Structural parameters affecting the kinetics of RNA hairpin formation

Abstract

There is little experimental knowledge on the sequence dependent rate of hairpin formation in RNA. We have therefore designed RNA sequences that can fold into either of two mutually exclusive hairpins and have determined the ratio of folding of the two conformations, using structure probing. This folding ratio reflects their respective folding rates. Changing one of the two loop sequences from a purine- to a pyrimidine-rich loop did increase its folding rate, which corresponds well with similar observations in DNA hairpins. However, neither changing one of the loops from a regular non-GNRA tetra-loop into a stable GNRA tetra-loop, nor increasing the loop size from 4 to 6 nt did affect the folding rate. The folding kinetics of these RNAs have also been simulated with the program 'Kinfold'. These simulations were in agreement with the experimental results if the additional stabilization energies for stable tetra-loops were not taken into account. Despite the high stability of the stable tetra-loops, they apparently do not affect folding kinetics of these RNA hairpins. These results show that it is possible to experimentally determine relative folding rates of hairpins and to use these data to improve the computer-assisted simulation of the folding kinetics of stem-loop structures.

Figure 1

Enzymatic structure probing of the JN2C and shorter JN2C-derived RNA fragments. (A) Schematic representation of the folding trajectories, in which the A nucleotides can base pair with B, resulting in the boldface/italics loop, or B with C, resulting in the boldface/underlined loop. In the kinetic experiments there should not be an interchange between the two mutually exclusive structures, while in the thermodynamic experiments they should be in equilibrium. The dashed lines delimit the size of the two smaller JN2C-derived 5′ and 3′ end hairpin fragments. ΔG values were calculated using RNA-fold () (B) Structure probing of JN2C and JN2C-derived fragments. CK is the control lane (without enzyme) for the kinetic probings and CT for the thermodynamic ones. K indicates the kinetic probing experiments and T the thermodynamic ones. Al is alkaline hydrolysis, T1D is digestion with RNase T1 under denaturing conditions, T1, V1 and T2 represent digestions with RNases T1, V1 and T2 under native conditions. Probing times are 5 and 15 min. The brackets indicate the position of the hairpin loops and single-stranded regions and the letters A, B and C indicate their respective positions in (A).

Figure 2

Enzymatic structure probing and barrier tree of the JN1LH sequence. (A) Schematic representation of the folding trajectories. The loop sequences of the double-hairpin structure are indicated by 1D and 2D, while R represents the loop of the rod-like structure. G* is the substitution of an A residue into a G, giving the stable tetra-loop sequence in the JN3LH RNA. (B) Barrier tree computed for JN1LH at 0°C. The black and grey parts represent the barrier trees of the double-hairpin and rod-like structure, respectively. Free energy values on the left hand axis are given in kcal mol⁻¹. Numbers indicate local minima ordered according to free energies. Numbers written along vertical lines give the free energies to the next higher saddle point. (C) Structure probing of the JN1LH structure. The 3h lanes are the probing patterns of the JN1LH kinetic samples after incubation for 3 h at 0°C. For other symbols see legend to Figure 1B.