HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 126

The interactions result in a sharp turn in the genomic ribozyme structure
from nucleotides G74 to A77 that forces C75 into close proximity with
the cleavage site while simultaneously jutting the nucleobase of the

unconserved residue at position 76 into solution.

3.2. Ribozyme folding
The HDV ribozymes form a tightly packed core structure that is solvent-
inaccessible in both cis - and trans -acting constructs. 48 Kinetics of folding
the genomic and antigenomic HDV ribozymes into the nested double
pseudoknots have not been described in detail, but the fast self-cleavage
kinetics and thermodynamic stability imply that the compaction is rapid.
The stability of the ribozymes is particularly striking given that in the
absence of Mg 2 þ , the ribozymes, particularly the genomic version at
0.4 M NaCl, do not reach a plateau of their melting curve at 95 C. 49 At
low concentration of NaCl and in the absence of Mg 2 þ , the ribozymes
do reach a melting curve plateau at 95 C, indicating that the ribozymes
can be fully thermally denatured in low ionic strength solvents. 50 The
HDV ribozymes are active in the presence of high concentrations of dena-
turants (18 M formamide or 8 M urea), which likely help resolve misfolded
ribozymes, as a higher fraction of the ribozymes self-cleave at high urea con-
centration. 2,50-52 In a measurement of secondary structure content using
ethidium fluorescence, both the precursor and product forms of the anti-
genomic HDV sequence have been shown to retain 50% secondary struc-
ture in 18 M formamide (95% vol), whereas a single-stranded DNA of the
same sequence showed much lower stability. 53 Similarly, studies have shown
that the HDV ribozymes are more active above physiological temperature,
with cleavage rate constants increasing up to 55 C even for trans -acting
ribozymes. 54
Ribozyme activity at elevated temperatures and denaturant concentra-
tions can be attributed to the denaturation of misfolded molecules. Such
kinetically trapped alternative conformations can form through improper
base-pairing interactions of flanking sequences with the ribozyme core or
even within the cores. 50,55-61 Alternative conformations result in slow over-
all cleavage rates, biphasic kinetics, or high fractions of uncleaved ribozymes.
Mutations that destabilize these ribozyme-disrupting interactions tend to
result in faster cleaving ribozymes. These results, however, are artifacts of
sample preparation, whereas ribozymes studied under cotranscriptional con-
ditions or purified without denaturation tend to exhibit fast, monophasic
self-scission and uniform hydrodynamic radii, respectively. 62,63 Overall,