Crystal Structures, Reactivity and Inferred Acylation Transition States for 2‘-Amine Substituted RNA

Abstract

Ribose 2'-amine substitutions are broadly useful as structural probes in nucleic acids. In addition, structure-selective chemical reaction at 2'-amine groups is a robust technology for interrogating local nucleotide flexibility and conformational changes in RNA and DNA. We analyzed crystal structures for several RNA duplexes containing 2'-amino cytidine (C(N)) residues that form either C(N)-G base pairs or C(N)-A mismatches. The 2'-amine substitution is readily accommodated in an A-form RNA helix and thus differs from the C2'-endo conformation observed for free nucleosides. The 2'-amide product structure was visualized directly by acylating a C(N)-A mismatch in intact crystals and is also compatible with A-form geometry. To visualize conformations able to facilitate formation of the amide-forming transition state, in which the amine nucleophile carries a positive partial charge, we analyzed crystals of the C(N)-A duplex at pH 5, where the 2'-amine is protonated. The protonated amine moves to form a strong electrostatic interaction with the 3'-phosphodiester. Taken together with solution-phase experiments, 2'-amine acylation is likely facilitated by either of two transition states, both involving precise positioning of the adjacent 3'-phosphodiester group.

Local RNA structural changes induced by crystallization are revealed by SHAPE

“…comm.). More recently, crystals of a self-complementary 16 base pair (bp) duplex containing one 29-amine substitution per strand were soaked in a solution containing an activated ester to produce a 29-amide at that position (Gherghe et al 2005). The three-dimensional structures of the unmodified and the modified RNA were compared to understand the molecular basis of this particular reactivity.…”

“…Many studies have mapped the structure of RNA molecules under different solution conditions (Gesteland et al 2005). However, to our knowledge, only one publication compares the chemical reactivity of a single modified nucleotide belonging to the same RNA in solution and in the crystal (Gherghe et al 2005). We used the recently developed selective 29-hydroxyl acylation analyzed by primer extension (SHAPE) method .…”

Ab initio determination of the flexibility of 2′‐aminoribonucleosides and 2′‐aminoarabinonucleosides inserted in duplexes

“…This discrepancy with the experimental result may come from interactions between the group in 2 0 and the atoms of the chain downstream on the 3 0 side that stabilize model 2. 26 Therefore model 1, in a full duplex, is probably energetically less favorable than model 2 and is not observed.…”

“…Recently, the crystal structure of a self complementary double stranded RNA containing a 2 0 -aminocytidine (A2M) on each chain has been reported (PDB id: 1YZD). 26 Fig. 7).…”

“…Moreover, the value of the puckering amplitude sat the east energy barrier (s 5 35.10) is greater than that in the B I and A conformations (s 5 32.60 and s 5 28.00, respectively). NMR 16 and X-ray 26 experiments showed that the puckering of 2 0 -aminoribonucleosides inserted into acid nucleic duplexes is N-type, so the B3LYP/6-31G* vacuum model is questionable and we therefore investigated the effect of the solvent as a continuous medium.…”