1H, 13C), ultraviolet absorption, and circular dichroism. It is concluded from these studies that the purine ring of the central residue (X; e.g., adenosine) forms a base-base stack exclusively with the purine or pyrimidine ring of the 2'-nucleotidyl unit (Y; 2'-residue). The residue attached to the central nucleoside via the 3'-5'-linkage (Z; 3'-residue) is "free" from the influence of the other two heterocyclic rings. The ribose rings of the central nucleoside and the 2'- and 3'-residues exist as equilibrium mixtures of C2'-endo (2E)-C3'-endo (3E) conformers. The furanose ring of the central nucleoside (e.g., A) when linked to a pyrimidine nucleoside via the 2'-5'-linkage shows a higher preference for the 2E pucker conformation (e.g., AUG, AUU, ACG, ca. 80%) than those linked to a guanosine nucleoside through the same type of bond (AGU, AGG, AGC, ca. 70%). This indicates some correlation between nucleotide sequence and ribose conformational equilibrium. The 2E-3E equilibrium of 2'-pyrimidines (Y) shows significant, sometimes exclusive, preference (70-100%) for the 3E conformation; 3'-pyrimidines and 2'-guanosines have nearly equal 2E and 3E rotamer populations; and the ribose conformational equilibrium of 3'-guanosines shows a preference (60-65%) for the 2E pucker. Conformational properties were quantitatively evaluated for most of the bonds (C4'-C5', C5'-O5', C2'-O2', and C3'-O3') in the branched "trinucleotides" AUU and AGG by analysis of 1H-1H, 1H-31P, and 13C-31P coupling constants. The C4'-C5' bond of the adenosine units shows a significant preference for the gamma + conformation. The dominant conformation about C4'-C5' and C5'-O5' for the 2'-and 3'-nucleotidyl units is gamma + and beta t, respectively, with larger gamma + and beta t rotamer populations for the 2'-unit. The increased conformational purity in the 2'-residue, compared to the 3'-residue, is ascribed to the presence of an ordered (adenine----2'-residue) stacked state. The favored rotamers about C3'-O3' and C2'-O2' are epsilon- and epsilon'-, respectively. The conformational features of AUU and AGG were compared to those of their constitutive dimers A3'p5'G, A2'p5'G, A3'p5'U, and A2'p5'U and monomers 5'pG and 5'pU.     

A theoretical investigation on the sequence selective binding of adriamycin to double-stranded polynucleotides.

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of adriamycin (ADM) to five self-complementary double-stranded hexanucleotides. Among the two regularly alternating hexanucleotides d (TATATA)2 and d (CGCGCG)2, a stronger binding is predicted for the former. The strongest complex is computed, however, for the mixed hexanucleotide d (CGTACG)2, containing the intercalation site between two CG base pairs and an adjacent TA base pair. The overall sequence preference is the result of an intricate interplay of sequence preferences of the constituents in particular of daunosamine and the 9-OH substituent. Altogether, the selective base pair recognition by adriamycin cannot be defined in terms of the two base pairs implicated in the intercalation site alone but must be expressed in terms of a triplet of base pairs.     

Recognition of nucleic acid double helices by homopyrimidine 2', 5'-linked RNA.

We have studied the effect of a 2',5'-RNA third strand backbone on the stability of triple helices with a 'pyrimidine motif' targeting the polypurine strand of duplex DNA, duplex RNA and DNA/RNA hybrids. Comparative experiments were run in parallel with DNA and the regioisomeric RNA as third strands adopting the experimental design of Roberts and Crothers. The results reveal that 2',5'-RNA is indeed able to recognize double helical DNA (DD) and DNA (purine):RNA (pyrimidine) hybrids (DR). However, when the duplex purine strand is RNA and the duplex pyrimidine strand is DNA or RNA (i.e. RD or RR), triplex formation is not observed. These results exactly parallel what is observed for DNA third strands. Based on T m data, the affinities of 2',5'-RNA and DNA third strands towards DD and DR duplexes were similar. The RNA third strand formed triplexes with all four hairpins, as previously demonstrated. In analogy to the arabinose and 2'-deoxyribose third strands, the possible C2'- endo pucker of 2',5'-linked riboses together with the lack of an alpha-2'-OH group are believed to be responsible for the selective binding of 2',5'-RNA to DD and DR duplexes, over RR and RD duplexes. These studies indicate that the use of other oligonucleotide analogues will prove extremely useful in dissecting the contributions of backbone and/or sugar puckering to the recognition of nucleic acid duplexes.     

Theoretical Design of Novel, 4 Base Pair Selective Derivatives of Mitoxantrone

Abstract

Mitoxantrone (MTX) is a recently synthesized antitumor intercalative molecule, currently in use in chemotherapy. Previous theoretical computations showed that the base pair selectivity of MTX is limited to the sole two base-pair sequence making up the intercalation site. In order to further extend the recognition site, we undertook, by means of theoretical computations, the design of novel MTX derivatives, in which the terminal hydroxyl group of each side chain is esterified with oligopeptides.

We compare in the present study the binding affinities of two derivatives, depsiGly-Lys(D) and depsiGly-Gly-Orn(L), for the palindromic sequences d(CCCGGG)₂, d(GCCGGC)₂, d(GGCGCC)₂, and d(CGCGCG)₂.

Major groove binding of the oligopeptide arms was shown to be significantly more favourable than either minor groove binding, or binding to the sole phosphate groups. With the two arms adopting two antiparallel directions, two distinct arrangements were investigated in the major groove: (a) the two oligopeptides are brought closer together by means of two hydrogen bonds involving the backbone of their second residue in a β-sheet like arrangement; (b) the two arms are remote from each other so as to reduce their mutual electrostatic repulsion. Whatever the disposition, the optimal binding configurations were invariably found to be those in which the cationic side chains of the terminal residues chelate N7/06 of two successive guanines, whenever present on a given strand. A distinct energetical preference for arrangement (a) was obtained with the depsiGly-Gly-Orn(L) derivative. Replacement of the central Gly residue by a Cys one, as in the sequence depsiGly-Cys-Orn(L), was proposed subsequently, so as to further stabilize such a β-sheet arrangement by means of a disulfide bridge between the two Cys residues.

The two investigated compounds were shown to preferentially bind sequences d(CCCGGG)₂ and d(GCCGGC)₂, with a tetrameric core CCGG rather than sequences d(GGCGCC)₂ and d(CGCGCG)₂, with a tetrameric core GCGC.     

A theoretical investigation of the base sequence preferences of monointercalating polymethylene carboxamide derivatives 9-aminoacridine.

Theoretical computations are performed of the comparative binding affinities of five polymethylene carboxamide derivatives of 9-aminoacridine to a series of double-stranded hexanucleotides. The purpose of this investigation is to ascertain whether minor groove recognition of a guanine base adjacent to the intercalation site can occur, and be preferentially stabilized, for a given length of the polymethylene side chain, encompassing from n = 2 up to n = 6 methylene groups. For that purpose, several representative sequences were investigated, in which intercalation of the 9-aminoacridine chromophore occurred at a central d(CpG) or d(TpA) step. Investigated were the self-complementary sequences d(CGCGCG)2, d(GCCGGC)2, d(TATATA)2 and d(ATTAAT)2, as well as the 'mixed' sequences d(ACTAAT) .d(ATTAGT) and d(TGTATA). d(TATACA). For n = 3 up to n = 6, such a recognition was enabled only when the guanine base was located downstream of the intercalation site, i.e. with steps d(CGG) and d(TAG). It occurred by means of a bidentate interaction involving, on the one hand, H(N2) and N3 of the base, and, on the other hand, the carbonyl oxygen and the cis amino hydrogen of the terminal formamide moiety of the ligand. Because of the flexibility of the side chain, however, alternative binding modes were also found to occur competitively, involving backbone-only interactions of the side chain. On the basis of the present computations, upon binding to the sequence d(GCCGGC)2, an optimal value of n = 5 could be derived, with the corresponding acridine derivative eliciting both a significant prevalence of the bidentate over backbone only binding mode, and the most favourable energy balance within the investigated series. This privileged value of n = 5 is fully consistent with the experimental results of Markovits et al. and Gaugain et al. The very flexibility of the side chain, however, hampered any preferential recognition of a triplet sequence with a downstream guanine, such as d(CGG) or d(TAG), to be elicited over sequences such as d(TAA), d(TAT) or d(TAC).     

7-Azido-actinomycin D: a photoaffinity probe of the sequence specificity of DNA binding by actinomycin D

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5'-dGpdC-3' sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5'-flanking base was a pyrimidine and the 3'-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5'-TGGG-3'.     

A theoretical investigation on the sequence selective binding of daunomycin to double-stranded polynucleotides

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of daunomycin (DNM) to six representative self-complementary double-stranded hexanucleotides: d(CGTACG)2,d(CGATCG)2,d(CITACI)2, d(TATATA)2, d(CGCGCG)2 and d(TACGTA)2. The conformational angles of the hexanucleotides are fixed in values found in the representative crystal structure of the d(CGTACG)2-DNM complex. The intermolecular DNM-hexanucleotide interaction energies and the conformational energy changes of DNM upon binding are computed and optimized in the framework of the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. Among the two regularly alternating hexanucleotides, d(TATATA)2 and d(CGCGCG)2, a stronger binding is predicted for the former, in agreement with experimental results obtained with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Altogether, however, among the six investigated sequences, the strongest complexes are computed for the mixed hexanucleotides d(CGATCG)2 and d(CGTACG)2, containing the intercalation site between two CG base pairs and an adjacent TA base pair. This situation may be related to the increased affinity of DNM for GC rich DNA's and to the situation in the crystal structure of the DNM-d(CGTACG)2 complex. Analysis of the intrinsic base sequence preferences expressed by the individual constituents of DNM, namely the daunosamine side chain, the chromophore ring and its two 9-hydroxyl and 9-acetoxy substituents, reveals that the overall sequence preference found is the result of a rather intricate interplay of intrinsic sequence preferences, in particular at the level of daunosamine and the 9-hydroxyl substituent.(ABSTRACT TRUNCATED AT 250 WORDS)     

A molecular mechanics investigation of RNA complexes. I. Ethidium intercalation in an HIV-1 TAR RNA sequence with an unpaired adenosine.

Nucleic acid complexes with ethidium intercalated into different sites in a segment of HIV-1 TAR RNA with an unpaired A base, along with corresponding complexes with a normal RNA sequence without an unpaired base were studied by molecular mechanics energy minimization methods. Different intercalation geometries as well as different orientations of the ethidium molecule in the intercalation sites were tested. A general binding affinity enhancement for the ethidium binding to the bulge sequence compared with the normal RNA segment was obtained. With the unpaired adenosine base stacked in the duplex, the binding site adjacent to the 3' side of the bulge was found to be the most energetically favorable binding site, and the intercalation site 5' to the bulge in the same sequence is much less favorable. Unique correlated backbone conformational changes on binding of ethidium to the intercalation site 3' to the bulge were found to relieve backbone strains caused by the stacking of the unpaired base into the helix. These backbone conformational changes present a plausible molecular basis for the experimentally observed ethidium binding preference in this bulge RNA segment (L.S. Ratmeyer, R. Vinayak, G. Zon and W.D. Wilson, J. Med. Chem. 35, 966, 1992).     

Energetics and stereochemistry of DNA complexation with the antitumor AT specific intercalators tilorone and m-AMSA.

Computations by the SIBFA method on the intercalative interaction energies of tilorone and m-AMSA with B-DNA representative oligonucleotides account for the specificity of these antitumor drugs for AT sites and minor groove intercalation. In tilorone this specificity is due to the strong preference of the side chains for the minor groove, which overcomes the preference of the chromophore for a GC intercalation site. In m-AMSA the specificity is due to the combined preference of both the chromophore and the anilino side chain for AT intercalation site and minor groove, respectively. o-AMSA is shown to manifest a similar (although significantly less pronounced specificity) as m-AMSA but a higher affinity for DNA. A comparison of the energetics and stereochemistry of intercalative binding to DNA of m-AMSA (AT minor groove specific) and 9-aminoacridine-4-carboxamide (GC major groove specific), which possess the same chromophore and differ only by the nature and position of the side chains, shows the possibility of important variations in the intercalative behaviour of chromophoric drugs as a function of the substituent groups attached to them.     

A molecular mechanical study of complexes formed between 4-nitroquinoline-N-oxide and dinucleoside phosphates.

Molecular mechanical calculations were done on complexes of 4-nitroquinoline-N-oxide (NQO) with various dinucleoside phosphates [(ApT)2, (CpG)2, (GpC)2, and (TpA)2]. Models built using proflavine (uniform C3' endo sugar puckers) and acridine orange (mixed C3' endo (3'-5') C2' endo sugar puckers) dinucleoside phosphate X-ray structures were used in the calculations. Relative binding energies, complex geometries, and various intercalator orientations in the complexes were studied. The results suggest qualitatively different geometries for pyr-(3'-5')-pur and pur-(3'-5')-pyr sequences. Specifically, we find marked distortion in some of the complexes (i.e. there is not a parallel coplanar relationship between the base pairs and intercalator), distortion of the NQO nitro group from planarity in the complexes and mobility of NQO in the intercalation site. We suggest that experimental studies of NQO-dinucleoside phosphate complexes may reveal intercalation complexes which deviate substantially more from a nearly parallel coplanar arrangement of bases and intercalator than has been previously observed.     

Theoretical studies on alpha-helix--DNA interactions

The interaction energies between (Ala)10 and alpha-helix fragment and different nucleotide sequences in right-handed B-form have been optimized using semi-empirical potential energy functions. The energies are calculated for two different orientations of the alpha-helix, viz., when the alpha-helix axis taken in the N----C direction is (i) parallel and (ii) antiparallel to the 5'-3' ascending strand of DNA, proximal to it. When both the DNA molecule as well as the alpha-helix are treated as rigid molecules it is found that a polyalanine alpha-helix has slightly more favourable contacts when it is in the proximity of a four nucleotide sequence of 5'-(N-A-T-N)-3' type, where N is either a purine or a pyrimidine. However, when the two interacting molecules are allowed to undergo local structural variations then the interaction energy appears to be independent of the base sequence confirming the non-specific nature of these interactions.