Role of loops in the guanine quadruplex formation by DNA/RNA hybrid analogs of G4T4G4.

CD spectroscopy, gel electrophoresis and absorption-based thermal stability were used to analyze quadruplex formation of RNA and RNA/DNA hybrid analogs of the deoxyoligonucleotide G4T4G4, which forms a well-characterized basket-type quadruplex. All RNA-containing dodecamers, g4u4g4, G4u4G4 and g4T4g4 (RNA lower-case, DNA capital letters), formed parallel, namely tetramolecular quadruplexes in Na+-containing solutions. The u4 loop forced DNA tetrads into the same conformation as adopted by g4u4g4. In contrast, the T4 loop destabilized the RNA tetrads. Potassium ions markedly stabilized parallel quadruplexes of RNA-containing analogs as well as their bimolecular folding. In the presence of K+, g4T4g4 formed exclusively bimolecular quadruplexes of both parallel and antiparallel types as indicated by CD. Thus, the T4 loop permits RNA strands to adopt an antiparallel arrangement. These findings may be useful for engineering particular quadruplex foldings in different quadruplex-forming sequences.

Guanine quadruplex formation by RNA/DNA hybrid analogs of Oxytricha telomere G(4)T(4)G(4) fragment.

Using circular dichroism spectroscopy, gel electrophoresis, and ultraviolet absorption spectroscopy, we have studied quadruplex folding of RNA/DNA analogs of the Oxytricha telomere fragment, G(4)T(4)G(4), which forms the well-known basket-type, antiparallel quadruplex. We have substituted riboguanines (g) for deoxyriboguanines (G) in the positions G1, G9, G4, and G12; these positions form the terminal tetrads of the G(4)T(4)G(4) quadruplex and adopt syn, syn, anti, and anti glycosidic geometries, respectively. We show that substitution of a single sugar was able to change the quadruplex topology. With the exception of G(4)T(4)G(3)g, which adopted an antiparallel structure, all the RNA/DNA hybrid analogs formed parallel, bimolecular quadruplexes in concentrated solution at low salt. In dilute solutions ( approximately 0.1 mM nucleoside), the RNA/DNA hybrids substituted at positions 4 or 12 adopted antiparallel quadruplexes, which were especially stable in Na(+) solutions. The hybrids substituted at positions 1 and 9 preferably formed parallel quadruplexes, which were more stable than the nonmodified G(4)T(4)G(4) quadruplex in K(+) solutions. Substitutions near the 3'end of the molecule affected folding more than substitutions near the 5'end. The ability to control quadruplex folding will allow further studies of biophysical and biological properties of the various folding topologies.

Towards a better understanding of the unusual conformations of the alternating guanine-adenine repeat strands of DNA.

Alternating guanine-adenine strands of DNA are known to self-associate into a parallel-stranded homoduplex at neutral pH, fold into an ordered single-stranded structure at acid pH, and adopt yet another ordered single-stranded conformer in aqueous ethanol. The unusual conformers melt cooperatively and exhibit distinct circular dichroism spectra suggestive of a substantial conformational order, but their molecular structures are not known yet. Here, we have probed the molecular structures using guanine and adenine analogs lacking the N7 atom, and thus unable of Hoogsteen pairing, or those restrained in the less-frequent syn glycosidic orientation. The studies showed that the syn glycosidic orientation of dA residues promoted the neutral homoduplex, whereas the syn orientation of dG was incompatible with the homoduplex. In addition, Hoogsteen pairing of dA seemed to be a crucial property of the homoduplex whereas dG did not pair in this way. The situation was the same in both single-stranded conformers with the dG residues. On the other hand, the presence of N7 was important with dA but its syn geometry was not favorable. The present data can be used as restraints to model the unusual molecular structures of the alternating guanine-adenine strands of DNA.

Factors influencing DNA expansion in the course of polymerase chain reaction.

We performed more than 3,500 polymerase chain reactions (PCRs) under various conditions with more than 400 DNA fragments of 4-150 nucleotides in length. Some of the PCRs provided expanded DNA molecules of kilobase lengths whereas others led to no expansion. Repetitiveness of the primary structure was mostly found to be necessary but not sufficient for the expansion. (A+T)-rich fragments expand better than (G+C)-rich ones and pyrimidine-rich fragments expand better than purine-rich fragments. Terminal nucleotides and the fragment length also are important for the expansion. Examples are presented when relatively small alterations of the DNA primary structure caused a dramatic change in the expansion. For example, A8T8 expanded a lot whereas T8A8 did not expand at all. The present work has implications for pathological expansions of microsatellites in the human genome as well as regarding the genome evolution in general.