Light-Triggered RNA Annealing by an RNA Chaperone

Article abstract of DOI:10.1002/anie.201501658

Non-coding antisense RNAs regulate bacterial genes in response to nutrition or environmental stress, and can be engineered for artificial gene control. The RNA chaperone Hfq accelerates antisense pairing between non-coding RNAs and their mRNA targets, by a mechanism still unknown. We used a photocaged guanosine derivative in an RNA oligonucleotide to temporally control Hfq catalyzed annealing. Using a fluorescent molecular beacon as a reporter, we observed RNA duplex formation within 15 s following irradiation (3 s) of photocaged RNA complexed with Hfq. The results showed that the Hfq chaperone directly stabilizes the initiation of RNA base pairs, and suggests a strategy for light-activated control of gene expression by non-coding RNAs.

Full text of DOI:10.1002/anie.201501658

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Chemie
International Edition: DOI: 10.1002/anie.201501658
German Edition: DOI: 10.1002/ange.201501658
RNA Chaperones
Light-Triggered RNA Annealing by an RNA Chaperone**
Subrata Panja, Rakesh Paul, Marc M. Greenberg,* and Sarah A. Woodson*
Abstract: Non-coding antisense RNAs regulate bacterial genes
in response to nutrition or environmental stress, and can be
engineered for artificial gene control. The RNA chaperone Hfq
accelerates antisense pairing between non-coding RNAs and
their mRNA targets, by a mechanism still unknown. We used
a photocaged guanosine derivative in an RNA oligonucleotide
to temporally control Hfq catalyzed annealing. Using a fluo-
rescent molecular beacon as a reporter, we observed RNA
duplex formation within 15 s following irradiation (3 s) of
photocaged RNA complexed with Hfq. The results showed that
the Hfq chaperone directly stabilizes the initiation of RNA base
pairs, and suggests a strategy for light-activated control of gene
expression by non-coding RNAs.
I
n bacteria, hundreds of small non-coding RNAs (sRNAs)
regulate the expression of genes involved in metabolism,
stress response, and virulence.[1] Many bacterial sRNAs act by
base pairing directly with an mRNA target, altering its
translation or its half-life.[2] The association of two comple-
mentary RNAs depends on their sequences and secondary
structures, and is typically inefficient at the low mRNA
concentrations in the cell. The bacterial RNA chaperone Hfq
increases the rate of base pairing with mRNA targets, and
stabilizes sRNA-mRNA complexes.[3] Herein, we investigate
the mechanism of Hfq-catalyzed annealing using a photo-
caged guanosine that provides rapid, light-dependent control
of RNA base pairing.
Figure 1. Photocaged control of RNA annealing. A) A working model
for Hfq-catalyzed RNA annealing. This work shows Hfq directly
stabilizes helix initiation complexes. B) Conversion of photocaged
guanosine (1) to guanosine (G) by UV irradiation. C) Target RNA
containing 1 does not anneal with a complementary molecular beacon
until after photochemical uncaging. The fluorescence intensity of the
FAM-labeled beacon reports the extent of annealing. D) Emission
spectra of the beacon–target complexes after irradiation with 295 nm
UV diodes.
Hfq forms a ring-shaped homo-hexamer that specifically
binds sRNAs and mRNAs.[4] An arginine patch on the rim of
the hexamer catalyzes RNA annealing and strand displace-
ment.[5] In our working model (Figure 1A), Hfq forms
a transient ternary complex with two RNA strands, increasing
helix initiation 103 to 104 times above the uncatalyzed rate.[5,6]
The remaining base pairs zipper, releasing double-stranded
RNA. Although previous experiments suggested Hfq helps
nucleate base pairing between RNA strands,[5,6] how it does so
is not understood.
reaction on the Hfq chaperone. Photocaged compounds have
found numerous applications in diverse fields of chemistry
and biology due to their ability to act as “ON/OFF” switches
regulated by a specific wavelength of light.[7–11] To be useful in
kinetic experiments, the uncaging reaction should be much
faster than the molecular process under investigation.
In the present work, the photocaged guanosine utilizes the
p-hydroxyphenacyl (pHP) photosolvolysis reaction (Fig-
ure 1B).[12] In contrast to the often used o-nitrobenzyl photo-
redox reaction, which proceeds through an intermediate that
can exist for seconds to a minute, pHP photosolvolysis
typically liberates its contents far more rapidly following
excitation. The deprotection rate of pHP correlates inversely
with the pKa of the conjugate acid of the leaving group. The
rate constant for release of phenolate (phenol pKa ꢀ 10) is
108 sÀ1. Although the rate constant for guanine (pKa ꢀ 9)
release is unknown, the similarity in pKa values between it
and phenol suggested that a pHP caged guanosine would
provide suitable temporal resolution for studying the effects
of Hfq on RNA hybridization. We anticipated that the altered
H-bonding pattern of the caged guanosine containing a pHP
group at the O6 position, combined with the steric bulk of
pHP group, would prevent RNA annealing (“OFF” state).
The syntheses of the photocaged guanosine nucleoside (1,
Scheme 1) and corresponding phosphoramidite (2, Support-
We synthesized a target RNA containing a photocaged
guanosine (1) that affords temporal control of the annealing
[*] Dr. S. Panja, Prof. S. A. Woodson
T.C. Jenkins Department of Biophysics, Johns Hopkins University
3400 N. Charles St., Baltimore MD 21218 (USA)
E-mail: swoodson@jhu.edu
Dr. R. Paul, Prof. M. M. Greenberg
Department of Chemistry, Johns Hopkins University
3400 N. Charles St., Baltimore MD 21218 (USA)
E-mail: mgreenberg@jhu.edu
[**] This work was supported by the NIH (R01 GM46686 to S.W. and R01
GM54996 to M.M.G.)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201501658.
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Figure 2. Hfq catalyzed annealing. A) Representation of the experimen-
tal procedure. 10 nm molecular beacon and 10 nm caged RNA were
incubated (Pre hn) with or without Hfq before 3 s UV flash (Post hn).
B) Samples were irradiated 10 s after mixing with no Hfq (gray), 10 nm
WT Hfq (black), and 10 nm inactive TM Hfq (light gray). C) Rate
constants for RNA annealing (Post hn) as in B versus [Hfq] hexamer.
The data points were fitted into a two-state binding equation with an
apparent Kd =6.5 nm. D) Kinetic traces of samples irradiated after
30 min of mixing; no Hfq, gray; 10 nm Hfq, black.
Scheme 1. Synthesis of the photocaged guanosine nucleoside 1.
a) DEAD, PPh3, 5, THF, À10 to 08C; b) OsO4, NaIO4, 2,6-lutidine,
dioxane/H2O, 258C; c) TABF, THF, 08C; d) NH3, MeOH then NaOMe,
MeOH, 0 to 258C.
ing information) began from 3. Various methods involving
coupling the corresponding a-hydroxyacetophenone with 3
were unsuccessful. Ultimately, the p-hydroxyphenacyl group
was introduced indirectly via a Mitsunobu reaction between 3
and allyl alcohol 5.[13] Nucleoside 1 was obtained from 6 via
exhaustive deprotection following transformation of the
terminal alkene (4) into the ketone (6) via a one-pot
osmylation/periodate oxidation.[14] Photolysis of monomeric
1 produced guanosine in 60% yield. Oligonucleotides con-
taining 1 were prepared via standard methods with the
exception that 2 was coupled manually.
target RNA (10 nm) with or without 10 nm Hfq hexamer and
recorded the fluorescence emission at 515 nm. After 10 s,
samples were flashed with UV light for 3 s, and the growth in
fluorescence recorded for a further 200 s (Figure 2A). The
uncaged target RNA annealed with the molecular beacon 35
times faster in the presence of Hfq (kobs = 0.1 sÀ1; Figure 2B)
than without the chaperone (kobs = 0.003 sÀ1; Figure 2B). A
small increase in fluorescence intensity before the flash
(black, Figure 2B) indicated that the molecular beacon and
target RNA bound Hfq during the 10 s pre-incubation, as
expected.[6]
To measure the RNA annealing kinetics, we used a FAM-
labeled molecular beacon and a complementary 16 nt target
sequence (Figure 1C and Experimental Section).[15] A 3’-A12
extension of the target binds the distal face of Hfq (KD
ꢀ 0.1 nm).[16] As shown previously,[6] Hfq protein accelerated
annealing of beacon and target RNAs from 0.06 sÀ1 to 2.1 sÀ1
(Supporting Information, Figure S12). To design a caged
RNA target, we tested three different G to A substitutions
(Supporting Information, Table S1) and found that a mis-
match at position 4 reduced the extent of base pairing to 5%,
with or without Hfq (Supporting Information, Figure S12). As
anticipated, placement of 1 at position 4 of the target RNA
also blocked duplex formation, judging from the small
increase in the fluorescence signal of the beacon when the
two RNAs were mixed (Figure 1D). Irradiation at 295 nm
sharply increased the fluorescence emission, owing to base
pairing with the uncaged target. Although the half-life for
uncaging was 20 s (Supporting Information, Figure S13),
subsequent experiments used a 3 s UV pulse, which provided
sufficient (1 nm) sensitivity and time resolution.
Hfq increased the RNA annealing rate and stabilized the
beacon-target duplex, as manifested by higher reaction
endpoints (Figure 2B). This enhancement depended on the
specific chaperone activity of Hfq, as no burst was observed in
experiments performed with an inactive triple mutant (TM)
lacking the arginine patch that catalyzes annealing (Fig-
ure 2B). The rate of RNA annealing after uncaging increased
with Hfq concentration, and reached saturation when the
concentration of Hfq equaled the concentration of target
RNA (Figure 2C; Supporting Information, Figure S14) in
agreement with previous measurements of the annealing
kinetics.[6] This required recruitment of Hfq to the RNA, as
the maximum annealing rate was three times lower when the
target RNA lacked the A12 binding site (Supporting Informa-
tion, Figure S15).
Surprisingly, when the target and beacon were equili-
brated for 30 min before the UV flash, base pairing occurred
within a few seconds after UV irradiation (Figure 2D). This
rapid annealing was observed with or without Hfq, although
Hfq increased the yield of double-stranded RNA as before
(Figure 2D; Supporting Information, Figure S14). Although
helix initiation is energetically unfavorable, we deduced the
To determine whether 1 blocks annealing by the Hfq
chaperone, we combined beacon (10 nm) and photocaged
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beacon and caged target slowly form partially base-paired
intermediates that are poised to anneal as soon as the caged
RNA is photolyzed. This partial base pairing does not unfold
the beacon, as the fluorescence remained low until the target
was uncaged (Supporting Information, Figure S16).
Hfq may lower the transition state free energy for RNA
annealing by creating stable helix initiation complexes. To test
this, we varied the pre-incubation time of the components
before the target RNA was photolyzed. Without Hfq, a burst
of annealing appeared as the pre-flash incubation was
lengthened (Supporting Information, Figure S17), and the
amplitude of the burst increased with kobs = 0.07 minÀ1 with
respect to pre-incubation time (black; Figure 3A). This is
RNA binding proteins such as Hfq have been proposed to
passively chaperone RNA interactions by neutralizing the
negative charge of the RNA or by simultaneously binding
more than one RNA strand.[17] By trapping helix initiation
complexes with photocaged RNA, we show that Hfq directly
overcomes the energetic barrier for nucleating the RNA
helix, explaining its potent activity as an RNA chaperone
(Figure 1A). That the complexes become more stable over
time, and that Hfq is required at the moment of uncaging,
suggest that Hfq also facilitates zippering of additional base
pairs, in contrast to previous models.[2,18] Future experiments
will test if the extent of zippering depends on the location of
the caged base. Bacterial sRNAs have modular structures that
can be targeted to new genes,[19,20] suggesting an approach for
activating Hfq-dependent sRNAs with light. Our results
demonstrate that photosolvolysis of pHP enables light-
activated control of rapid biopolymer transitions such as
base pairing, providing new insight into the mechanism of
biological regulation.
Experimental Section
Compound 1 and caged RNA, 5’-GUG1UCAGUCGAGUGGA12,
were synthesized and characterized as described in Supporting
Information. The molecular beacon was 5’-6-FAM-GGUCCCCCA-
CUCGACUCACCACCGGACC-DABCYL (Trilink). Light-trig-
gered RNA annealing was performed in a Fluorolog-3 (Horiba)
spectrofluorometer modified as described in the Supporting Infor-
mation. Molecular beacon and caged target RNA (10 nm each) were
combined in 500 mL 10 mm Tris-HCl pH 7.5, 50 mm NaCl, 50 mm KCl
(TNK) at room temperature before uncaging with 3 s irradiation at
295 nm (35 mW). FAM emission was recorded at 515 nm (see the
Supporting Information) and normalized to the maximum change
after 1 min irradiation. The time origin was defined by the moment of
mixing. For protease treatment, 5 mL (4 U) proteinase K was added at
the times indicated. Hfq concentrations are given per hexamer.
Figure 3. Hfq stabilizes helix initiation. A) Burst of annealing following
uncaging, after different pre-incubation times, was fit to an exponential
rate equation with kobs =0.07 minÀ1, no Hfq; >10 minÀ1, 10 nm Hfq.
B) Samples treated with proteinase K (PK) before uncaging; no treat-
ment, black; after 10 s Pre hn, blue; before adding Hfq, red.
equal within error to kobs for annealing uncaged target RNA
(Supporting Information, Figure S12A). In the presence of
Hfq, the amplitude of the burst phase rose dramatically after
just 10 s pre-incubation (red; Figure 3A and Supporting
Information, Figure S18), indicating that most of the RNA-
Hfq complexes were competent to base pair once the target
was uncaged. Since 30 min was needed to reach a similar
fraction of reactive intermediate without Hfq, this corre-
sponds to a 180-fold increase in the effective rate of helix
initiation.
Keywords: Hfq · non-coding RNA · photocaged nucleotides ·
p-hydroxyphenacyl · RNA chaperones
How to cite: Angew. Chem. Int. Ed. 2015, 54, 7281–7284
Angew. Chem. 2015, 127, 7389–7392
To ask whether Hfq is still needed to facilitate base pairing
after the helix initiation takes place, we removed Hfq with
proteinase K (PK) at different stages of the annealing
reaction. The target and beacon RNAs were pre-incubated
with Hfq for 10 s as usual to form the helix initiation
complexes (Figure 3B). When proteinase K was added for
10 s before photolysis, the fluorescence intensity dropped to
the no protein background, suggesting that Hfq was no longer
bound to the RNAs (blue; Figure 3B). After the UV flash,
only a small increase in fluorescence was observed, showing
that the protease destroyed the helix initiation complexes.
Proteinase K had no effect on the RNA in the absence of Hfq
(Supporting Information, Figure S19A). A control reaction in
which proteinase K was added to the RNAs before Hfq also
yielded little RNA duplex (red; Figure 3B). Semi-native
PAGE confirmed that Hfq was digested under these con-
ditions (Supporting Information, Figure S19B). Thus, Hfq
must remain bound to the target RNA at the time of
photolysis, to convert the helix initiation complexes into
duplex product.
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Received: February 19, 2015
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Published online: May 8, 2015
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