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JAN LIPHARDT |
Unfolding RNA by Force |
We have used laser tweezers to measure the forces necessary to unfold
single RNA molecules. The forces required, plus the change in length on
unfolding the RNA, provide an estimate of the free energy of unfolding.
If the force is kept constant in the unfolding region, the length fluctuates
between the folded form and the unfolded form. For a 22 base pair RNA hairpin
at room temperature, a force of about 15 pN causes jumps of about 24 nm
between hairpin and single strand. The average lifetime (of order 200 ms)
of each form gives the kinetics of the transition. The ratio of times spent
in each form is the equilibrium constant. Both the kinetics and the equilibrium
can be measured as a function of force and extrapolated to zero force
to compare with thermal melting. |
RNAs with tertiary structure, such as the Tetrahymena thermophila group
I intron (L21) and its subdomains (P4-P6 and P5abc), introduce new phenomena.
The transitions are significantly dependent on Mg, and they are no longer
in reversible equilibrium in the transition region. The unfolding of L21
has distinct transitions that are assigned to the unfolding of P4-P6 and
PSabc. |
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