Investigation of the DNA-dependent cyclohexenyl nucleic acid polymerization and the cyclohexenyl nucleic acid-dependent DNA polymerization.

DNA polymerases from different evolutionary families [Vent (exo-) DNA polymerase from the B-family polymerases, Taq DNA polymerase from the A-family polymerases and HIV reverse transcriptase from the reverse transcriptase family] were examined for their ability to incorporate the sugar-modified cyclohexenyl nucleoside triphosphates. All enzymes were able to use the cyclohexenyl nucleotides as a substrate. Using Vent (exo-) DNA polymerase and HIV reverse transcriptase, we were even able to incorporate seven consecutive cyclohexenyl nucleotides. Using a cyclohexenyl nucleic acid (CeNA) template, all enzymes tested were also able to synthesize a short DNA fragment. Since the DNA-dependent CeNA polymerization and the CeNA-dependent DNA polymerization is possible to a limited extend, we suggest CeNA as an ideal candidate to use in directed evolution methods for the development of a polymerase capable of replicating CeNA.

Deoxythreosyl phosphonate nucleosides as selective anti-HIV agents.

Out of a series of eight new phosphonate nucleosides with an l-threose and an l-2-deoxythreose sugar moiety, two new compounds were identified (PMDTA and PMDTT) that showed potent anti-HIV-1 (HIV-2) activity [EC50 = 2.53 microM (PMDTA) and 6.59 microM (PMDTT)], while no cytoxicity was observed at the highest concentration tested [CC50 > 316 microM (PMDTA) and > 343 microM (PMDTT)]. The kinetics of incorporation of PMDTA into DNA (using the diphosphate of PMDTA as substrate and HIV-1 reverse transcriptase as catalyst) was similar to the kinetics observed for dATP, while the diphosphate of PMDTA was a very poor substrate for DNA polymerase alpha. The incorporated PMDTA fits very well in the active site pocket of HIV-1 reverse transcriptase.

Nucleotide analogues containing 2-oxa-bicyclo[2.2.1]heptane and l-alpha-threofuranosyl ring systems: interactions with P2Y receptors.

The ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y(1) receptor has been successfully substituted with a rigid methanocarba ring system, leading to the conclusion that the North (N) ring conformation is preferred in receptor binding. Similarly, at P2Y(2) and P2Y(4) receptors, nucleotides constrained in the (N) conformation interact equipotently with the corresponding ribosides. We now have synthesized and examined as P2Y receptor ligands nucleotide analogues substituted with two novel ring systems: (1) a (N) locked-carbocyclic (cLNA) derivative containing the oxabicyclo[2.2.1]heptane ring system and (2) l-alpha-threofuranosyl derivatives. We have also compared potencies and preferred conformations of these nucleotides with the known anhydrohexitol-containing P2Y(1) receptor antagonist MRS2283. A cLNA bisphosphate derivative MRS2584 21 displayed a K(i) value of 22.5 nM in binding to the human P2Y(1) receptor, and antagonized the stimulation of PLC by the potent P2Y(1) receptor agonist 2-methylthio-ADP (30 nM) with an IC(50) of 650 nM. The parent cLNA nucleoside bound only weakly to an adenosine receptor (A(3)). Thus, this ring system afforded some P2Y receptor selectivity. A l-alpha-threofuranosyl bisphosphate derivative 9 displayed an IC(50) of 15.3 microM for inhibition of 2-methylthio-ADP-stimulated PLC activity. l-alpha-Threofuranosyl-UTP 13 was a P2Y receptor agonist with a preference for P2Y(2) (EC(50)=9.9 microM) versus P2Y(4) receptors. The P2Y(1) receptor binding modes, including rotational angles, were estimated using molecular modeling and receptor docking.

Influence of threose nucleoside units on the catalytic activity of a hammerhead ribozyme.

TNA (alpha-L-threose nucleic acids) is potentially a natural nucleic acid, that might have acted as an evolutionary alternative of RNA. We determined the catalytic activity of hammerhead ribozymes containing a threofuranosyl-modified nucleoside at position U4 and U7, and compared these results with those obtained from HNA (hexitol nucleic acids) insertion into the same ribozyme. Our experiments showed that, although the threofuranosyl-modified ribozymes still cleave the substrate strand, cleavage activity is highly decreased. It, therefore, seems that TNA can play a functional role in the RNA world, but only to a limited extent.

Cleavage of DNA without loss of genetic information by incorporation of a disaccharide nucleoside.

A ribose residue inserted between the 3'-OH of one nucleotide and the 5'-phosphate group of the next nucleotide, functions as a site-specific cleavage site within DNA. This extra ribose does not interrupt helix formation and it protects duplex DNA against cleavage by restriction enzymes. Cleavage can be obtained with periodate and all ribose fragments can be removed with sodium hydroxide. As a result of this, an intact natural oligodeoxynucleotide is obtained after ligation reaction, which means that site-specific cleavage and recovering of intact DNA occurs without loss of genetic information.

Recognition of threosyl nucleotides by DNA and RNA polymerases.

Alpha-L-threose nucleic acids (TNA) are potentially natural nucleic acids that could have acted as an evolutionary alternative to RNA. We determined whether DNA or RNA polymerases could recognize phosphorylated threosyl nucleosides. We found that for both the Vent (exo-) DNA polymerase and HIV reverse transcriptase K(m) values were increased and kcat values decreased for the incorporation of tTTP in comparison to their natural counterparts. Our results suggest that TNA may have played a role in the evolution of the DNA-RNA-protein world. Thus, TNA may be a candidate for further studies in evolutionary chemistry and biology.