Do the 16mer, 5'-GUGGUCUGAUGAGGCC-3' and the 25mer, 5'-GGCCGAAACUCGUAAGAGUCACCAC-3', form a Hammerhead Ribozyme structure in physiological conditions? An NMR and UV thermodynamic Study

Anna Trifonova, Parag Acharya, Johan Isaksson, E. Zamaratsky,
A. Földesi, T. Maltseva, J. Chattopadhayaya*

Department of Bioorganic Chemistry, Box 581, Biomedical Center, University of Uppsala,
S-751 23 Uppsala, Sweden. E-mail: jyoti@bioorgchem.uu.se;  Fax: +4618554495

Key words: 2H double-labelled nucleoside, Hammerhead Ribozyme Structure


 



Figure 1. The secondary structures of the 16mer (A) and 25mer (B) RNA strands, and the hammerhead ribozyme construct (C) for which X-ray data is available1. The nomenclature used is also shown and the deuterated residues are printed in bold font. In order to prevent self-cleavage, 2'-O-methyl cytidine (C*) has been incorporated at the cleavage-site.

Figure 2. (A) Two transitions are observed in UV for the isolated 16mer RNA in 2.0M NaCl phosphate buffer. The main transition is still RNA concentration independent (Tm = 71.8 ± 0.4 °C), whereas the second transition appears to be a bimolecular process (see 16mer NMR assignment below). (B) The relative amount of isolated 16mer RNA going through the second transition is increasing with increased salt concentration in the phosphate buffer (pH 7.5). The RNA concentration is 8 µM in all samples.

Figure 3. Expandable pdf figures in colour are available at our website [http://bioorgchem.uu.se/]. (A) The imino region of the 16mer (0°C) shows the internal basepairings, including the characteristic G-U mismatch, giving rise to misleading basepaired imino resonances in 1D titration experiments (data not shown). (B) 16mer assignment (20°C). The solid lines trace the aromatic-H1 connections in the 16mer hairpin structure. The dashed lines trace a secondary duplex structure that only exists to any large extent in salt concentrations above 100 mM NaCl and temperatures below 40°C. The breaks in inter-residue connectivity in the duplex trace between residues 7-12 suggest a flexible bulge at the 4 central residues, between the two 8G-11G mismatches. (C) The sequential H6/H8-H1 assignment of the 25mer RNA (20°C). A secondary uni-molecular structure, possibly a different fold, also exists in approximately 4:6 ratio. (D) The H6/H8-H1 region of the mixture of 16mer and 25mer resembles the superimposition of the spectra of the two components isolated. Thus, there is no significant change of conformation taking place, meaning that no hammerhead-like structure is formed when the two oligo RNAs are mixed in a 100 mM NaCl phosphate buffer (20°C).