Studying sparsely populated conformational states in RNA combining chemical synthesis and solution NMR spectroscopy

Abstract

Using chemical synthesis and solution NMR spectroscopy, RNA structural ensembles including a major ground state and minor populated excited states can be studied at atomic resolution. In this work, atom-specific C labeled RNA building blocks - a 5-C-uridine and a 2,8-C-adenosine building block - are used to introduce isolated C-H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5-C-uridine 2'-O-TBDMS-phosphoramidite building block was introduced into a 21 nucleotide (nt) tP5c stem construct of the tP5abc subdomain of the Tetrahymena group I ribozyme. Then, the 2,8-C-adenosine 2'-O-TBDMS-phosphoramidite building block was incorporated into a 9 kDa and a 15 kD construct derived from the epsilon (ε) RNA element of the duck Hepatitis B virus. The 2,8-C-adenosine resonances of the 9 kDa 28 nt sequence could be mapped to the full-length 53 nt construct. The isolated NMR active nuclei pairs were used to probe for low populated excited states (<10%) via C-Carr-Purcell-Meiboom-Gill (CPMG)-relaxation dispersion NMR spectroscopy. TheC-CPMG relaxation dispersion experiment recapitulated a secondary structure switching event in the P5c hairpin of the group I intron construct previously revealed by N relaxation dispersion experiments. In the ε-HBV RNA an unfolding event occurring on the millisecond time scale was found in the upper stem in-line with earlier observations. This unpaired conformational state is presumed to be important for the binding of the epsilon reverse transcriptase (RT) enzyme. Thus, a full description of an RNA's folding landscape helps to obtain a deeper understanding of its function, as these high energy conformational states often represent functionally important intermediates involved in (un)folding or ribozyme catalysis.

RNA Dynamics by NMR Spectroscopy

“…The 5-13 Cu ridine and 2,8-13 Ca denine labels were subsequently used for studies of a2 8-mere psilon RNA element of duck hepatitis Bv irus, detecting an unfolding event on am illisecond scale, in agreement with previous R 11 measurements. [17] Chemical shifts of both protons and carbons for ab ulge of Asite ES werep robed with combination of 2',8-13 C-ATP;1 ',6-13 C-CTP;1',8-13 C-GTP;2 ',6-13 C-UTP labels and similar life times of ES were obtained with 13 Ca nd 1 HC PMG experiments. [83] Experimentsw ere expanded to include amethylene 1 H, 13 CTROSY-detected CPMG pulse sequence that was successfully tested for iron responsive element [83] (Figure 14).…”

“…Examples: R 11 relaxation dispersion measurements have uncovered some of the basic principles of miss-matches and rare tautomeric forms in RNA that are important for understanding of translationa nd replication errors. [6,8,17,94] Kimsey et al have Figure 17. Concepto fR 11 experiments.…”

“…understanding on sequence-dependent imino exchange rates. [57][58][59][60][61][62][63][64] Obtaining nucleotide-resolution data on imino exchange rates was also used extensively to assess the effect of naturallyo ccurring or artificially incorporated modifications on the stabilityo fm odified RNAs [17] or the effecto fp rotein binding on RNA stability. [37] Several systems that were characterized with methods accessing as imilar time-scale of motion( EXSY or real-timeN MR) needed to take into an accountc ontributions of imino exchange to thee xtracted dynamic parameters and complemented their study with measurements of imino exchange rates.…”

“…[17,40,41,75,84,85] Examples are C2'/C4'-labeling of ribose, [88] 2'-O-13 CH 3 -uridine, 6-13 Curidine and 6-13 C-cytidine, [41] [8][9][10][11][12][13] Cp urines, [75,84] 5-13 C-uridine and a2 ,8-13 C 2 -adenine. [17] 3.3. R 11 (relaxationi nthe rotating frame) Theory:S imilart ou sing CPMG in order to detect exchange contributions on top of R 2 relaxation times, it is possible to re- place the 1808 train of hard pulses by as pin lock pulse and detect exchange as contribution to detected R 11 values.…”

“…Energy landscape of RNA exemplified by tP5abc. [15][16][17] Motions correspondtor eorientation of bonds andm olecular tumbling on ps-ns scale, baseflipping on ms-ms scale and large structural reorientations,such as (re-)folding on sscale.S everal NMRm ethodsa ccessmotionsr anging from timescales of ps to days. RNA structures are reprinted with permission from refs.…”