1. Stability of Three-Stranded DNA Structure

    1. Biological Background and Appplications
      Triplex DNA was firstly discovered in a complex of poly(A) and poly(U) by Felsenfeld, Davis and Rich in 1957. Arnott and coworkers established the triplex DNA model by X-ray diffraction analyses in 1974. In last ten more years, studies of triplex DNA have been paid much more attention because of its importance as a tool for DNA sequencing, gene control and therapeutic applications. The site-specific character of triplex formation offers viable biochemical, pharmacological and therapeutic applications by acting as repressors at the transcriptional (antigene) level, which also provides means to design powerful artificial endonuclease when the third strand is coupled with a cleaving agent. The triplex forming activity also holds strong promise in the areas of genome mapping. Oligonucleotide-directed triple-helix formation in a gene's promoter region has been shown to downregulate the expression of that gene through the inhibition of mRNA synthesis. The intermolecular triplex has recently been shown to inhibit gene expression in vivo, including a case demonstrating the inhibition of HIV-1 transcription in infected human cells. Medically related issues have been vigorous motivators of triple-stranded research in the past five years.

      Triple helix formation is sensitive to the length of the third strand, single base mismatches, pH, cation concentration and valence, temperature, and backbone composition (DNA or RNA) of the three strands. The ability to target a broad range of DNA sequences and the high specificity and stability of the resulting local triple helical structures make this a powerful technique for the recognition of single sites within megabase segments of double helical DNA. Significant progress has been made in understanding characteristics of the triplex as a gene control tool. The ability to design oligonucleotides which will recognize any unique base pair sequence and form a stable triplex in a promoter region of the human genome, however, still remains a challenge. In general, the stable triplex structure has been restricted to a homopurine-homopyrimidine duplex in which the additional third strand binds to the homopurine strand of the duplex. In order to extend this third strand binding to any random base pair sequence, comprehension of the parameters necessary for the stability of triple-stranded systems is crucial.

    2. Structure of Triplex-DNA

      The third pyromidine strand binds to the major groove of DNA pyromidine.purine duplex DNA to form a triple-stranded DNA.

    3. Theoretical Results
      1. DNA Loop and Water Effects on Stability of Triplex-DNA
        Based on the results of computational simulations, I found that the loops, which are important in nucleic acid structures, have minor effect on the stability of intra-triplex DNA. However, the interaction of the triplex with water molecules is a central issue. The water molecules appreciably increase the triplex stability by screening the electrostatic repulsion of phosphate groups along the DNA backbone. Furthermore, water molecules connect two DNA strands by forming hydrogen bonds between adjacent bases to enhance the stability of entire triplex DNA.

      2. Effects of Substitution on Stability of Triplex-DNA
        The studies of three hairpin triplex DNA with substitution of 5-bromocytosine for cytosine occurring in different strands have shown that substitution in strand I dramatically increases the triplex stability, while the substitution in strand III or both in strand I and III slightly decrease the stability of the triplex. The counterions of Na+ are also demonstrated to greatly enhance the stability of triplex DNA. In addition, the results from both the experimental and theoretical studies indicated that many triplexes exhibit polymorphic conformation because of multiple sugar puckers, an intermediate conformation between the ideal C3'-endo and C2'-endo.

      3. Stability of Braid-like DNA
        Another type of three-stranded DNA, termed as braid-like DNA, is also investigated using molecular mechanics. Based on the experimental data of scanning tunneling microscopy (STM), models of three braid-like DNAs composed by three different base triplets AAA, TAT, and GCA were constructed, respectively. The results showed that the conformational energy of braid-like DNA is higher than that of corresponding triplex DNA. Thus, braid-like DNA is much more difficult to form. The normal Watson-Crick or Hoogsteen hydrogen-bonds were broken in the braid-like DNA. However, the three strands in braid-like DNA were found to be equivalent, while those of triplex are not. Each period of braid-like DNA has 18 nucleotides, half of which is right-handed, the other half is left-handed. For comparison, triplex DNA has 11 base-pairs in each period.

  2. Duplex DNA Interaction with Ligands

    1. Biological Backgrounds
      Interaction of DNA with drugs is of particular pharmacological importance. Owing to DNA's central role in biological replication and protein biosynthesis, modification by drug interaction greatly alters cell metabolism, diminishing and in some cases terminating cell growth. Most drugs, carcinogens, and ligands are characterized by extended hetero-cyclic aromatic chromophores. Applications of thess compounds in medicine has been greatly expended in recent years. Studies of ligands binding to DNA are important in the design of new and efficient drugs targeted to DNA. Usually, molecules which possess large planar ring systems can be liganded to DNA by inserting the planar ring between two adjacent base-pair of the DNA. Both therapeutic (anticancer, antiviral, and antibacterial drugs) and toxic (mutagenic, probably carcinogen) behavior has been observed for compounds which intercalate to DNA.

    2. Theoretica Results
      We studied the interactive modes of a host+guest system targeting to the duplex DNA. In the host+guest system of rhodamine~B-ethylenediamine-beta-cyclodextrins (RhB-beta-CDen) and borneol, the cyclodextrins (CD) are cyclic oligosaccharides composed of six or more D-glucopyramose unit, which play a role as typical host, and the RhB-beta-CDen, which has a three-aromatic-ring group, can probe DNA. This mechanism can be used to control a DNA sensory system like an on/off switch using host+guest recognition.
      The theoretical results showed that the borneol can insert into the CD cavity and push the three-aromatic-ring group out of the cavity toward the bulk water environment. The geometric shape of borneol and the van der Waals interaction between host and guest are both the important factors that influence the binding ability. Two interactive modes of host+guest system targeting to DNA were identified: the first one is CD cavity binding along the minor groove of duplex DNA with hydrogen bonds bridging the ligand and DNA, the second one is the benzene ring of the rhodamine B group intercalating into the DNA minor groove parallel to the base pairs, and no direct hydrogen bonds between the ligand and DNA. It is also found that many water molecules bridge between ligand and DNA using hydrogen bonds to stabilize the entire system. Our results demonstrated that there are more than one interactive modes for ligands targeting to duplex DNA. The electrostatic interaction between the ligand and DNA plays an central role in the binding mode, while the van der Waals interaction is important in the intercalating mode. This conclusion will contribute to the design of new and efficient drugs targeted to DNA.