Multistep mechanism of G-quadruplex resolution during DNA replication.

G-quadruplex (or G4) structures form in guanine-rich DNA sequences and threaten genome stability when not properly resolved. G4 unwinding occurs during S phase via an unknown mechanism. Using Xenopus egg extracts, we define a three-step G4 unwinding mechanism that acts during DNA replication.

Structural Effects of Incorporation of 2'-Deoxy-2'2'-Difluorodeoxycytidine (Gemcitabine) in A- and B-Form Duplexes.

We report the structural effect of 2'-deoxy-2',2'-difluorocytidine (dFdC) insertions in the DNA strand of a DNA : RNA hybrid duplex and in a self-complementary DNA : DNA duplex. In both cases, the modification slightly destabilizes the duplex and provokes minor local distortions that are more pronounced in the case of the DNA : RNA hybrid. Analysis of the solution structures determined by NMR methods show that dFdC is an adaptable derivative that adopts North type sugar conformation when inserted in pure DNA, or a South sugar conformation in the context of DNA : RNA hybrids.

Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy.

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge.

Structural properties and gene-silencing activity of chemically modified DNA-RNA hybrids with parallel orientation.

We report, herein, a new class of RNAi trigger molecules based on the unconventional parallel hybridization of two oligonucleotide chains. We have prepared and studied several parallel stranded (ps) duplexes, in which the parallel orientation is achieved through incorporation of isoguanine and isocytosine to form reverse Watson-Crick base pairs in ps-DNA:DNA, ps-DNA:RNA, ps-(DNA-2'F-ANA):RNA, and ps-DNA:2'F-RNA duplexes. The formation of these duplexes was confirmed by UV melting experiments, FRET and CD studies.

Stabilization of i-motif structures by 2'-β-fluorination of DNA.

i-Motifs are four-stranded DNA structures consisting of two parallel DNA duplexes held together by hemi-protonated and intercalated cytosine base pairs (C:CH(+)). They have attracted considerable research interest for their potential role in gene regulation and their use as pH responsive switches and building blocks in macromolecular assemblies. At neutral and basic pH values, the cytosine bases deprotonate and the structure unfolds into single strands.

G-quadruplexes with (4n - 1) guanines in the G-tetrad core: formation of a G-triad·water complex and implication for small-molecule binding.

G-quadruplexes are non-canonical structures of nucleic acids, in which guanine bases form planar G-tetrads (G·G·G·G) that stack on each other in the core of the structure. G-quadruplexes generally contain multiple times of four (4n) guanines in the core. Here, we study the structure of G-quadruplexes with only (4n - 1) guanines in the core.

Locked 2'-Deoxy-2',4'-Difluororibo Modified Nucleic Acids: Thermal Stability, Structural Studies, and siRNA Activity.

2'-Deoxy-2',4'-difluorouridine (2',4'-diF-rU) was readily incorporated into DNA and RNA oligonucleotides via standard solid phase synthesis protocols. NMR and thermal denaturation (Tm) data of duplexes was consistent with the 2',4'-diF-rU nucleotides adopting a rigid North (RNA-like) sugar conformation, as previously observed for the nucleoside monomer. The impact of this modification on Tm is neutral when incorporated within RNA:RNA duplexes, mildly destabilizing when located in the RNA strand of a DNA:RNA duplex, and highly destabilizing when inserted in the DNA strand of DNA:RNA and DNA:DNA duplexes.

Rigid 2',4'-difluororibonucleosides: synthesis, conformational analysis, and incorporation into nascent RNA by HCV polymerase.

We report on the synthesis and conformational properties of 2'-deoxy-2',4'-difluorouridine (2',4'-diF-rU) and cytidine (2',4'-diF-rC) nucleosides. NMR analysis and quantum mechanical calculations show that the strong stereoelectronic effects induced by the two fluorines essentially "lock" the conformation of the sugar in the North region of the pseudorotational cycle. Our studies also demonstrate that NS5B HCV RNA polymerase was able to accommodate 2',4'-diF-rU 5'-triphosphate (2',4'-diF-rUTP) and to link the monophosphate to the RNA primer strand.

Structure of the human telomere in Na+ solution: an antiparallel (2+2) G-quadruplex scaffold reveals additional diversity.

Single-stranded DNA overhangs at the ends of human telomeric repeats are capable of adopting four-stranded G-quadruplex structures, which could serve as potential anticancer targets. Out of the five reported intramolecular human telomeric G-quadruplex structures, four were formed in the presence of K(+) ions and only one in the presence of Na(+) ions, leading often to a perception that this structural polymorphism occurs exclusively in the presence of K(+) but not Na(+). Here we present the structure of a new antiparallel (2+2) G-quadruplex formed by a derivative of a 27-nt human telomeric sequence in Na(+) solution, which comprises a novel core arrangement distinct from the known topologies.

Dramatic effect of furanose C2' substitution on structure and stability: directing the folding of the human telomeric quadruplex with a single fluorine atom.

Human telomeric DNA quadruplexes can adopt different conformations in solution. We have found that arabinose, 2'F-arabinose, and ribose substitutions stabilize the propeller parallel G-quadruplex form over competing conformers, allowing NMR structural determination of this particularly significant nucleic acid structure. 2'F-arabinose substitution provides the greatest stabilization as a result of electrostatic (F-CH---O4') and pseudo-hydrogen-bond (F---H8) stabilizing interactions.

The solution structure of double helical arabino nucleic acids (ANA and 2'F-ANA): effect of arabinoses in duplex-hairpin interconversion.

We report here the first structure of double helical arabino nucleic acid (ANA), the C2'-stereoisomer of RNA, and the 2'-fluoro-ANA analogue (2'F-ANA). A chimeric dodecamer based on the Dickerson sequence, containing a contiguous central segment of arabino nucleotides, flanked by two 2'-deoxy-2'F-ANA wings was studied. Our data show that this chimeric oligonucleotide can adopt two different structures of comparable thermal stabilities.

Differential stability of 2'F-ANA*RNA and ANA*RNA hybrid duplexes: roles of structure, pseudohydrogen bonding, hydration, ion uptake and flexibility.

Hybrids of RNA with arabinonucleic acids 2'F-ANA and ANA have very similar structures but strikingly different thermal stabilities. We now present a thorough study combining NMR and other biophysical methods together with state-of-the-art theoretical calculations on a fully modified 10-mer hybrid duplex. Comparison between the solution structure of 2'F-ANA*RNA and ANA*RNA hybrids indicates that the increased binding affinity of 2'F-ANA is related to several subtle differences, most importantly a favorable pseudohydrogen bond (2'F-purine H8) which contrasts with unfavorable 2'-OH-nucleobase steric interactions in the case of ANA.