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The remarkable binding properties of LNA (Locked Nucleic Acid) and alpha-L-LNA (the alpha-L-ribo configured diastereoisomer of LNA) are summarized, and hybridization results for LNA/2'-O-Me-RNA chimera and LNAs with a "dangling" nucleotide are introduced. In addition, results from NMR investigations on the furanose conformations of the individual nucleotide monomers in different duplexes are presented. All these data are discussed with focus on the importance of conformational steering of unmodified nucleotides in partly modified LNA and alpha-L-LNA sequences in relation to the unprecedented binding properties of LNA and alpha-L-LNA.

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 structure of a DNA duplex containing one 1-(2-O,3-C-ethylene-beta-D-arabinofuranosyl)-thymidine nucleoside (T5) modification was investigated by use of two-dimensional 1H NMR spectroscopy at 750 MHz. The structure of the d(CCGCT5AGCG):d(CGCTAGCGG) duplex (CT5AG) containing one of this 2'-O,3'-C-linked bicycloarabino conformational restricted modification has been determined. We obtained inter-proton distance bounds from NOESY spectra by including a complete relaxation matrix analysis. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. We also analyzed the fine structure of the cross peaks in a selective DQF-COSY spectra to determine the sugar conformations of the nucleotides. Forty final structures were generated for CT5AG from A-form and B-form dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.92A. The structures were observed to be markedly irregular compared to canonical B-DNA, especially in terms of large variations in propeller twist and buckle. Also, lack of stacking of two bases near the modification site is observed. The sugar conformations of all the unmodified nucleotides are close to pure C2'-endo conformation. The structural feature of CT5AG was discussed in relation to the thermal stability and resistance towards exonucleolytic degradation.

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.

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.

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.

The thiazole orange dye 1,1'-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-bis-4-[(3-methyl-2,3-dihydro-2(3H)-benzo-1 ,3-thiazolylidene)methyl]quinolinium tetraiodide (TOTO) binds to double-stranded DNA (dsDNA) in a sequence selective bis-intercalation. We have examined the binding of derivatives of TOTO with different substituents on the benzothiazole ring. The analogues are the following: 1,1'-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-[4-[3-(benzyl-2, 3-dihydro-2-(3H)-benzothiazolylidene)methyl]quinolinium]-[4-[3-(++ +methy l-2, 3-dihydro-2-(3H)-benzothiazolylidene)methyl]quinolinium]tetraio dide (TOTOBzl) and 1,1'-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-bis-4-[(3-ethyl-2,3-dihydro-2(3H)-benzo-1, 3-thiazole)methyl]quinolinium tetraiodide (TOTOEt). In this paper, we report the synthesis of TOTOBzl and TOTOEt together with the one- and two-dimensional 1H NMR investigations of complexes between these TOTO analogues and the dsDNA oligonucleotide d(CGCTAGCG)2. Both analogues yield extremely stable complexes in which each chromophore is sandwiched between two base pairs in a (5'-CpT-3'):(5'-ApG-3') site. The linker spans over two base pairs in the minor groove. The benzyl group in TOTOBzl and the ethyl groups in TOTOEt is pointing outward in the major groove.

We have used one and two dimensional 1H NMR spectroscopy to characterize the binding of a homodimeric oxazole yellow dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4-( 3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene)-quinoliniu m tetraiodide (YOYO), to oligonucleotides containing the (5'-CTAG-3')2 and the (5'-CCGG-3')2 binding sites in either different oligonucleotides or in the same oligonucleotide. YOYO bis-intercalates strongly in all the oligonucleotides used and binds preferentially to a (5'-CTAG-3')2 binding site in the oligonucleotide d(CGCTAGCG)2 (1). YOYO also binds preferentially to a (5'-CCGG-3')2 sequence in the oligonucleotide d(CGCCGGCG)2 (2) but slightly less favorably than to the (5'- CTAG-3')2 sequence in 1. The binding of YOYO to the d(CGCTAGCCGGCG):d(CGCCGGCTAGCG) (3) oligonucleotide, containing two preferential binding sites, was also examined. YOYO forms mixtures of 1:1 and 1:2 complexes with oligonucleotide 3 in ratios dependent on the relative amount of YOYO and the oligonucleotides in the sample. The binding of YOYO to the oligonucleotide 3 occur sequence selective in the (5'-CTAG-3')2 site and the (5'- CCGG-3')2 site. We have also used two dimensional 1H NMR spectroscopy to determine the solution structure of the DNA oligonucleotide d(5'-CGCTAGCG-3')2 complexed with YOYO. The determination of the structure was based on a total relaxation matrix analysis of the NOESY cross peaks intensities. DQF-COSY spectra were used to obtain coupling constants for the deoxyribose ring protons. The coupling constants were transformed into angle estimates. The NOE derived distance and dihedral restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the YOYO-complex from both A-form and B-form dsDNA starting structures giving a total of 40 final structures. Since many NOE contacts were observed between YOYO and dsDNA the resulting structure has a fairly high resolution and allows determination of local features in the dsDNA structure after YOYO binding. The root-mean-square (rms) deviation of the coordinates for the forty structures of the complex was 0.39 A. The local DNA structure is distorted in the complex. The helix is unwound by 106 degrees and has an overall helical repeat of 13 base pairs caused by the bis-intercalation of YOYO. The polypropylene amine linker chain is located in the minor groove of dsDNA. Even though the YOYO chromophore contains an oxygen atom instead of the larger sulphur atom in the corresponding compound, TOTO, the structures establish that YOYO require more space than TOTO in the intercalation sites. This is probably caused by the more rigid and planar chromophores in YOYO compared to TOTO.

The thiazole orange dye 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis[4-[3-methy l-2, 3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO) binds to double-stranded DNA (dsDNA) in a sequence selective bisintercalation. Each chromophore is sandwiched between two base pairs in a (5'-CpT-3'):(5'-ApG-3') site, and the linker spans over two base pairs in the minor groove. The binding of analogs of TOTO in which the linker has been modified is examined. The aim of the study is to utilize the sequence selectivity of the TOTO chromophores to enhance and/or alter the overall selectivity of the binding. One- and two-dimensional 1H-NMR investigations of complexes between TOTO analogs and various dsDNA oligonucleotides are reported. The following analogs were synthesized and used: 1,1'-(4,4,8,8-tetramethyl-4,8-diazadodecamethylene) -bis[4-[3-methyl-2,3-dihydro- (benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO10), 1,1'-(5,5,9,9-tetramethyl-5,9-diazatridecamethylene)-bis[4-[3-meth yl-2, 3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO11), and 1,1'-(6,6,10,10-tetramethyl-6,10-diazapentadecamethylene)-bis[4-[3 -methyl-2, 3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO13). The results show that with a longer linker the dyes can bisintercalate into two (5'-CpT-3'):(5'-ApG-3') sites separated by one or two base pairs. Bisintercalation in two such "isolated" binding sites yields non-nearest-neighbor bisintercalation in which the linker spans over more than two base pairs. The investigations also showed that an exact length of the linker is not crucial for the site selectivity since TOTO, TOTO10, and TOTO11 are almost equally suitable in binding selectively to the (5'CTAG-3')2 sequence. Fluorescence measurements show that TOTO10, TOTO11, and TOTO13 have higher fluorescence quantum yields than TOTO when bound to d(CGCTAGCG)2. This indicates that the length of the linker in TOTO may not be the optimum one in terms of using the dye as a fluorescence marker.

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)-quinol inium 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.

Two-dimensional 1H NMR spectroscopy was used to determine the solution structure of the double-stranded DNA oligonucleotide d(5'-CGCATATAGCC-3'): d(5'-GGCTAXATGCG-3'), where X is 1-(2-O-methyl-beta-D-arabinofuranosyl)thymine. The structure determination was based on a total relaxation matrix analysis of NOESY cross-peak intensities using the MARDIGRAS program. The improved RANDMARDI procedure was used during the calculations to include the experimental "noise" in the NOESY spectra. The NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the modified DNA duplex from both A-form and B-form DNA starting structures. The root-mean-square deviation of the coordinates for the 40 structures was 0.82 A. The duplex adopts a normal B-DNA-type helix, and the spectra as well as the structure show that the modified nucleotide X adopts a C2'-endo (S) sugar conformation. There are no significant changes in the helix originating from the modified nucleotide. The CH3O group on X is directed toward the major groove, and there seems to be free space for further modifications at this position.

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.

The palindromic deoxyribonucleotides 5'-CGCGA-TATCGCG-3' and 5'-CGCGTTAACGCG-3' have been characterized by 1H NMR spectroscopy. The NMR data identified both B-DNA duplex conformations and hairpin conformations, the latter with loop regions consisting of the four central nucleotides. The resonances of the various conformations were assigned by use of two-dimensional NMR methods. The relative stability of the various conformations was investigated as a function of temperature, ionic strength and nucleotide concentration. The duplexes were found to be stabilized at high ionic strength and at low temperature, while the hairpins were stabilized at low ionic strength and at medium temperature. The thermodynamics of the duplex-hairpin and the hairpin-random coil transitions were examined, and compared to the other two oligonucleotide in the palindromic d(CGCG(A/T)4CGCG) oligonucleotide family. The relative stabilities of the duplex conformations with respect to the random coil conformations are similar for the d(CGCGAATTCGCG), d(CGCGATATCGCG) and d(CGCGTATACGCG) oligonucleotides. The duplex conformation of d(CGCGTTAACGCG) is less stable. The hairpin of d(CGCGTTAACGCG) seems also to be less stable relative to the random coil conformation than in the case of the other oligonucleotides at an equal oligonucleotide concentration. A cruciform intermediate between the duplex and hairpin conformations is suggested to explain some discrepancies observed in this work in case of the d(CGCGTTAACGCG) oligonucleotide. This is similar to what has been reported for the d(CGCGTATACGCG) oligonucleotide.

We have used two-dimensional 1H NMR spectroscopy to determine the solution structure of the DNA oligonucleotide d(5'-CGCTAGCG-3')2 complexed with the bis-intercalating dye 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis[4-(3-methyl -2,3- dihydrobenzo-1,3-thiazolyl-2-methylidene)qui nolinium] tetraiodide (TOTO). The determination of the structure was based on total relaxation matrix analysis of the NOESY cross-peak intensities using the program MARDIGRAS. Improved procedures to consider the experimental "noise" in NOESY spectra during these calculations have been employed. The NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the TOTO complex from both 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 1.45 A. The local DNA structure is distorted in the complex. The helix is unwound by 60 degrees and has an overall helical repeat of 12 base pairs, caused by bis-intercalation of TOTO. The poly(propylenamine) linker chain is located in the minor groove of dsDNA. Calculations indicate that the benzothiazole ring system is twisted relative to the quinoline in the uncomplexed TOTO molecule. The site selectivity of TOTO for the CTAG-CTAG site is explained by its ability to adapt to the base pair propeller twist of dsDNA to optimize stacking and the hydrophobic interaction between the thymidine methyl group and the benzothiazole ring. There is a 3000-fold fluorescence enhancement upon binding of TOTO to dsDNA. Rotation about the cyanine methine bonds is possible in free TOTO, allowing relaxation nonradiatively. When bound to dsDNA, the benzothiazole ring and the quinolinium ring are clamped by the nucleobases preventing this rotation, and the chromophore loses excitation energy by fluorescence instead.

We have used one and two dimensional 1H NMR spectroscopy to characterize 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)-quinolin ium tetraiodide (TOTO), to various double stranded DNA oligonucleotides. TOTO binds strongly to all the oligonucleotides used, but usually more than one complex is observed and exchange between different binding sites broadens the lines in the NMR spectra. Complete precipitation occurs when TOTO is bound to small oligonucleotides. Binding to larger oligonucleotides occurs by bis-intercalation. The 1:1 complex of TOTO with the oligonucleotide d(CCGACTGATGC):d (GCATCAGTCGG) gave only one complex that was shown to be a bis-intercalation in the CTGA:TCAG binding site. The binding to this site was also characterized by studying the TOTO complex with the d(CCGCTGAGC):d(GCTCAGCGG) oligonucleotide. NOE connectivities and molecular modelling were used to characterize the complex. The 1:1 complex of TOTO with the oligonucleotide d(CCGCTAGCG):d(CGCTAGCGG) containing a CTAG:CTAG binding site was similarly characterized by NMR. It was concluded that the binding of TOTO to larger oligonucleotides is site selective with CTAG:CTAG as the preferred binding site.

NMR spectroscopy combined with molecular modeling was used to characterize a heterodimeric complex with Dst and 2-ImN bound in the minor groove of d(GCCTAACAAGG).d(CCTTGTTAGGC) (1:1:1 2-ImN.Dst.DNA complex). The imidazole-pyrrole-pyrrole ligand 2-ImN spans 5'-GTTA-3' of the TAACA.TGTTA binding site with the imidazole nitrogen specifically recognizing the guanine amino group. The Dst ligand lies along the 5'-AACA-3' sequence and complements the 2-ImN ligand in the formation of the antiparallel side-by-side heterodimeric complex. Titrations of the same site with Dst or 2-ImN alone yield homodimeric complexes (2:1 ligand.DNA) of lower stability than the 1:1:1 2-ImN.Dst.DNA complex. Dst and 2-ImN binding to d(CGCAAACTGGC).d(GCCAGTTTGCG) was also investigated. The 1:1:1 2-ImN.Dst.DNA complex is again the most stable complex with the AAACT.AGTTT site and is similar to the TAACA.TGTTA complex. No monomeric binding of either 2-ImN or Dst was observed to either site.

Sodium-23 spin-lattice relaxation rate (the reciprocal relaxation time) measurements have been used to study the intercalation of 9-aminoacridine in calf thymus DNA. The results are analyzed by a two state model based on the counterion condensation theory and a theory for the quadrupolar relaxation of counterions in polyelectrolyte solutions. It is shown that change of the solvent from H2O to D2O has a negligible effect on the intercalation process. Furthermore, an attempt is made to analyze the dependence of the 7Li spin-lattice relation rate on intercalation of 9-aminoacridine in LiDNA. It is shown that both quadrupolar and dipolar mechanisms contribute to the bound 7Li relation rate, and that both these contributions are reduced upon intercalation of 9-aminoacridine.

A series of 9-(arylamino)-1,2,3,4-tetrahydroacridines, including the tetrahydro m-AMSA [N-[4-(acridin-9-yl-amino)-3- methoxyphenyl]methanesulfonamide] derivative, has been synthesized. 23Na NMR spin-lattice relaxation rate (1/T1) measurements have been used to study whether these hydrogenated acridines were capable of intercalative binding to calf thymus DNA. The results have been compared to corresponding measurements for 9-aminoacridine, m-AMSA, and MgCl2. All compounds studied were capable of intercalative binding to DNA. However, it was found that the interaction was strongly influenced by substituents on the 9-arylamino group. Thus, tetrahydro m-AMSA was found to intercalate much more weakly with DNA than m-AMSA. Removal of the 3'-methoxy substituent of the 9-arylamino group resulted in intercalation in DNA that was almost as strong as that for m-AMSA.

23Na spin-lattice relaxation rate (1/T1 = R1) measurements have been used to study the intercalation of a series of 9-aminoacridine derivatives in DNA. The 23Na relaxation rate is strongly dependent upon the amount of intercalator added to a sodium DNA solution. The results are analyzed by a combined use of the ion condensation theory and the quadrupolar relaxation theory of polyelectrolyte solutions. This interpretation shows that the major effect in lowering the relaxation rate by intercalation is not due to the release of sodium ions but is caused by a substantial decrease in the relaxation rate Rb for the remaining bound sodium ions. Likewise, titration of NaDNA solutions with MgCl2 shows that condensation of Mg2+ on the DNA double helix reduces Rb. A good agreement between experiment and theory is found if the average lengthening following intercalation of a 9-aminoacridine moiety is assumed to be approximately 2.7 A. The distinction between mono- and bis-intercalation is clearly indicated by the results. The two bis-intercalating drugs examined are found to bis-intercalate only up to r less than or equal to 0.02. For r greater than 0.02 the drugs apparently mono-intercalate.