The molecular structure of Okazaki fragment r(CGCA)d(AAAAAGCG):d(CGCTTTTTTGCG) in solution

The hetero duplex molecule, r(CGCA)d(AAAAAGCG):d(CGCTTTTTTGCG) which corresponds to Okazaki fragment was synthesized and its molecular structure has been analyzed by NMR study. The RNA strand of RNA-DNA hybrid region adopts A-form and DNA strand of the same region deviates from the standard B-form. The conformation of DNA-DNA duplex segment belongs to B-form. The hybrid-DNA duplex junction shows a structural discontinuities, A-B junction. The same conformational characteristic of oligo(dA): oligo(dT) tract as that of DNA oligomer which has same base sequence has been observed.     

Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon

A molecular beacon approach was developed to directly determine the association constant of RNA-DNA hybrid formation. The molecular beacon was composed of a 15-nt loop structure containing the antisense sequence that can hybridize with the AUG translational start site of the HER2/neu gene, which is overexpressed in a significant proportion of breast, ovarian, and lung tumors. The equilibrium association constant (Ka) of DNA binding to the RNA oligonucleotide was 6.4 +/- 0.14 x 10(7) M(-1) in the presence of 150 mM NaCl at 22 degrees C. The free energy change (AG) associated with RNA-DNA hybrid formation was -10.7 kcal/mole. The melting temperature (Tm) of RNA-DNA hybrid was 64.4 degrees C +/- 1 degree C in the presence of 150 mM NaCl. The RNA-DNA hybrid was more stable than the corresponding DNA-DNA duplex in 150 mM NaCl, as judged by both Ka and Tm data. We also determined the Ka, deltaG, and Tm values of RNA-DNA and DNA-DNA duplex formation in the presence of three monovalent cations, Li+, K+, and Cs+. The feasibility of this method was also investigated using a phosphorothioate molecular beacon. The information generated through this new approach for thermodynamic measurements might be useful for the design of oligonucleotides for antisense therapeutics.     

Repair efficiency of (5′S)-8,5′-cyclo-2′-deoxyguanosine and (5′S)-8,5′-cyclo-2′-deoxyadenosine depends on the complementary base

5′-R and 5′-S diastereoisomers of 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24–32 bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a]P-N²-dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N²-dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.     

Energy and structural analysis of double nucleic acid triplets

In order to investigate the energy and structural character of RNA-RNA triplets and RNA-DNA duplex base triplets, 64 sets of three-dimensional models of RNA-DNA duplex base triplets and mRNA-tRNA triplex base triplets were constructed and optimized by homologous modeling method using the software InsightII. The comparative statistical method and cluster analysis were adopted to study these features. The result showed: (i) all energy parameters of monomer RNA-DNA hybrid triplets and ternary complexes appeared significantly different; and some parameters related with overall molecules such as overall energy, bond energy and coulomb energy have statistically significant correlations between the structures in vacuum and aquatic solutions while other parameters, including theta energy, phi energy, hydrogen bond energy and non-bond energy, changed significantly, but not continuously. (ii) However, the case of mRNA-tRNA triplets was much more complicated in that only the bond energy's correlation coefficient is -0.8. Typically, the main contribution of GC pairs and G/A/U bases were interesting. The models of RNA-DNA hybrid triplets and mRNA-tRNA triplet should be helpful for the study of base pairing in codons and the biological effectiveness of antisense nucleic acids.     

Specific recognition of napthyridine-based ligands toward guanine-containing bulges in RNA duplexes and RNA-DNA heteroduplexes

Mismatched bulges in nucleic acid constructs are important in the recognition event between biological molecules. Herein, it is observed that napthyridine dimer 2 is able to specifically bind G-G mismatches in all nucleic acid constructs comprising of RNA-RNA, RNA-DNA and DNA-DNA duplexes. However, the binding affinity of 2 is strongest toward DNA duplex, followed by RNA-DNA heteroduplex and RNA duplex being the weakest binding partner. Nonetheless, this binding behavior suggests that the binding process primarily occurs between the guanine base pairs and the napthyridine moiety, and is independent of the tertiary structure of the nucleic acid duplexes.