Bis-intercalation of a homodimeric thiazole orange dye in DNA in symmetrical pyrimidine-pyrimidine-purine-purine oligonucleotides.

One- and two-dimensional 1H NMR spectroscopy were used to characterize the binding of a homodimeric thiazole orange dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis-4-(3 -methyl-2,3-dihydro-(benzo- 1,3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO), to various double-stranded DNA oligonucleotides containing symmetric (5'-pyr-pyr-pu-pu-3')2 or (5'-pu-pu-pyr-pyr-3')2 sequences. It was found that TOTO binds preferentially to oligonucleotides containing a (5'-CTAG-3')2 or a (5'-CCGG-3')2 sequence. Binding to the (5'-CCGG-3')2 sequence is less favored than to the (5'-CTAG-3')2 sequence. The complexes of TOTO with d(CGCTAGCGCTAGCG)2 (10) and d(CGCTAGCCGGCG):d(CGCCGGCTAGCG) (11) oligonucleotides, each containing two preferential binding sites, was also examined. In both cases TOTO forms mixtures of 1:1 and 1:2 dsDNA-TOTO complexes in ratios dependent on the relative amount of TOTO and the oligonucleotides in the sample. Binding of TOTO to the two oligonucleotides is sequence selective at the (5'-CTAG-3')2 and (5'-CCGG-3')2 sites. The 1H NMR spectra of both the 1:2 complexes and the three different 1:1 complexes have been assigned. A slight negative cooperativity is observed in formation of the 1:2 complexes. The ratio between the two different 1:1 complexes formed with oligonucleotide 11 is 2.4 in favor of binding to the (5'-CTAG-3')2 site. This is very similar to results obtained when the two sites are in different oligonucleotides. Thus the distribution of TOTO among the (5'-CTAG-3')2 and (5'-CCGG-3')2 sites is independent of whether the two sites are in the same or two different oligonucleotides.     

On the sequence selective bis-intercalation of a homodimeric thiazole orange dye in DNA.

The thiazole orange dye 1,1'-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-bis-4-[(3-methyl-2,3-dihydro(benzo-1, 3-thiazolyl)-2-methylidene]quinolinium tetraiodide (TOTO) binds sequence selectively to double-stranded DNA (dsDNA) by bis-intercalation. Each chromophore is sandwiched between two base pairs in a d(5'-py-p-py-3'):d(5'-pu-p-pu-3') site, and the linker spans over two base pairs in the minor groove. We have examined the binding of TOTO to various dsDNA oligonucleotides containing variations of the 5'-CTAG-3' binding motif by introducing inosine (I = inosine, 2-desaminoguanosine) and 5-methylcytosine ((me)C). A one- and two-dimensional NMR spectroscopy characterization yielded detailed structural information on the binding mode and for the well-defined TOTO-complexes competition experiments allowed determination of the relative binding strengths resulting from the various structural alterations. The experimentally observed base pair preference of TOTO in the palindromic sequences investigated is (me)CG > CG > CI > TA for the flanking base pair and (me)CI > CI > TA > CG > UA for the central base pair. The best binding site observed so far is the d(5-C(me)CIG-3')(2) site. This site is much more favorable than the d(5'-CTAG-3')(2) site formerly believed to be the best binding site. The present paper discusses these results in terms of different contributions to the binding affinity and offers some explanations for the site selectivity of TOTO.     

Concerted intercalation and minor groove recognition of DNA by a homodimeric thiazole orange dye

The thiazole orange dye TOTO binds to double-stranded DNA (dsDNA) by a sequence selective bis-intercalation. Each chromophore is sandwiched between two base pairs in a (5'-CpT-3'):(5'-ApG-3') site, and the linker spans two base pairs in the minor groove. We have used one- and two-dimensional NMR spectroscopy to examine the dsDNA binding of an analogue of TOTO in which the linker has been modified to contain a bipyridyl group (viologen) that has minor groove binding properties. We have investigated the binding of this analogue, called TOTOBIPY, to three different dsDNA sequences containing a 5'-CTAG-3', a 5'-CTTAG-3', and a 5'-CTATAG-3' sites, respectively, demonstrating that TOTOBIPY prefers to span three base pairs. The many intermolecular NOE connectivities between TOTOBIPY and the d(CGCTTAGCG):d(CGCTAAGCG) oligonucleotide in the complex shows that the bipyridyl-containing linker is positioned in the minor groove and spans three base pairs. Consequently, we have succeeded in designing and synthesizing a ligand that recognizes an extended recognition sequence of dsDNA as the result of a concerted intercalation and minor groove binding mode.     

The solution structure of a locked nucleic acid (LNA) hybridized to DNA

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2'-O, 4'-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional 1H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32A. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3 A. The average twist over the sequence are 35.9 degrees +/- 0.3 degrees. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by 1H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5'-CT(L)AG-3' site than in the unmodified 5'-CTAG-3' site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.     

Dynamic Bis-Intercalation of a Homodimeric Thiazole Orange Dye in DNA: Evidence of Intercalator Creeping

Abstract

We have used one and two dimensional exchange ¹H NMR spectroscopy to characterize the dynamics of the binding of a homodimeric thiazole orange dye, 1,1′-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO), to double stranded DNA (dsDNA). The double stranded oligonucleotides used were d-(CGCTAGCG)₂ ( 1 ) and d(CGCTAGCTAGCG)₂ ( 2 ). TOTO binds preferentially to the (5′-CTAG-3′)₂ sites and forms mixtures of 1:1 and 1:2 dsDNA-TOTO complexes with 2 in ratios dependent on the relative amount of TOTO and the oligonucleotide in the sample. The dynamic exchange between preferential binding sites in the case of a 2:1 1 -TOTO mixture is an intermolecular exchange process between two binding sites on different oligonucleotides. In the case of the 1:1 2 -TOTO complex an intramolecular exchange process occur between two different binding sites on the same strand. Both processes were studied. The results demonstrate the ability of TOTO to migrate along a dsDNA strand in an intramolecular exchange process. The migration process (“creeping”) along the DNA strand is 6 times faster than the rate of intermolecular exchange between sites in two different oligonucleotides.     

The Solution Structure of a Locked Nucleic Acid (LNA) Hybridized to DNA

Abstract

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2′-0, 4′-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional'H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32Å. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3Å. The average twist over the sequence are 35.9° ± 0.3°. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by ¹H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5′-CT^LAG-3′ site than in the unmodified 5′-CT^LAG-3′ site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.     

Solution structure and energy calculation of bis-intercalation of homodimeric thiazole orange dye derivatives in DNA: effects of modifying the linker.

We have used two-dimensional (1)H NMR spectroscopy obtained at 750 MHz to determine a high-resolution solution structure of the double-stranded DNA oligonucleotide d(5'-CGCTAGCG-3')(2) complexed with the bis-intercalating dye 1,1'-(5,5,9,9-tetramethyl-5, 9-diazatridecamethylene)-bis-4-[3-ethyl-2,3-dihydro(benzo-1, 3-thiazolyl)-2-methylidene]quino-linium tetraiodide (TOTO11Et). The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross-peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the TOTO11Et complex from A-form and B-form dsDNA starting structures. The root-mean-square (rms) deviation of the coordinates for the 40 structures of the complex was 0.52 A. A conformational analysis of the deoxyribose rings based on coupling constants obtained from selective DQF-COSY spectra revealed that all ring conformations were almost pure S-type. The structure of the TOTO11Et complex was compared with the structure of a similar DNA complex with a dye containing a shorter linker (TOTOEt). Substantial differences were observed between the two structures because of the difference in the length of the linker. Most prominent was a large difference in the degree of unwinding of the dsDNA part in the two complexes. Unwinding of 73 degrees and 22 degrees relative to the free dsDNA was observed for the complexes with TOTOEt and TOTO11Et, respectively. The AMBER94 force field together with the GB/SA solvation model was used for energy calculations on both of the two complexes. In the calculations, the complex formation was divided into two steps: (i) unwinding of the free oligonucleotide and (ii) association of the bis-intercalators to the unwound oligonucleotide. The complex formation was in favor of TOTO11Et, mainly because the dsDNA is distorted less in the complex with TOTO11Et than in the complex with TOTOEt.     

Interaction of dimeric intercalating dyes with single-stranded DNA.

The unsymmetrical cyanine dye thiazole orange homodimer (TOTO) binds to single-stranded DNA (ssDNA, M13mp18 ssDNA) to form a fluorescent complex that is stable under the standard conditions of electrophoresis. The stability of this complex is indistinguishable from that of the corresponding complex of TOTO with double-stranded DNA (dsDNA). To examine if TOTO exhibits any binding preference for dsDNA or ssDNA, transfer of TOTO from pre-labeled complexes to excess unlabeled DNA was assayed by gel electrophoresis. Transfer of TOTO from M13 ssDNA to unlabeled dsDNA proceeds to the same extent as that from M13 dsDNA to unlabeled dsDNA. A substantial amount of the dye is retained by both the M13 ssDNA and M13 dsDNA even when the competing dsDNA is present at a 600-fold weight excess; for both dsDNA and ssDNA, the pre-labeled complex retains approximately one TOTO per 30 bp (dsDNA) or bases (ssDNA). Rapid transfer of dye from both dsDNA and ssDNA complexes is seen at Na+ concentrations > 50 mM. Interestingly, at higher Na+ or Mg2+ concentrations, the M13 ssDNA-TOTO complex appears to be more stable to intrinsic dissociation (dissociation in the absence of competing DNA) than the complex between TOTO and M13 dsDNA. Similar results were obtained with the structurally unrelated dye ethidium homodimer. The dsDNA- and ssDNA-TOTO complexes were further examined by absorption, fluorescence and circular dichroism spectroscopy. The surprising conclusion is that polycationic dyes, such as TOTO and EthD, capable of bis-intercalation, interact with dsDNA and ssDNA with very similar high affinity.     

Two-dimensional 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex and a quinoxaline ((MeCys3, MeCys7]TANDEM) drug duplex complex

Assignment of the 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex, d(CCCGATCGGG), and its quinoxaline ((MeCys3, MeCys7]TANDEM) drug duplex complex has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. The 31P chemical shifts of this 10 base pair oligonucleotide follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. While the 31P chemical shifts show sequence-specific variations, they also do not generally follow the Calladine "rules" previously demonstrated. 31P NMR also provides a convenient monitor of the phosphate ester backbone conformational changes upon binding of the drug to the duplex. Although the quinoxaline drug, [MeCys3, MeCys7]TANDEM, is generally expected to bind to duplex DNA by bis-intercalation, only small 31P chemical shift changes are observed upon binding the drug to duplex d(CCCGATCGGG). Additionally, only small perturbations in the 1H NMR and UV spectra are observed upon binding the drug to the decamer, although association of the drug stabilizes the duplex form relative to the other states. These results are consistent with a non-intercalative mode of association of the drug. Modeling and molecular mechanics energy minimization demonstrate that a novel structure in which the two quinoxaline rings of the drug binds in the minor groove of the duplex is possible.     

Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides.

Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.     

Soluble expression and purification of tumor suppressor WT1 and its zinc finger domain

Full length murine WT1 and its zinc finger domain were separately inserted into Escherichia coli expression vectors with various fusion tags on either terminus by Gateway technology (Invitrogen) and expression of soluble protein was assessed. Fusion proteins including the four zinc finger domains of WT1 were used to optimize expression and purification conditions and to characterize WT1:DNA interactions in the absence of WT1:WT1 interactions. Zinc finger protein for in vitro characterization was prepared by IMAC purification of WT1 residues 321-443 with a thioredoxin-hexahistidine N-terminal fusion, followed by 3C protease cleavage to liberate the zinc fingers and cation exchange chromatography to isolate the zinc fingers and reduce the level of the truncated forms. Titration of zinc finger domain with a binding site from the PDGFA promoter gave a K(d) of 100±30nM for the -KTS isoform and 130±40nM for the +KTS isoform. The zinc finger domain was also co-crystallized with a double-stranded DNA oligonucleotide, yielding crystals that diffract to 5.5?. Using protocols established for the zinc finger domain, we expressed soluble full-length WT1 with an N-terminal thioredoxin domain and purified the fusion protein by IMAC. In electro-mobility shift assays, purified full-length WT1 bound double-stranded oligonucleotides containing known WT1 binding sites, but not control oligonucleotides. Two molecules of WT1 bind an oligonucleotide presenting the full PDGFA promoter, demonstrating that active full-length WT1 can be produced in E. coli and used to investigate WT1 dimerization in complex with DNA in vitro.     

DNA binding properties of key sandramycin analogues: systematic examination of the intercalation chromophore

The examination of a key series of chromophore analogues of sandramycin (1) is detailed employing surface plasmon resonance to establish binding constants within a single high affinity bis-intercalation binding site 5'-d(GCATGC)2, and to establish the preference for sandramycin binding to 5'-d(GCXXGC)2 where XX=AT, TA, GC, and CG. From the latter studies, sandramycin was found to exhibit a preference that follows the order: 5'-d(GCATGC)2 > 5'-d(GCGCGC)2, delta deltaG(o)= 0.4 kcal/mol > 5'-d(GCTAGC)2, delta deltaG(o) = 0.9 kcal/mol> or =5'-d(GCCGGC)2, delta delta G(o) = 1.0 kcal/mol although it binds with high affinity to all four deoxyoligonucleotides. The two highest affinity sequences constitute repeating 5'-PuPy motifs with each intercalation event occurring at a 5'-PyPu step. The most effective sequence constitutes the least stable duplex, contains the sterically most accessible minor groove central to the bis-intercalation site, and the ability to accept two gly-NH/T C2 carbonyl H-bonds identified in prior NMR studies. Similarly, the contribution of the individual structural features of the chromophore were assessed with the high affinity duplex sequence 5'-d(GCATGC)2. In addition to the modest affinity differences, one of the most distinguishing features of the high affinity versus lower affinity bis-intercalation or mono-intercalation directly observable by surface plasmon resonance was the temporal stability of the complexes characterized by the exceptionally slow off-rates.     

Interaction of platinum compounds with short oligodeoxynucleotides containing guanine and cytosine

The products resulting from reaction of cis-Pt(NH3)2Cl2 with d(CpCpGpG), d(GpCpG), d(pCpGpCpG), d(pGpCpGpC) and d(CpGpCpG) and from reaction of [Pt(dien)Cl]Cl with d(CpCpGpG) and d(GpCpG) have been characterized with the aid of proton NMR spectroscopy, circular dichroic spectroscopy and Pt analysis. The binding sites of the Pt compounds were determined by pH-dependent NMR spectroscopy. Binding of the two Pt compounds invariably occurs at the guanine N7 atoms. In all compounds containing [cis-Pt(NH3)2]2+ chelates are formed by coordination of platinum to two guanines of the same oligonucleotide. The resulting intrastrand-cross-linked oligonucleotides contain either d(GpG) . cisPt units, or d(GpCpG) . cisPt units. In the latter case the middle cytosine is not coordinated to platinum. As a result the conformational changes originating from these two chelates are different from each other. In the case of [Pt(dien)Cl]Cl as a starting product, two types of oligonucleotide adducts are formed, i.e. those with one Pt atom/molecule and those with two Pt atoms/molecule. The NMR spectra of the adducts containing only one Pt(dien)2+ show that only one adduct is formed, although two guanine bases are present. This indicates a preference for one of the N7 atoms in the molecule.     

Hybridization of antisense oligonucleotides with the 3'part of tRNA(Phe)

The interaction of antisense oligodeoxyribonucleotides with yeast tRNA(Phe) was investigated. 14-15-mers complementary to the 3'-terminal sequence including the ACCA end bind to the tRNA under physiological conditions. At low oligonucleotide concentrations the binding occurs at the unique complementary site. At higher oligonucleotide concentrations, the second oligonucleotide molecule binds to the complex due to non-perfect duplex formation in the T-loop stabilized by stacking between the two bound oligonucleotides. In these complexes the acceptor stem is open and the 5'-terminal sequence of the tRNA is accessible for binding of a complementary oligonucleotide. The results prove that the efficient binding of oligonucleotides to the 3'-terminal sequence of the tRNA occurs through initial binding to the single-stranded sequence ACCA followed by invasion in the acceptor stem and strand displacement.     

Measuring the Binding Stoichiometry of HIV-1 Gag to Very-Low-Density Oligonucleotide Surfaces Using Surface Plasmon Resonance Spectroscopy

The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions, and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)(n) oligonucleotides using surface plasmon resonance (SPR) spectroscopy. We found a single NC protein is able to bind to more than one immobilized oligonucleotide, provided that the oligonucleotides are close enough together. As NC is believed to be the nucleic acid binding domain of Gag, we might expect Gag to show the same complex behavior. We wished to analyze the stoichiometry of Gag binding to oligonucleotides without this complication due to tertiary complex formation. We have therefore analyzed Gag binding to extremely low oligonucleotide density on SPR chips. Such low densities of oligonucleotides are difficult to accurately quantitate. We have determined by Fourier transform ion cyclotron (FTICR) mass spectrometry that four molecules of NC bind to d(TG)(10) (a 20-base oligonucleotide). We developed a method of calibrating low-density surfaces using NC calibration injections. Knowing the maximal response and the stoichiometry of binding, we can precisely determine the amount of oligonucleotide immobilized at these very-low-density surfaces (<1 Response Unit). Using this approach, we have measured the binding of Gag to d(TG)(10). Gag binds to a 20-mer with a stoichiometry of greater than 4. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules can bind to this complex.     

Effect of protein binding on ultrafast DNA dynamics: characterization of a DNA:APE1 complex

Synthetic oligonucleotides with a fluorescent coumarin group replacing a basepair have been used in recent time-resolved Stokes-shift experiments to measure DNA dynamics on the femtosecond to nanosecond timescales. Here, we show that the APE1 endonuclease cleaves such a modified oligonucleotide at the abasic site opposite the coumarin with only a fourfold reduction in rate. In addition, a noncatalytic mutant (D210N) binds tightly to the same oligonucleotide, albeit with an 85-fold reduction in binding constant relative to a native oligonucleotide containing a guanine opposite the abasic site. Thus, the modified oligonucleotide retains substantial biological activity and serves as a useful model of native DNA. In the complex of the coumarin-containing oligonucleotide and the noncatalytic APE1, the dye's absorption spectrum is shifted relative to its spectrum in either water or within the unbound oligonucleotide. Thus the dye occupies a site within the DNA:protein complex. This result is consistent with modeling, which shows that the complex accommodates coumarin at the site of the orphaned base with little distortion of the native structure. Stokes-shift measurements of the complex show surprisingly little change in the dynamics within the 40 ps-40 ns time range.     

Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand

We have used DNase I footprinting to examine the binding of five different 17-mer oligonucleotides to a 53-base oligopurine tract containing four pyrimidine interruptions. Although all the expected triplexes formed with high affinity (K(d) approximately 10-50 nM), one oligonucleotide produced a footprint at a second site with about 20-fold lower affinity. We have explored the nature of this secondary binding site and suggest that it arises when each end of the third strand forms a 7-mer triplex with adjacent regions on the duplex, generating a contiguous 14-base triplex with a bulge in the center of the third strand oligonucleotide. This unusual binding mode was examined by use of oligonucleotides that were designed with the potential to form different length third-strand loops of various base composition. We find that triplexes containing single-base bulges are generally more stable than those with dinucleotide loops, though triplexes can be formed with loops of up to nine thymines, generating complexes with submicromolar dissociation constants. These structures are much more stable than those formed by adding two separate 7-mer oligonucleotides, which do not generate DNase I footprints, though a stable complex is generated when the two halves are covalently joined by a hexa(ethylene glycol) linker. MPE produces less clear footprints, presumably because this cleavage agent binds to triplex DNA, but confirms that the oligonucleotides can bind in unexpected places. These results suggest that extra care needs to be taken when designing long triplex-forming oligonucleotides so as to avoid triplex formation at shorter secondary sites.     

NMR investigation of the interaction of mithramycin A with d(ACCCGGGT)2.

The binding of mithramycin A to d(ACCCGGGT)2 has been investigated by one- and two-dimensional 1H NMR spectroscopy. Titration of the drug into the octamer solution results in loss of the oligonucleotide C2 symmetry at stoichiometric ratios less than 4 drug molecules per duplex. However, at a ratio of 4:1 (drug/duplex), the C2 symmetry of the oligonucleotide is restored. From these data it is evident that more than one complex forms at ratios less than 4:1 while only one complex predominates at the ratio 4:1. This is the first report of a DNA octamer which binds 4 large drug molecules. These results are compared to those we have recently reported for mithramycin binding to d(ATGCAT)2, where only a single, bound complex is observed, with a stoichiometry of 2:1.     

1H NMR studies of tris(phenanthroline) metal complexes bound to oligonucleotides: characterization of binding modes

The binding of Ru(phen)3(2+), Rh(phen)3(3+), and Co(phen)3(3+) to the oligonucleotides d(GTGCAC)2 and 5'-pd(CGCGCG)2 has been examined by 1H NMR spectroscopy as a function of temperature, concentration, and chirality of the metal complex. The duplex oligonucleotides act as chiral shift reagents for the metal complexes; phenanthroline protons associated with each enantiomer are resolved upon binding to the oligomer. The spectral titrations, consistent with photophysical studies, indicate that the complexes bind to the oligomer through two modes: one assigned as intercalation favoring the delta-isomer, and the other assigned as the surface-bound interaction favoring the lambda-isomer. The ligand protons are perturbed in a manner that implies sensitivity of particular protons to binding mode; specifically, the H4,7 protons appear to be altered most for the lambda-enantiomer while the H5,6 protons are perturbed more for the delta-enantiomer. The NMR chemical shift variations appear particularly sensitive to this surface-bound interaction, which, on the basis of a comparison of binding and photophysical parameters for Ru(phen)3(2+), appears more prominant in binding to oligonucleotides than that to polynucleotides. With respect to oligonucleotide proton shifts, the adenine H2 proton, positioned in the minor groove of the helix, shows the largest upfield shifts with metal binding, and more dramatically with lambda-isomers. The major groove thymine methyl protons (TMe) shift downfield to a lesser extent, and more so for delta-isomers. The different binding modes also differ with respect to their dynamics of association; the longitudinal relaxation rates of delta- and lambda-4,7 phenanthroline protons of Rh(phen)3(3+) are 0.88 and 1.14 s, respectively, in the presence of d(GTGCAC)2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding, annealing and strand exchange between oligonucleotides in different sites of the RecA protein filament

The efficiency of single-stranded (ss) oligonucleotides binding at the secondary site of the RecA protein filament is demonstrated to depend on the strandedness of DNA bound at the primary site. When the primary site is occupied by a ss-oligonucleotide, the binding of another ss-oligonucleotide at the secondary site is characterized by higher affinity and a lower rate of dissociation than is the case when the primary site is occupied by a double-stranded oligonucleotide. In contrast to a DNA strand exchange reaction suppressed by a heterologous oligonucleotide bound at the secondary site of the RecA filament, the occupation of the secondary site by a heterologous oligonucleotide does not prevent renaturation between the oligonucleotides bound at the primary site and complementary oligonucleotides from solution demonstrating that the binding of a DNA strand in the secondary site is not a necessary intermediate step in RecA-promoted DNA renaturation.