Crystallographic studies of chemically modified nucleic acids: a backward glance

Abstract

Chemically modified nucleic acids (CNAs) are widely explored as antisense oligonucleotide or small interfering RNA (siRNA) candidates for therapeutic applications. CNAs are also of interest in diagnostics, high-throughput genomics and target validation, nanotechnology and as model systems in investigations directed at a better understanding of the etiology of nucleic acid structure, as well as the physicochemical and pairing properties of DNA and RNA, and for probing protein-nucleic acid interactions. In this article, we review research conducted in our laboratory over the past two decades with a focus on crystal-structure analyses of CNAs and artificial pairing systems. We highlight key insights into issues ranging from conformational distortions as a consequence of modification to the modulation of pairing strength, and RNA affinity by stereoelectronic effects and hydration. Although crystal structures have only been determined for a subset of the large number of modifications that were synthesized and analyzed in the oligonucleotide context to date, they have yielded guiding principles for the design of new analogs with tailor-made properties, including pairing specificity, nuclease resistance, and cellular uptake. And, perhaps less obviously, crystallographic studies of CNAs and synthetic pairing systems have shed light on fundamental aspects of DNA and RNA structure and function that would not have been disclosed by investigations solely focused on the natural nucleic acids.

Fig. 1

Structures of DNA, RNA and phosphorothioate DNA (PS-DNA).

Fig. 2

The pseudo-rotation phase angle (P) cycle and conformations of the sugar moiety. The 4′-oxygen is highlighted as a red sphere.

Fig. 3

Idealized, diamond lattice models of (a) RNA with the sugar adopting a C3′-endo pucker (A-DNA similar) and (b) DNA with the sugar adopting a C2′-endo pucker. Space filling models of (c) A- and (d) B-form DNA duplexes.

Fig. 4

Space filling models of DDD duplexes with thymidines replaced by (a) 2′-deoxy-6′-α-hydroxy- and (b) 2′-deoxy-6′-α-methyl-carbocyclic Ts. The views are into the central portion of the minor groove and carbon, oxygen, nitrogen and phosphorus atoms are colored gray, red, blue and orange, respectively. The exocyclic O6′ hydroxyl oxygen and C7′ methyl carbon atoms are highlighted in magenta and green, respectively, and water molecules are depicted as cyan spheres. Note the differences in the minor groove width in the two duplexes; to accommodate the four methyl groups the minor groove is expanded in the duplex on the right and a water molecule is trapped between them.

Fig. 5

RNA hydration (adapted from [60]). (a) Superimposition of 16 nucleotides from the crystal structure of [r(CCCCGGGG)]₂ reveals four groups of water molecules (crosses, highlighted with ellipsoids) around ribose 2′-hydroxyl groups. The blue group (water molecules linking O2′ to N3(G) or O2(C)), the red group (water molecules linking O2′ to phosphates) and the green group (water molecules located in the directed of the 3′-adjacent ribose) together with the O2′-C2′ bond form approximate tetrahedral geometries. (b) Major groove hydration; fused ribbons of pentagons involving four water molecules and a phosphate oxygen are highlighted in green and red and water molecules bridging O6 from adjacent guanines are highlighted in yellow. (c) Minor groove hydration; formation of tandem-water bridges across the groove whereby 2′-hydroxyl groups serve as bridgeheads. A more irregular pattern in the lower half of the duplex (spheres highlighted in cyan and yellow) arises as a consequence of inter-duplex lattice contacts.

Fig. 6

Structures of selected 2′-O-modifications.

Fig. 7

Structures of the (a) 2′-O-MOE-, (b) 2′-O-NMA-, (c) 2′-O-NMC and (d) 2′-O-DMAEOE-RNA modifications. Carbon, oxygen, nitrogen and phosphorus atoms are colored green, red, blue and orange, respectively. Carbon atoms of the 2′-O-substituents are colored in gray and water molecules are shown as cyan spheres.

Fig. 8

(a) The [d(CGCGAA)-U*U*-d(CGCG)]₂ duplex with 2′-S-methylated uridines (U*) viewed into the central minor groove. (b) The native DDD duplex [d(CGCGAATTCGCG)]₂ viewed roughly along the same direction. The color code is the same as in Fig. 7 and carbon and sulfur atoms of modified residues are colored in gray and sulfur, respectively. Thin solid lines represent the helical axis.

Fig. 9

Conformations of (a) FANA- and (b) ANA-modified duplexes exhibiting different degrees of bending into the major groove. The color code is the same as in Fig. 7 and 2′-fluorine and 2′-oxygen atoms are highlighted as green and red spheres, respectively.

Fig. 10

Crystal structures of (a) bicyclo-DNA and (b) tricyclo-DNA.

Fig. 11

Comparison between the overall geometries of (a) the homo-DNA [(β-D-2′,3′-dideoxyglucopyranosyl)] nucleic acid duplex [dd(CGAATTCG)]₂ (viewed into the major groove) and a canonical B-form DNA duplex of the same sequence (viewed across the major and minor grooves). (b) Structure of homo-DNA. (c) Structure of (L)-α-threofuranosyl (3′→2′) nucleic acid (TNA).

Fig. 12

(a) Anomeric effect between the N3′ lone electron pair (green) and the antibonding σ* orbital of the P-O5′ bond in N3′→P5′ phosphoramidate DNA. (b) Putative conjugation between the N5′ lone pair (green) and the antibonding σ* orbital of the P-O3′ bond with P→N5′ phosphoramidate DNA. Provided the furanose adopts an A-type pucker, this arrangement would result in a steric repulsion between the 5′-amino hydrogen and a 2′-hydrogen of the 2′-deoxyribose (red arrow).

Fig. 13

Crystal structure of the guanyl G-clamp (a cytosine analogue), confirming formation of five hydrogen bonds indicated by thin solid lines. Carbon atoms of the modification are colored in gray.

Fig. 14

Comparison of DNA duplexes capped by a stilbenediether (Sd) linker. (a) Structure of Sd. (b) Face-to-face and (c) edge-to-face orientations of Sd relative to the adjacent G:C base pair. Carbon atoms of Sd are colored in gray.

Fig. 15

Structures of (a) an RNA difluorotoluene (rDFT):A base pair (b) an RNA difluorotoluene (rDFT):G base pair.