Biophysical and Biochemical Studies on the Formation of Nucleic Acids Duplexes Containing Ndna Oligonucleotides, a Novel Nucleic Acids with Site-Specific Methyl Phosphotriester Backbone Linkages.

Project: A - Government Institutionb - Ministry of Science and Technology

Project Details

Description

Formation of nucleic acids duplexes is the corner stone of every nucleic acids based biotechnologies. Therefore, the progress in the understanding and manipulations of the formation of nucleic acid duplexes will be valuable literally. Two contrary factors govern the formations of nucleic acid duplexes. The base-pairing of the complementary strands brings in the favorable hydrogen-bonding and base staking interactions. On the other hands, the repulsive forces between the negatively charged backbones of the complementary strands are disruptive to the formation of nucleic acid duplexes. The presence of cations can reduce the repulsive interactions of the complementary strands, and stabilize the resultant nucleic acid duplexes. To meet the needs of biotech applications, researchers of chemical biology have synthesized many chemically modified nucleic acid oligonucleotides (herein as oligos) to address problems which could not be dealt with satisfactorily by using regular DNA and RNA oligonucleotides. Among those, the LNA (locked nucleic acids), PNA (peptide nucleic acids), and MPO (methyl phosphonate oligos) are particularly interesting, because they contain modifications on the nucleic acid backbones, leaving the base-pairing of complementary strands unaffected. While the studies of MPO are limited, many reports indicate that the LNA and PNA only enhance the thermal stability of the nucleic acid duplexes. Although LNA and PNA have been promisingly tested in many biomedical applications, it would be very interesting to have other backbone-modified oligos which may differentially destabilize/stabilize nucleic acid duplexes under some desired conditions.By collaborating with Prof. W-Y Chen (Department of Chemical and Materials Engineering, National Central University), we recently obtained novel oligos, the nDNA oligos. The nDNA oligos are chemically synthesized by the Helios Biomedical Electronic Technology Company (瀚源生醫電子科技) using a proprietary protocol. The nDNA oligos contain site-specific methyl phosphotriester (MPTE) backbone linkages. At the modified sites of nDNA, the backbone phosphodiester linkages are methylated to form MPTE, neutralizing the negative charges of the phosphoryl oxide. In the nucleic acid duplexes containing nDNA oligos, the reduction of the backbone negative charges may enhance the duplex stability, while the presence of hydrophobic methyl groups may destabilize the duplexes. Therefore it is very interesting to study the formation of nucleic acid duplexes with nDNA oligos.We have conducted initial characterizations of the nDNA, using the methods of native gel electrophoresis, CD spectrometer, isothermal titration calorimetry, and the measurements of melting temperature (Tm) of nucleic acid duplexes. The preliminary results indicated that nDNA oligos form duplexes with their complementary strands by releasing more heat than the regular oligos do. However, it appears that the nucleic acid duplexes with nDNA have lower thermal stability and less favorable G of duplex formation. Also, the single nucleotide polymorphisms (SNP) can be better differentiated by using the nDNA oligos than by the regular DNA oligos.Nevertheless our preliminary studies were not conclusive, leaving many questions. For example, regarding to the discrimination of SNP with nDNA, what is the optimal number and spacing of MPTE? Also, how does nDNA compare with LNA and PNA? Theses practical questions should be addressed.
StatusFinished
Effective start/end date8/1/1811/1/19

Keywords

  • DNA
  • RNA
  • nDNA
  • oligonucleotides
  • nucleic acid chemistry
  • thermodynamics
  • physical chemistry
  • biophysics