Time-resolved RNA SHAPE chemistry

Article abstract of DOI:10.1021/ja8061216

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry yields quantitative RNA secondary and tertiary structure information at single nucleotide resolution. SHAPE takes advantage of the discovery that the nucleophilic reactivity of the ribose 2′-hydroxyl group is modulated by local nucleotide flexibility in the RNA backbone. Flexible nucleotides are reactive toward hydroxyl-selective electrophiles, whereas constrained nucleotides are unreactive. Initial versions of SHAPE chemistry, which employ isatoic anhydride derivatives that react on the minute time scale, are emerging as the ideal technology for monitoring equilibrium structures of RNA in a wide variety of biological environments. Here, we extend SHAPE chemistry to a benzoyl cyanide scaffold to make possible facile time-resolved kinetic studies of RNA in～1 s snapshots. We then use SHAPE chemistry to follow the time-dependent folding of an RNase P specificity domain RNA. Tertiary interactions form in two distinct steps with local tertiary contacts forming an order of magnitude faster than long-range interactions. Rate-determining tertiary folding requires minutes despite that no non-native interactions must be disrupted to form the native structure. Instead, overall folding is limited by simultaneous formation of interactions～55 A distant in the RNA. Time-resolved SHAPE holds broad potential for understanding structural biogenesis and the conformational interconversions essential to the functions of complex RNA molecules at single nucleotide resolution. Copyright

Full text of DOI:10.1021/ja8061216

Published on Web 11/08/2008
Time-Resolved RNA SHAPE Chemistry
Stefanie A. Mortimer and Kevin M. Weeks*
Department of Chemistry, UniVersity of North Carolina, Chapel Hill, North Carolina 27599-3290
Received August 4, 2008; E-mail: weeks@unc.edu
Selective 2′-hydroxyl acylation analyzed by primer extension
SHAPE) chemistry is emerging as a powerful approach for
quantitative analysis of the equilibrium structures of diverse
(
1-3
biological RNAs.
SHAPE chemistry exploits the discovery that
the nucleophilic reactivity of the ribose 2′-hydroxyl position is
strongly gated by the underlying nucleotide flexibility (Figure 1).
Flexible nucleotides preferentially adopt conformations that react
with a hydroxyl-selective electrophile to form a 2′-O-adduct, while
base paired or otherwise conformationally constrained nucleotides
are unreactive. The electrophile is concurrently consumed by
hydrolysis making a specific quench step unnecessary, provided
the adduct-forming reaction with RNA is allowed to continue until
1
all reagent is consumed. SHAPE works extremely well for
Figure 1. Mechanism of RNA SHAPE chemistry with BzCN. BzCN reacts
both with 2′-hydroxyl groups at flexible positions in RNA to form a 2′-O-
adduct and also undergoes inactivation by hydrolysis.
interrogating the equilibrium structure of RNA motifs from a few
nucleotides to large catalytic RNAs and viral genomes spanning
hundreds to thousands of nucleotides.
1
-4
To date, SHAPE chemistry has been based on electrophilic
reagents derived from an isatoic anhydride (IA) scaffold. Reactivity
can be tuned by varying the electron withdrawing potential of the
functional groups on the IA scaffold. We have created a suite of
secondary structure. Sites of 2′-O-adduct formation were identified
as stops to primer extension using fluorescently labeled DNA
2,4
primers, resolved by capillary electrophoresis. Absolute SHAPE
reactivities observed for the RNase P RNA using BzCN are very
similar to those obtained with 1M7. Both reagents react preferen-
tially with loops and, in both cases, loops that participate in tertiary
1
,2,3c
reagents with hydrolysis half-lives from 14 to 430 s.
In our
view, the most reactive of these reagents, 1-methyl-7-nitroisatoic
2
anhydride (1M7), represents a near-ideal probe for nucleotide-
interactions are protected from forming 2′-O-adducts in the presence
resolution analysis of RNA structures at equilibrium. The intrinsic
2+
of Mg
(Figure 2). There is a very strong linear correlation
2
+
reactivity of 1M7 is insensitive to solution conditions like Mg
(
Pearson’s r g 0.80) between 1M7 and BzCN reactivities, both in
concentration and temperature. Complete degradation of the reagent
requires 70 s: this moderate level of reactivity makes the reagent
easy to handle in the laboratory but means that RNA structures are
2+
the presence and absence of Mg (data not shown). Thus, like
1
2
M7, BzCN reactivity measures local nucleotide flexibility and is
selective for the 2′-OH group. BzCN therefore meets the first of
the two criteria for time-resolved SHAPE because it accurately
2
averaged on the minute time scale.
A full understanding of structure-function relationships in RNA
biology requires a nucleotide-resolution view of the time-resolved
mechanisms by which RNA molecules fold, interconvert between
reports the secondary and tertiary structure of the RNase P
2+
specificity domain in the presence and absence of Mg
.
5
We next determined the rate of BzCN hydrolysis by monitoring
the competition between hydrolysis versus 2′-O-adduct formation
using the model nucleotide, 3′-phosphoethyl-5′-adenosine mono-
phosphate (pAp-ethyl) (Figure 3A). Hydrolysis is rapid and was
quenched at defined time points with dithiothreitol (see filled circle,
Figure 3B). The rate constant for BzCN hydrolysis was determined
by fitting the fraction adduct formed as a function of reaction time
distinct states, and function in ribonucleoprotein complexes. The
reactivity of IA derivatives is circumscribed by the electron
withdrawing potential of the substituents that can be introduced
into this reagent. The limits of this useful scaffold have likely been
reached.
We therefore sought to develop a new chemical framework to
interrogate RNA structure on the seconds time scale. Benzoyl
cyanide (BzCN) reacts with hydroxyl functional groups to yield a
stable ester. Formation of the stable cyanide ion leaving group
renders the reaction irreversible (Figure 1). As with all SHAPE
electrophiles, this scaffold should satisfy two important criteria:
(
Figure 3B). BzCN undergoes hydrolysis with a half-life of 0.25 s
at 37 °C (Figure 3A).
The reaction between BzCN and RNA is complete in ∼1 and
2
.5 s at 37 and 25 °C (Figure 3). BzCN is thus an ideal reagent for
following RNA folding on the seconds time scale.
(
1) react broadly with RNA 2′-hydroxyl groups in a structure-
selective manner and (2) undergo rapid inactivation by hydrolysis
with water (Figure 1).
We then used BzCN to analyze the folding behavior of the RNase
P RNA in a time-resolved SHAPE experiment. The RNase P RNA
(154 nt) forms several classes of tertiary structure modules: a
U-shaped T-loop motif that docks into the P7-P10 helices through
an A-minor interaction, numerous stacking interactions (including
A130 on A230 in P9), two large internal loops (J11/12 and
J12/11) which form extensive noncanonical interactions, and a
We used BzCN to assess the structure of the specificity domain
of Bacillus subtilis ribonuclease P (RNase P), whose structure has
2
,3c
been well characterized using IA-based reagents.
RNA was treated with BzCN under conditions that stabilize the
native tertiary fold (10 mM MgCl , 100 mM NaCl, pH 8.0) and
where the RNA forms only its
The RNase P
2
2
+
6
also in the absence of Mg
tetraloop-receptor interaction (involving L12 and P10.1).
1
6178 9 J. AM. CHEM. SOC. 2008, 130, 16178–16180
10.1021/ja8061216 CCC: $40.75  2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Histograms comparing absolute SHAPE reactivities for the RNase
2
+
P specificity domain in the presence and absence of Mg obtained using
M7 (top) and BzCN (bottom). Loop (Lx) and joining structure (Jx)
1
landmarks are emphasized.
Figure 3. Reaction half-life of BzCN. (A) Reaction of BzCN with pAp-
ethyl. (B) Time-dependent reaction of BzCN with pAp-ethyl at 37 °C. Half-
life of BzCN in water was determined by fitting the fraction 2′-O-adduct
formed to 1 - exp[(0.02)(exp(-khydrolysist) - 1)].
Figure 4. Formation of tertiary interactions in the RNase P specificity
domain. (A) Processed capillary electrophoresis traces obtained for the
reaction of BzCN with the RNase P specificity domain from 5 s to 20 min.
The difference in peak intensity between the (+) and (-) BzCN channels
reports the extent of 2′-O-adduct formation at each point. Nucleotides
involved in tertiary interactions show a decrease in reactivity over time.
Nucleotide 178 undergoes this transition slowly, while nucleotide 190
undergoes this transition more rapidly (blue and red lines, respectively).
Tertiary folding of the RNase P RNA was initiated by adding
2
MgCl (to 10 mM) to a solution of RNA (0.5 µM) preincubated at
3
7 °C in 100 mM NaCl (at pH 8.0). Aliquots were removed at
time intervals between 5 and 1200 s and added directly to BzCN.
This experiment provides 1 s snapshots of RNase P structure over
(
B) Folding rate constants monitored by time-resolved SHAPE. All
2
+
2
0 min (Figure 4A). Within 5 s after addition of Mg , there is a
nucleotides show a decrease in SHAPE reactivity as a function of time,
indicating an increase in structural constraints as the RNA forms tertiary
interactions. Nucleotides folding in the fast and slow phases are indicated
by open and closed symbols, respectively.
rapid ordering of the RNA secondary structure, observed as a
decrease in SHAPE reactivity throughout the RNA. We focused
our analysis on the tertiary folding that occurs after this step. After
data normalization, significant changes in SHAPE reactivity occur
at 16 nucleotides in the RNA. These nucleotides fell into two
kinetically distinct categories (Figure 4B). The first category is a
The time-resolved SHAPE data make possible modeling of
kinetic intermediates in the folding pathway for the RNase P RNA.
Consistent with the two categories of reactivity changes, we observe
two distinct folding steps. The first tertiary folding step is
characterized by significant protections in the structural module
involving the J11/12 and J12/11 loops (residues 185-196 and
217-225) and also in the interaction of A229 with C134 and C232
(in red, Figure 5). The second, slower, folding step involves
-
1
fast phase that folds with a rate constant of 0.06 s ; the second
category is a slower phase that folds with a rate constant of 0.004
-
1
s
. The nucleotides that fall into the first, fast, phase also exhibit
conformational changes consistent with the slow phase, suggesting
that the slow phase involves a whole-scale tightening of the RNA
structure (see Supporting Information, Table S1).
J. AM. CHEM. SOC. 9 VOL. 130, NO. 48, 2008 16179

C O M M U N I C A T I O N S
Figure 5. Mechanism for folding of the RNase P specificity domain. Fast-forming tertiary interactions found in the intermediate are red, interactions that
9
form in the rate-determining step are in blue. Structure is drawn to approximate the arrangement of helices in three-dimensional space.
formation of the core of the specificity domain: docking of the
T-loop (U175-A179) at P7-P10, stacking of A230 on A130 in P9,
and docking of the GAAA tetraloop (P12) into its receptor in P10.1
Acknowledgment. We are indebted to Dorothy Erie and Scott
Kennedy for use of and extensive assistance with quench-flow
instrumentation. The NSF supported this work (MCB-0416941 to
K.M.W.).
(
in blue, Figure 5).
There are several critical features of this folding mechanism.
Supporting Information Available: Experimental methods and one
table. This material is available free of charge via the Internet at http://
pubs.acs.org.
Although it is becoming clear that RNA folding is not obligatorily
1b,3b,7
hierarchical,
in the case of the RNase P RNA, tertiary structure
folding does clearly follow the near-complete formation of the
secondary structure. Second, tertiary interactions involving local
interactions between the J11/12 and J12/11 loops and involving a
base stacking interaction at A229 form first (in red, Figure 5). Third,
the rate-limiting folding step, despite being on the order of minutes,
does not involve the breaking of non-native contacts. Instead, the
rate-determining step involves formation of tertiary interactions
located 55 Å apart (in blue, Figure 5).
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