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

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 ¹³C labeled RNA building blocks - a 5-¹³C-uridine and a 2,8-¹³C₂-adenosine building block - are used to introduce isolated ¹³C-¹H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5-¹³C-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 2,8-¹³C₂-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 2,8-¹³C₂-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 ¹³C-Carr-Purcell-Meiboom-Gill (CPMG)-relaxation dispersion NMR spectroscopy. The ¹³C-CPMG relaxation dispersion experiment recapitulated a secondary structure switching event in the P5c hairpin of the group I intron construct previously revealed by ¹⁵N 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.