Phase diagrams for DNA crystallization systems

Phase diagrams for several oligonucleotide duplex-spermine systems have been constructed. These diagrams characterize the duplex and spermine concentrations ranges in which crystalline precipitates are formed. All of them are wedge-like form. The slope of the upper branch of the diagram is determined by the oligonucleotide length. The position of the lower branch depends on both the nucleotide sequence and its length. The position of the lower branch depends on both the nucleotide sequence and its length. It has been shown that the addition to the system of MgCl2 and NaCl salts and MPD results in specific changes in the diagrams. A model for oligonucleotide duplex-spermine system has been suggested which explains the main characteristic features of the obtained phase diagrams. The experimental phase diagrams for the (pGpT)n (pApC)n-spermine system (n = 2,3,4) have been analyzed ion terms of this model and the values of the binding constants of spermine and Mg2+ ions binding to duplexes have been determined. It permitted to identify the complexes that precipitated in different regions of the phase diagrams under various conditions. The diagram obtained in the presence of a cobalt hexammine counterion is also considered. It has been shown that this phase diagram, in general, is similar to those obtained for the oligonucleotide duplex-spermine system.     

The use of phase diagrams in the crystallization of oligonucleotide duplexes. I. A model of crystallization of the (pGpT)n.(pApC)n + spermine system

A set of experimental phase diagrams revealing the region of existence of microcrystals in mixture "(pGpT)n.(pApC)n+spermine", n = 2,3,4, was obtained. All diagrams are wedge-like with the slope of the upper branch and the level of the lower one depending on the oligonucleotidd length. The presence of MPD, MgCl2 and NaCl changes the form of the diagrams in a different manner. A model explaining the peculiar features of the diagrams for mixture "oligonucleotide duplex+spermine" is proposed. The analysis of the diagrams was carried out on the basis of this model and the values of the binding constants for binding of spermine and Mg2+ to duplexes were estimated. Some conclusions about the types of complexes, which may form microcrystals in different regions of diagrams were made.     

The mixing behavior of pseudobinary phosphatidylcholine-phosphatidylglycerol mixtures as a function of pH and chain length

The miscibility of phosphatidylcholine (PC) and phosphatidylglycerol (PG) with different chain lengths (n = 14, 16) was examined by differential scanning calorimetry (DSC) at pH 2 and pH 7. The determination of the coexistence curves of the phase diagrams was performed using a new procedure, namely the direct simulation of the heat capacity curves as described recently (Johann et al. 1996, Garidel et al. 1997). From the simulations of the heat capacity curves first estimates for the nonideality parameters for nonideal mixing as a function of composition were obtained and phase diagrams were constructed using temperatures for the onset and offset of melting which were corrected for the broadening effect caused by a decrease in cooperativity of the transition. In most cases, the composition dependence of the nonideality parameters indicated nonsymmetric mixing behavior. The phase diagrams were further refined by simulations of the coexisting curves using a four-parameter model to account for nonideal and nonsymmetric mixing in the gel as well as in the liquid-crystalline phase. The mixing behavior of the systems was analyzed as a function of pH and chain length difference to elucidate the effect of these two parameters on the shape of the phase diagrams. At pH 7 the phase boundaries are much closer together and a narrower coexistence range is obtained compared to the corresponding phase diagrams at pH 2. For DPPC/DMPG at pH 2, the shape of the phase diagram and the strongly positive nonideality parameter ρ ₁ for the liquid-crystalline phase indicates an upper azeotropic point. This indicates an unusual behavior of the system, namely more pronounced clustering of like molecules in the liquid-crystalline phase compared to the gel phase. Received: 17 March 1997 / Accepted: 4 July 1997     

Invasion of complementary oligonucleotides into (CA/TG)31 repetitive region of linear and circular DNA duplexes

(CA/TG)_n repeats belong to microsatellite DNA. They are the most abundant among the other dinucleotide repeats in mammals, constituting approximately 0.25% of the entire genome. These repeats are recombination hot spots; however, the corresponding mechanisms are yet vague. We postulated that one of the reasons underlying an increase in the recombination frequency in the repetitive region could be the con-formational characteristics of duplex resulting from a specific geometry of base-stacking contacts, providing for initiation of a single-stranded DNA invasion in th e duplex homologous regions. This work for the first time demonstrates a DNA-DNA interaction of the d(CA)₁₀ and d(TG)₁₀ oligonucleotides with linear and circular duplexes containing (CA/TG)₃₁ repeats during their coincubation in a protein-free water solution at 37°C. Using radioactively labeled oligonucleotides, we demonstrated that the duplex—oligonucleotide interaction intensity depended on the molar ratio of duplex-to-oligonucleotide at a duplex concentration of 30 nM. A decrease in this concentration to 3 nM had no effect on the intensity of oligonucleotide invasion. It was demonstrated that over 1% of the duplexes yet much less than 10% were involved in the interaction with oligonucleotides assuming that one oligonucleotide molecule interacted with one molecule of the duplex. Analysis of the kinetics showed that d(CA)₁₀ invasion commenced from the first minute of incubation with duplexes, while d(TG)₁₀ interacted with the duplex even at a higher rate. The role of conformational plasticity of CA/TG repeats in the discovered interaction is discussed as well as its biological significance, in particular, the role of CA microsatellites in the initiation of homologous recombination.     

2D 1H and 31P NMR Spectra and Distorted A-DNA-Like Duplex Structure of a Phosphorodithioate Oligonucleotide

Abstract

Assignment of the ¹H and ³¹P NMR spectra of a phosphorodithioate modified oligonucleotide decamer duplex, d(CGCTTpS^? ₂AAGCG)₂ (10-mer-S; a site of dithioate substitution is designated with the symbols pS^? ₂), was achieved by two-dimensional homonuclear TOCSY, NOES Y and ¹H-³¹P Pure Absorption phase Constant time (PAC) heteronuclear correlation spectroscopy. In contrast to the parent palindromic decamer sequence (1) which has been shown to exist entirely in the duplex B-DNA conformation under comparable conditions (100 mM KCI), the dithiophosphate analogue forms a hairpin loop. However, the duplex form of the dithioate oligonucleotide can be stabilized at lower temperatures, higher salt and strand concentration. The solution structure of the decamer duplex was calculated by an iterative hybrid relaxation matrix method (MORASS) combined with 2D NOESY-distance restrained molecular dynamics. These backbone modified compounds, potentially attractive antisense oligonucleotide agents, are often assumed to possess similar structure as the parent nucleic acid complex. Importantly, the refined structure of the phosphorodithioate duplex shows a significant deviation from the parent unmodified, phosphoryl duplex. An overall bend and unwinding in the phosphorodithioate duplex is observed. The structural distortion of the phosphorodithioate duplex was confirmed by comparison of helicoidal parameters and groove dimensions. Especially, the helical twists of the phosphorodithioate decamer deviate significantly from the parent phosphoryl decamer. The minor groove width of phosphorodithioate duplex 10-mer-S varies between 8.4 and 13.3 Å which is much wider than those of the parent phosphoryl decamer d(CGCTTAAGCG)₂ (4.2～9.4Å). The larger minor groove width of 10-mer-S duplex contributes to the unwinding of the backbone and indicates that the duplex has an overall A-DNA-like conformation in the region surrounding the dithiophosphate modification.     

Controlling nucleic acid secondary structure by intercalation: effects of DNA strand length on coralyne-driven duplex disproportionation

Small molecules that intercalate in DNA and RNA are powerful agents for controlling nucleic acid structural transitions. We recently demonstrated that coralyne, a small crescent-shaped molecule, can cause the complete and irreversible disproportionation of duplex poly(dA)·poly(dT) into triplex poly(dA)·poly(dT)·poly(dT) and a poly(dA) self- structure. Both DNA secondary structures that result from duplex disproportionation are stabilized by coralyne intercalation. In the present study, we show that the kinetics and thermodynamics of coralyne-driven duplex disproportionation strongly depend on oligonucleotide length. For example, disproportionation of duplex (dA)₁₆·(dT)₁₆ by coralyne reverts over the course of hours if the sample is maintained at 4°C. Coralyne-disproportioned (dA)₃₂· (dT)₃₂, on the other hand, only partially reverts to the duplex state over the course of days at the same temperature. Furthermore, the equilibrium state of a (dA)₁₆·(dT)₁₆ sample in the presence of coralyne at room temperature contains three different secondary structures [i.e. duplex, triplex and the (dA)₁₆ self-structure]. Even the well-studied process of triplex stabilization by coralyne binding is found to be a length-dependent phenomenon and more complicated than previously appreciated. Together these observations indicate that at least one secondary structure in our nucleic acid system [i.e. duplex, triplex or (dA)_n self-structure] binds coralyne in a length-dependent manner.     

A double-headed nucleotide with two cytosines: DNA with condensed information and improved duplex stability

Double-headed nucleotide monomers are capable of condensing the genetic information of DNA. Herein, a double-headed nucleotide with two cytosine bases (C_C) is constructed. The additional cytosine is connected through a methylene linker to the 2′-position of arabinocytidine. The nucleotide is incorporated into oligonucleotides and its effect on duplex stability is studied. For single incorporations, a thermal stabilization of 4.0?°C is found as compared to the unmodified duplex and it is shown that both nucleobases of C_C participate in Watson-Crick base pairing. In combination with the previously published U_T monomer, it is also shown that multiple incorporations are tolerated. For instance, a 16-mer sequence is targeted by a 13-mer oligonucleotide by using one C_C and two U_T monomers without compromising the overall duplex stability. Finally, the potential of double-headed nucleotides in triplex-forming oligonucleotides is studied, however, with the conclusion that the present design is not well-suited for this function.     

Characteristics of the interactions of cortisol-apolipoprotein A-I and tetrahydrocortisol-apolipoprotein A-I complexes with eukaryotic DNA

Primary hepatocyte culture has been used to demonstrate that the cortisol-apolipoprotein A-I complex does not affect the DNA and protein biosynthesis rates, whereas the tetrahydrocortisol-apolipoprotein A-I complex (THC-apoA-I) substantially increases the rates of ³H-thymidine and ¹⁴C-leucine incorporation into DNA and protein, respectively. Small-angle X-ray scattering data show that only THC-apoA-I effectively interacts with eukaryotic DNA, which is accompanied by local DNA melting. The (GCC)_n repeat, a component of many human and other eukaryotic genes, is the most probable region of the interaction of this complex with DNA. An oligonucleotide (duplex) of this type has been synthesized. Its interaction with THC-apoA-I yields a larger complex, which breaks up, giving rise to complementary oligonucleotide strands. They also interact with THC-apoA-I. The equilibrium kinetics of this multiphase process is described. The interaction of the cortisol-apolipoprotein A-I complex with the duplex is less specific and does not cause its breakup or the formation of complementary oligonucleotides.     

Interaction of spermine and DNA

The effect of spermine upon the denaturation temperature (T_m) of DNA's of various base compositions has been found to depend upon both the base composition of the DNA and the pH of the solution. Measurement of the hydrogen ion titration curve of spermine as a function of temperature reveals that the net charge of the spermine molecule is undergoing a rapid change with temperature in the range of temperatures at which DNA denatures. Since the value of T_m depends upon base composition, the correlation of the effect of spermine upon T_m with the base composition of the DNA used may be explainable in terms of the changing degree of ionization of spermine. The binding of spermine to native DNA has also been studied by dialysis equilibrium. There is no significant variation either in the number of strongly binding sites or strength of binding with base composition. It is concluded that there is no evidence of correlation between the binding of spermine and the base composition of DNA.     

Oligonucleotide–Minor Groove Binder Conjugates and Their Complexes with Complementary DNA: Effect of Conjugate Structural Factors on the Thermal Stability of Duplexes

Synthetic polycarboxamide minor groove binders (MGB) consisting of N‐methylpyrrole (Py), N‐methylimidazole (Im), N‐methyl‐3‐hydroxypyrrole (Hp) and β‐alanine (β) show strong and sequence‐specific interaction with the DNA minor groove in side‐by‐side antiparallel or parallel orientation. Two MGB moieties covalently linked to the same terminal phosphate of one DNA strand stabilize DNA duplexes formed by this strand with a complementary one in a sequence‐specific manner, similarly to the corresponding mono‐conjugated hairpin structures. The series of conjugates with the general formula Oligo‐(L‐MGB‐R)_m was synthesized, where m = 1 or 2, L = linker, R = terminal charged or neutral group, MGB = –(Py)_n–, –(Im)_n– or –[(Py/Im)_n–(CH₂)₃CONH–(Py/Im)_n–] and 1 < n < 5. Using thermal denaturation, we studied effects of structural factors such as m and n, linker L length, nature and orientation of the MGB monomers, the group R and the backbone (DNA or RNA), etc. on the stability of the duplexes. Structural factors are more important for linear and hairpin monophosphoroamidates than for parallel bis‐phosphoroamidates. No more than two oligocarboxamide strands can be inserted into the duplex minor groove. Attachment of the second sequence‐specific parallel ligand [–L(Py)₄R] to monophosphoroamidate conjugate CGTTTATT–L(Py)₄R leads to the increase of the duplex Tm, whereas attachment of [–L(Im)₄R] leads to its decrease. The mode of interaction between oligonucleotide duplex and attached ligands could be different (stacking with the terminal A:T pair of the duplex or its insertion into the minor groove) depending on the length and structure of the MGB.     

Interaction of methyl green with an oligonucleotide in intramolecular duplex and triplex conformations

Interaction of methyl green with the oligonucleotide 5′-dGGAAAAGG-[T₄]-GGAAAAGG-[T₄]-CCTTTTCC (where [T₄] is a nucleotide sequence of four thymines) in hairpin duplex and in intramolecular triplex structures has been studied by circular dichroism. We found that methyl green binding to the duplex form shows a complex pattern, exhibiting an exciton contribution when the number of bound molecules increases. Differences between this pattern and previously published results on other DNAs reveals the presence of different types of complexes. In contrast to previous findings with the triple helix poly(dA).2poly(dT) we show that the methyl green is not totally excluded from this triplex structure made of Pur:Pur:Pyr triplets. Received: 15 July 1997 / Accepted: 22 September 1997     

SYNTHESIS AND CHARACTERIZATION OF DNA DUPLEXES CONTAINING AN N3T-ETHYL-N3T INTERSTRAND CROSSLINK IN OPPOSITE ORIENTATIONS

DNA duplexes containing an ethyl interstrand crosslink that bridges the N³ atoms of thymidines on the opposite strands have been synthesized using an approach that combines conventional solid phase oligonucleotide synthesis and the selective removal of protecting groups of a crosslinked thymidine dimer. This approach allows for the assembly of a crosslinked duplex directly on the solid support. Duplexes that contain a N³T-ethyl-N³T interstrand crosslink in a staggered orientation at either a -TA- or -AT- step in a duplex have been prepared. When placed in an -AT- step of a duplex the effect was stabilizing relative to the non-crosslinked control duplex (ΔT_m=+24°C) and this crosslinked duplex was found to efficiently form multimers in the presence of T4 ligase. In the case of the -TA- crosslinked duplex the stabilizing effect was less pronounced (ΔT_m=+6°C) and likewise did not undergo self ligation under identical conditions. Molecular modeling studies suggested that the -AT- containing lesion had little deviation in structure relative to the non-crosslinked duplex DNA control, whereas the -TA- crosslinked duplex exhibited significant buckling of the base pairs flanking the lesion.     

Parallel-Stranded DNA with Natural Base Sequences

Noncanonical parallel-stranded DNA double helices (ps-DNA) of natural nucleotide sequences are usually less stable than the canonical antiparallel-stranded DNA structures, which ensures reliable cell functioning. However, recent data indicate a possible role of ps-DNA in DNA loops or in regions of trinucleotide repeats connected with neurodegenerative diseases. The review surveys recent studies on the effect of nucleotide sequence on preference of one or other type of DNA duplex. (1) Ps-DNA of mixed AT/GC composition was found to have conformational and thermodynamic properties drastically different from those of a Watson–Crick double helix. Its stability depends strongly on the specific sequence in a manner peculiar to the ps double helix, because of the energy disadvantage of the AT/GC contacts. The AT/GC boundary facilitated flipping of A and T out of the ps double helix. Proton acceptor groups of bases are exposed into both grooves of the ps-DNA and are accessible to solvent and ligands, including proteins. (2) DNA regions containing natural minor bases isoguanine and isomethylisocytosine were shown to form ps-DNA with transAT-, trans isoGC, and transiso^5meCG pairs exceeding in stability a related canonical duplex. (3) Nucleotide sequence dG(GT)₄G from yeast telomeres and microsatellites was demonstrated to form novel ps-DNA with GG and TT base pairing. Unlike d(GT) _n - and d(G _n T _m ) sequences able to form quadruplexes, the dG(GT)₄G sequence formed no alternative double- or multistranded structures in a wide range of experimental conditions, thus suggesting that the nucleotide context governs the observed structural polymorphism of the d(GT) _n sequence. The possible biological role of ps-DNA and the prospects of its study are discussed.     

Disturbing effect of perdeuterated fatty acids used as probes in phospholipid monolayers

Surface pressure-area per molecule isotherms have been obtained for tetradecanoic acid (C₁₄H) and perdeuterated tetradecanoic acid (C₁₄²H) and their mixtures at air/water interface. The perdeuterated fatty acid was then used as a probe to evaluate the consequent disturbing effect of perdeuteration in dimyristoyl (DMPC) and dipalmitoyl (DPPC) phosphatidylcholine monolayers used as model membranes. It appears from the analysis of the transition pressure variation versus mole fraction of the probe that C₁₄H/C₁₄²H mixtures behave ideally, whereas mixtures of DMPC-C₁₄²H or DMPC-C₁₄H and DPPC-C₁₄²H lead to negative azeotropic phase diagrams showing that the disturbing isotopic effect of the probe is really negligible with respect to the hydrocarbon chain structure as can be seen from the phase diagram analysis. According to these data, it seems that a perdeuterated lipid is suitable as a really almost non perturbing probe only if the latter constitutes the deuterated homologous of the system forming molecules under study.     

Micro thin-layer techniques for rapid sequence analysis of P-labeled RNA: Double digestion and pancreatic ribonuclease analyses

Double digestion of oligonucleotides obtained from ribonuclease T₁ or pancreatic ribonuclease A fingerprints results in the following series of products: (Ap)_0-nCp, (Ap)_0-nUp, and (Ap)_0-nGp. A new micromethod is described for the rapid analysis of these digests. The procedure consists of two-dimensional chromatography on a small PEI thin-layer plate. In the first dimension, the oligonucleotides are separated independent of their Ap content into three groups, which represent the Cp-, Gp-, and Up- 3′-terminal oligonucleotide series, respectively. In the second dimension, the products are fractionated according to their chain length. This method, which allows direct identification of even the longer Ap tracts in a double digest of an oligonucleotide or an RNA chain, is very rapid and highly sensitive and can be applied to the simultaneous analysis of a large number of samples in a single run. The procedure has also been adapted to the analysis of pancreatic ribonuclease A digests of small RNA fragments.     

Selectivity at a three-base bulge site in the DNA binding of ΔΔ-[{Ru(phen)2}2(μ-dppm)]4+ [dppm is 4,6-bis(2-pyridyl)pyrimidine; phen is 1,10-phenanthroline]

The binding of the stereoisomers of [{Ru(phen)₂}₂(μ-bpm)]⁴⁺, [{Ru(phen)₂}₂(μ-dppm)]⁴⁺ and [{Ru(phen)₂}₂(μ-bb)]⁴⁺ {phen is 1,10-phenanthroline; bpm is 2,2′-bipyrimidine, dppm is 4,6-bis(2-pyridyl)pyrimidine, bb is 1,2-bis[4-(4′-methyl-2,2′-bipyridyl)]ethane} to an oligonucleotide duplex [d(GCATCGAAAGCTACG)•d(CGTAGCCGATGC)] containing a three-base bulge has been studied using a fluorescence intercalator displacement assay. Of the dinuclear ruthenium complexes, the dppm-linked species showed the strongest binding to the oligonucleotide, with the ΔΔ isomer binding slightly more strongly than the meso isomer and the ΛΛ isomer exhibiting the weakest binding. In order to determine whether the ΔΔ-[{Ru(phen)₂}₂(μ-dppm)]⁴⁺ metal complex specifically bound at the three-base bulge site, a ¹H NMR study of the binding of the metal complex to the oligonucleotide duplex d(GCATCGAAAGCTACG)•d(CGTAGCCGATGC) was carried out. Although a detailed picture of the metal complex–oligonucleotide association could not be determined from the NMR results owing to the broadening of the resonances from the metal complex and nucleotide residues at the bulge site, the NMR results do indicate that the metal complex specifically binds at the three-base bulge site. The combined results of this study suggest that the dppm-bridged dinuclear ruthenium complexes have considerable potential as probes for the unusual secondary structure obtained by the insertion of a three-base bulge within duplex DNA.     

The solid-liquid phase diagrams of binary mixtures of consecutive, even saturated fatty acids: differing by four carbon atoms

The complete solid-liquid phase diagrams for four binary mixtures of saturated fatty acids are presented, for the first time, in this work. These mixtures are formed by caprylic acid (C_8:0) + lauric acid (C_12:0), capric acid (C_10:0) + myristic acid (C_14:0), lauric acid (C_12:0) + palmitic acid (C_16:0) and myristic acid (C_14:0) + stearic acid (C_18:0). The phase diagrams were obtained by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). FT-Raman spectrometry and polarized light microscopy were used to complement the characterization for a complete understanding of the phase diagram. All of the phase diagrams here reported show the same global behavior that is far more complex than previously accepted. They present not only peritectic and eutectic reactions, but also metatectic reactions, due to solid-solid phase transitions common in fatty acids, and regions of solid solution not previously reported. This work contributes to the elucidation of the phase behavior of these important biochemical molecules with implications in various industrial applications.     

Poly(L-Lysine)-Graft-Dextran Copolymer Remarkably Promotes Pyrimidine-Motif Triplex Formation at Neutral Ph: Thermodynamic and Kinetic Studies

Abstract

The binding constant of a homopyrimidine oligonucleotide with its target duplex for the pyrimidine-motif triplex formation at neutral pH was analyzed and found to be remarkably higher in the presence of the poly (L-lysine)-graft-dextran copolymer than that without any triplex stabilizer or in the presence of spermine. The promoting effect mainly results from the considerable increase in the association rate constant.     

Non-ideal mixing and fluid–fluid immiscibility in phosphatidic acid–phosphatidylethanolamine mixed bilayers

Differential scanning calorimetry (DSC) was used to study the miscibility of phosphatidic acids (PAs) with phosphatidylethanolamines (PEs) as a function of chain length (n = 14, 16) and degree of ionization of PAs at pH 4, pH 7, and pH 12. Phase diagrams were constructed using temperature data for onset and end of the phase transition obtained from the direct simulation of the heat-capacity curves. The phase diagrams were analyzed by simulations of the coexistence curves utilizing a four-parameter regular solution model. For PA–PE mixtures, the non-ideality parameters are a function of composition indicating non-symmetric non-ideal mixing behavior. At pH 7, where the PA component is negatively charged, the systems DMPA:DMPE and DPPA:DPPE have positive non-ideality parameters ρ ₁ in both phases, indicating a preferred aggregation of like molecules. In contrast, DMPA:DPPE and DPPA:DMPE mixtures had negative ρ ₁ values. Measurements at pH 4 showed that mixed pair formation is favored when PA is protonated. At pH 12 where PA is doubly charged, highly positive ρ _l1 parameters are obtained for the liquid-crystalline phase except for the system DPPA:DPPE (ρ ₁ < 0). This indicates clustering of like molecules and possibly domain formation in the liquid-crystalline phase. DPPA:DMPE at pH 12 even shows a miscibility gap in the liquid-crystalline phase. Obviously, despite the presence of doubly charged PA a fluid–fluid immiscibility is induced.     

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.