Use of Both Direct and Indirect ¹³C Tags for Probing Nitrogen Interactions in Hairpin Ribozyme Models by ¹⁵N NMR

Abstract

We have used the synthesis and 15N NMR study of separate loop A and loop B domains of the hairpin ribozyme to demonstrate that multiple 15N atoms can be incorporated into an RNA strand and be unambiguously distinguished through a combination of direct and indirect tagging by 13C atoms. Absence of 15N chemical shift changes shows that the G8N1 in loop A does not become deprotonated up to pH 8, and that the G21N7 of loop B does not bind to Mg2+.

Isotope-Labeled RNA Building Blocks for NMR Structure and Dynamics Studies

“…Several groups have also developed strategies to obtain 13 C/ 15 N-labeled amidites. Initial efforts were developed by the Pitsch [ 62 ] and Jones [ 63 , 64 , 65 , 66 ] research groups. More recently, the Micura [ 67 , 68 ] and Kreutz [ 17 , 61 , 69 , 70 , 71 ] research groups have dramatically improved the efficiency and scalability of amidite synthesis for NMR analysis.…”

Solid-Phase Chemical Synthesis of Stable Isotope-Labeled RNA to Aid Structure and Dynamics Studies by NMR Spectroscopy

“…Incorporation of 15 N-labels into RNA provides information on both canonical Watson-Crick and non-canonical base pairs in RNA [56,62,78,79,80]. [1,NH 2 - 15 N 2 ]- and [2- 13 C-1,NH 2 - 15 N 2 ]-guanosine were position-specifically incorporated into short (8–12 nts) RNA fragments to compare spectral properties of GU to GC base pairs [79].…”

“…Similarly, [2- 13 C-1,NH 2 - 15 N 2 ]-guanosine and [7,NH 2 - 15 N 2 ]-adenosine were position-specifically incorporated into short (8 nts) RNAs to study face-to-face and sheared GA base pairs [78]. Additionally, [2- 13 C-1,7,NH 2 - 15 N 3 ]-guanosine and [8- 13 C-1,7,NH 2 - 15 N 3 ]-adenosine were incorporated into loop A and B domains (14–24 nts) from the hairpin ribozyme found in the satellite RNA of tobacco ringspot virus [80]. In these examples, the 13 C isotopes were used as tags to distinguish between the residues in doubly labeled samples since 1 J H2C2 or 1 J H8C8 splits the 1 H resonances for residues that contain 13 C directly coupled to 15 N. Wenter and Pitsch incorporated [1- 15 N]-adenosine and -guanosine and [3- 15 N]-uridine and -cytidine labels into 5 sites of the 32 nt bistable RNA [65].…”