Crystal structure of intercalated four-stranded d(C3T) at 1.4 A resolution.

Abstract

The crystal structure of d(C3T), solved at 1.4 A resolution, reveals that the molecule forms a four-stranded intercalated complex. It consists of two parallel-stranded duplexes, each of which Is held together by cytosine-protonated cytosine base pairs. The two duplexes are intercalated with each other and have opposite strand orientation. The molecule has a flat, lath-like appearance, and the covalently bonded cytosines have a slow right-handed twist of 17.1°. However, there is considerable asymmetry. On one of the flat sides, the phosphate groups are rotated away from the center of the molecule. They are held In this orientation by bridging water molecules that bind the NH of cytosine and a phosphate group of an opposite chain. There is also considerable microheterogeneity in the structure. The cytosine hemiprotonation occurs even at pH 7 where stable crystals form.For some time it has been known that nucleic acids containing stretches of cytidine residues can form parallel strands held together by cytosine-protonated cytosine base pairs (C-C+) (1-4). In an NMR analysis of d(TC5), Gudron and his associates (5) proposed an unusual structure in which two such parallel-stranded duplexes, held together by C'C+ base pairs, intercalate with each other in opposite polarity to form a four-stranded molecule. The evidence for the structure was based on strong interactions between the Hi' protons on different strands, suggesting that the backbones were close together. Recent crystal structures of d(TAACCC) (C.K., I.B., C.L., R.R., R.M., and A.R.), (unpublished data) and of d(C4) (6) were in agreement with the general conclusions deduced by the NMR studies. In telomeres at the end of chromosomes, stretches of cytosine residues are found, usually linked to thymine residues. Here, we present a crystallographic analysis of the structure of d (C3T) Rotation searches of all the models indicated a molecule with the helical axis approximately parallel to the crystallographic b axis, as we expected. Subsequent translation searches of some of the models yielded positions for the molecule that were free of close van der Waals contacts. Rigid body refinement followed by intensive simulated annealing using the program XPLOR (7) improved the density around the phosphate groups ofthe backbone and allowed us to build another model with more accurate helical twist based on the phosphate positions. We used this improved molecule as a starting model and molecular replacement was performed again. From the very beginning, we could identify two thymine residues stacked on top of six C-C+ layers and added those to the model. At this stage, simulated annealed omit maps (6) clearly showed the location of the remaining two thymine residues stacked perpendicular to the helix axis. Positional refinement followed by refinement of the temperature factors led to a final R factor of 17.7% for 5013 reflections above the 2cr level (based on F.) between 10 and 1.4 A. The free R factor (8) value based on a random 10%o subset of reflections is 22.5%...

Base-tetrad swapping results in dimerization of RNA quadruplexes: Implications for formation of thei-motif RNA octaplex

“…Backbone torsion angles (°) for selected nucleotides in the present structure Boldface type indicates values of torsions ␣ and ␥ that are greatly different from those in the usual A-RNA duplexes. as two quadruplexes of r(UG) 4 that are incorporated in the antiparallel fashion. The twist (22°) and the rise (7.3 Å) derived from the base step U1-G2 in the r(UG) 4 of the present crystal structure made it possible to construct a model of an intercalated octaplex.…”

“…as two quadruplexes of r(UG) 4 that are incorporated in the antiparallel fashion. The twist (22°) and the rise (7.3 Å) derived from the base step U1-G2 in the r(UG) 4 of the present crystal structure made it possible to construct a model of an intercalated octaplex. The length of the octaplex can be extended by repeat translating 7.3 Å and rotating 22°of the r(UG) 4 unit along the central axis of octaplex.…”

“…In addition to the usual double helices, nucleic acids also can form triple (1) and quadruple helices (2). In DNA, guanine-rich sequences readily form quadruple helices by stacking of guanine base tetrads (2), whereas cytosine-rich sequences adopt the i-motif quadruplex by intercalation of protonated C⅐CH ϩ base pairs (2)(3)(4)(5). In RNA, four consecutive guanine residues can form quadruplex in both solution (6) and crystalline state (7).…”

Ultrafast excited-state dynamics of RNA and DNA C tracts

“…This antiparallel arrangement of bond dipoles may contribute to the overall electrostatic stabilization. Stacking of exocyclic groups allows the hemiprotonated base pairs to approach to a distance of 3.1 Å [52], much closer than the ~3.4 Å typically found in double-stranded nucleic acids.…”

“…It has been assumed in the past that poly(rC) and poly(dC) form hemiprotonated duplexes [36], but there is no decisive structural proof. About fifteen years ago, NMR [27,50] and later x-ray crystal structures [51,52] showed that dC-rich oligonucleotides adopt a quadruplex form ("i-motif DNA") composed of two intercalated duplexes, each of which consists of CH + ·C base pairs. Crystals of d(CT) 3 grown at neutral pH adopt the quadruplex structure [52], showing that i-motif structures do not require acidic conditions for deoxycytidylates.…”