DNA-catalyzed hydrolysis of esters and aromatic amides

Article abstract of DOI:10.1021/ja4077233

We previously reported that DNA catalysts (deoxyribozymes) can hydrolyze DNA phosphodiester linkages, but DNA-catalyzed amide bond hydrolysis has been elusive. Here we used in vitro selection to identify DNA catalysts that hydrolyze ester linkages as well

Full text of DOI:10.1021/ja4077233

Communication
pubs.acs.org/JACS
DNA-Catalyzed Hydrolysis of Esters and Aromatic Amides
Benjamin M. Brandsen, Anthony R. Hesser, Marissa A. Castner, Madhavaiah Chandra,
and Scott K. Silverman*
Department of Chemistry, University of Illinois at Urbana−Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, United
States
S
* Supporting Information
ribozyme was also evolved to cleave an amide bond, using a
ABSTRACT: We previously reported that DNA catalysts
(deoxyribozymes) can hydrolyze DNA phosphodiester
linkages, but DNA-catalyzed amide bond hydrolysis has
been elusive. Here we used in vitro selection to identify
DNA catalysts that hydrolyze ester linkages as well as
DNA catalysts that hydrolyze aromatic amides, for which
the leaving group is an aniline moiety. The aromatic
amide-hydrolyzing deoxyribozymes were examined using
linear free energy relationship analysis. The hydrolysis
reaction is unaffected by substituents on the aromatic ring
(ρ ≈ 0), suggesting general acid-catalyzed elimination as
the likely rate-determining step of the addition−
elimination hydrolysis mechanism. These findings establish
that DNA has the catalytic ability to achieve hydrolysis of
esters and aromatic amides as carbonyl-based substrates,
and they suggest a mechanism-based approach to achieve
DNA-catalyzed aliphatic amide hydrolysis.
guanosine nucleophile to attack the amide analogue of the
natural phosphodiester substrate, but extending this specific
finding to broader peptide cleavage is difficult.9 Hydrolysis of a
cholesteryl carbonate derivative was catalyzed by a ribozyme
that was identified indirectly by aptamer selection using a
transition state analogue,10 but the strategy was not applied to
amide hydrolysis, and the approach has not proven widely
applicable.
In the present study, we systematically and directly pursued
DNA-catalyzed hydrolysis of two related types of carbonyl-
based substrate, esters and amides. Both esters and amides
should be cleavable by hydrolytic addition−elimination
mechanisms, and the ease of amide hydrolysis should be
tunable by changing the nitrogen substituent. Our results reveal
robust DNA-catalyzed ester hydrolysis as well as DNA-
catalyzed amide hydrolysis when the nitrogen is substituted
with an aromatic ring; i.e., the substrate is an aromatic amide, or
anilide. Insight into the mechanism of DNA-catalyzed aromatic
amide hydrolysis was obtained from linear free energy
relationship analysis. The findings directly demonstrate the
feasibility of DNA-catalyzed hydrolysis of carbonyl-based
substrates, including for the first time an (aromatic) amide.
Our results also suggest mechanism-based approaches to
overcome the continuing challenge of DNA-catalyzed hydrol-
ysis of a simple aliphatic amide bond.
DNA-catalyzed hydrolysis of phosphodiester bonds emerged
in our previous report despite the availability of several amide
bonds in the selection substrate,2 although many such
deoxyribozymes from that and later studies function well with
all-DNA substrates.5c Therefore, here we applied a stringent
selection pressure to allow the survival of DNA sequences only
if they catalyze ester or amide hydrolysis but not
phosphodiester cleavage (Figure 1). A 40-nucleotide random
region (N40) was provided with a covalently attached substrate
that presented either an ester or an amide linkage embedded
between two DNA oligonucleotide segments, with Watson−
Crick base pairs holding the substrate in place. The population
of random DNA sequences (each with attached substrate) was
incubated under conditions anticipated to be conducive to ester
or amide hydrolysis. DNA-catalyzed cleavage of the ester or
amide linkage revealed a free carboxylic acid (CO2H) group,
which was captured by splinted reaction with an amino-
oligonucleotide and the coupling reagent DMT-MM11 (Figure
eoxyribozymes have been shown to catalyze numerous
D
chemical reactions, many of which involve cleavage or
ligation of substrates at phosphodiester linkages.1 Most of the
earliest deoxyribozymes were identified to catalyze RNA
cleavage by transesterification, in a reaction analogous to that
catalyzed by ribonuclease protein enzymes. Previously, we
showed that DNA can catalyze DNA phosphodiester
hydrolysis,2 which is a very challenging reaction because the
uncatalyzed half-life (i.e., the t1/2 at near-neutral pH and in the
absence of divalent metal ion cofactors at 25 °C) is ∼30 million
years.3 Sequence-specific hydrolysis by DNA catalysts identified
using in vitro selection4 in the presence of Zn2+ and Mn2+
cofactors was shown to proceed with a half-life on the order of
15 min, corresponding to 1012 rate enhancement.2 Sub-
sequently, we showed that Zn2+ alone can be the cofactor,
with a half-life on the order of 0.5 min and nearly 1014 rate
enhancement.5 Although amide (peptide) bond hydrolysis has
a much shorter uncatalyzed half-life on the order of hundreds of
years,6 our efforts to achieve DNA-catalyzed amide hydrolysis
by in vitro selection have to date been fruitless, instead leading
to DNA-catalyzed DNA phosphodiester hydrolysis in our initial
report2 and no DNA-catalyzed amide hydrolysis in several
unpublished follow-up efforts. Therefore, DNA-catalyzed amide
bond hydrolysis is a major unsolved problem, especially
considering the practical value of engineered and artificial
protease enzymes.7 Others have sought nucleic acid catalyzed
hydrolysis of carbonyl-based substrates. The group I intron
ribozyme has modest aminoacyl esterase activity.8 This
Received: July 26, 2013
Published: October 15, 2013
© 2013 American Chemical Society
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studies. During the key selection step of each round, the
incubation conditions were defined either as (A) 70 mM
HEPES, pH 7.5, 1 mM ZnCl2, 20 mM MnCl2, 40 mM MgCl2,
and 150 mM NaCl at 37 °C or as (B) 50 mM CHES, pH 9.0,
40 mM MgCl2, and 150 mM NaCl at 37 °C. Each of the Zn2+,
Mn2+, and Mg2+ ions have been effective deoxyribozyme
cofactors in many prior selection experiments, but Zn2+ and
Mn2+ cannot be used above pH 7.5 due to precipitation and
oxidation, respectively.
Both of the selections for ester hydrolysis using substrate 1
led to strong catalytic activity (see all selection progressions in
Figure S3), despite a nontrivial background ester cleavage rate
under both incubation conditions (4% background hydrolysis
in 14 h under conditions A, and 4% background hydrolysis in 1
h under conditions B). Under conditions A, after 10 rounds in
which each selection step was performed for 14 h, the
hydrolysis yield of the DNA pool reached 49%. Under
conditions B, after 13 rounds with each selection step
performed for 1 h, the pool yield was 38%. Both pools were
cloned, and individual deoxyribozymes were characterized as
described below. Separately, of the two selections for aromatic
amide hydrolysis with substrate 2, only the effort using
conditions A led to activity. After 8 rounds with each selection
step performed for 14 h, the pool yield was 36%, and individual
deoxyribozymes were cloned and characterized. In contrast, the
experiment using conditions B led to no detectable activity
(<0.5%) after 17 rounds. Finally, neither of the two selections
with the Ala-Phe-Ala substrate 3 led to detectable activity
(<0.5%) after 17 rounds. Sequences of all deoxyribozymes are
provided in Figure S4.
Individual deoxyribozymes were characterized for each of the
three selection experiments that led to substantial catalytic
activity, with product identities established by mass spectrom-
etry (Figure S5). Eleven and three unique ester-hydrolyzing
deoxyribozymes that cleave substrate 1 were identified from the
selections under respective conditions A (pH 7.5 with Zn2+/
Mn2+/Mg2+) and B (pH 9.0 with Mg2+).13 The 14 sequences
are essentially unrelated to one another (Figure S4A). The
single-turnover rate constants were as high as kobs = 0.54 h−1
(10ZA8 under conditions A) and 3.1 h−1 (13ZB38 under
conditions B), with hydrolysis yields up to 90% (Figures 3 and
S6). These kobs values correspond to rate enhancements of up
to 240, computed relative to the uncatalyzed ester hydrolysis
rate constants under the corresponding incubation conditions.
The eleven deoxyribozymes for conditions A all responded
similarly to changes in pH by showing maximal yield at pH 7.5,
although in some cases activity was maintained at pH 7.2 or 7.8
(Figure S7A). The three deoxyribozymes for conditions B were
all faster at higher pH over the range 7.5 through 10 (Figure
S7B). The metal ion dependence of each deoxyribozyme was
also examined (Figure S8). Of the eleven deoxyribozymes for
conditions A, all required Zn2+ for optimal activity; seven
additionally required Mn2+, and two additionally required both
Mn2+ and Mg2+ for optimal activity. Each of the three
deoxyribozymes for conditions B required Mg2+ (noting that
conditions B omit both Zn2+ and Mn2+).
Figure 1. In vitro selection of ester- and amide-hydrolyzing
deoxyribozymes. DNA-catalyzed hydrolysis is followed by capture of
the revealed carboxylic acid using an amino-oligonucleotide, which
results in an upward PAGE shift for the active DNA sequences. See
Figure S1 for details. The dashed loop on the left enables the selection
process but is dispensable for catalysis.
1; see also Figure S1). Control experiments verified that
phosphodiester hydrolysis near the ester or amide linkage did
not result in subsequent DMT-MM-activated capture. The
amino-oligonucleotide induced a substantial increase in length
of the captured deoxyribozyme product, which was readily
separated by PAGE and amplified by PCR to enter the next
selection round.
Using the strategy of Figure 1, three hydrolysis substrates
were subjected to in vitro selection, each under two incubation
conditions. The three substrates (Figure 2) included either an
Figure 2. Structures of the ester and amide substrates 1−6 used in this
study. The horizontal black bars represent DNA oligonucleotide
segments. Ester and amide linkages are colored red. See Figure S2 for
more detailed structures.
ester linkage (1), an aromatic amide (anilide) linkage for which
the hydrolytic leaving group is an aniline moiety (2), or the Ala-
Phe-Ala tripeptide (3). The tripeptide substrate 3 was evaluated
because of the possibility that simply providing an aromatic ring
near the intended reaction site would improve the ability of
DNA to interact with the substrate, as has been observed for
the “flexizyme” aminoacylation ribozymes.12 Substrate 2 also
includes an aromatic ring, but in addition, the aromatic nitrogen
substituent changes the electronic structure of the amide
linkage (particularly the nitrogen basicity) and, via ring
substitution, provides a derivatization site for mechanistic
Five anilide-hydrolyzing deoxyribozymes that cleave sub-
strate 2 emerged from the selections under conditions A (pH
7.5 with Zn2+/Mn2+/Mg2+; 36% pool yield at round 8).
Sequence alignment (Figure S4B) shows two regions of
substantial conservation flanking a central variable region.
The two DNA catalysts with the highest yields, 8ZC9 and
8ZC30, had single-turnover kobs values of 0.13 0.01 h−1 (n =
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8ZC9 was partially randomized (25% per nucleotide) and
reselected for hydrolysis of 2, after 6 rounds (33% pool yield) a
set of 13 related sequences was identified, showing the same
conservation pattern as for the parent 8ZC deoxyribozymes
(Figure S4B). These reselected DNA catalysts had kobs values
up to 3-fold higher than that of 8ZC9 (data not shown).
We recently showed that, for other DNA-catalyzed reactions,
the outcome of in vitro selection depends critically on the
length of the random region; e.g., larger random regions
explore more complex structures but are sampled less
thoroughly.14 Here we evaluated N20, N30, N50, and N60
regions with anilide substrate 2 and conditions A (pH 7.5
with Zn2+/Mn2+/Mg2+), rather than N40 as used to identify the
8ZC deoxyribozymes. The N50 and N60 selections gave no
activity through round 11 and were discontinued. In contrast,
the N20 and N30 selections led to 49% cleavage at round 9 and
33% cleavage at round 8, respectively. The new N20
deoxyribozymeswhich among themselves shared a largely
conserved sequencehad no conservation when compared to
the N40-derived 8ZC deoxyribozymes (Figure S4C); kobs values
were similar to those for 8ZC9 (data not shown), as was the
yield optimum at pH 7.5 (Figure S11A). The new N20
deoxyribozymes functioned well with Zn2+ as the sole divalent
metal ion, without requiring either Mn2+ or Mg2+. In parallel,
the eight new N30 deoxyribozymes retained both conserved
sequence elements of the N40-derived 8ZC deoxyribozymes
(Figure S4C). The N30 kobs values were similar to those of the
N40 deoxyribozymes (data not shown), and the yield was
optimal at pH 7.5 (Figure S11B). The N30 deoxyribozymes also
shared the requirement for Zn2+ and Mn2+ (but not Mg2+) with
most of their N40 counterparts. In summary, of the N20−N60
experiments, only the smaller N20, N30, and N40 random regions
led to catalytic activity, and the sequences and metal ion
dependences of the N20 DNA catalysts were distinct from those
of the N30 and N40 deoxyribozymes.
Figure 3. PAGE images and kinetic plots for ester-hydrolyzing
deoxyribozymes. S = substrate 1; P = cleavage product. PAGE images
show time points at t = 30 s, 30 min, and 12 h for one representative
deoxyribozyme from each set. (A) Deoxyribozymes identified from the
selection under conditions A. kobs values: 0.54, 0.34, 0.15 h−1; kbkgd
0.0024 h−1. See Figure S6 for data for the other eight 10ZA
deoxyribozymes. (B) Deoxyribozymes identified from the selection
under conditions B. kobs values: 3.1, 1.7, 0.56 h−1; kbkgd 0.013 h−1. See
Figures S7 and S8 for detailed pH and metal ion requirements.
Each of the 8ZC deoxyribozymes was evaluated with
substrate 4 (Figure 2) that retains the aromatic ring of 2 but
includes a methylene group between the aromatic ring and the
former anilide nitrogen atom (i.e., the leaving group is
benzylamine rather than aniline). Each 8ZC deoxyribozyme
was also tested with substrate 5 that replaces the benzene ring
of 2 with cyclohexane. In all cases, no activity was observed
(<0.5%; data not shown). Unsurprisingly given these results,
aliphatic amide 6which is structurally analogous to ester 1
but has an aliphatic amide moiety in place of the esteris also
not cleaved by these DNA catalysts. Substrate 2 offers two
distinct amide bonds for cleavage: an aromatic amide for which
the leaving group is aniline, and a benzamide for which the
leaving group is a primary aliphatic amine. The only observed
hydrolysis reaction for 2 was cleavage of its aromatic amide
(Figure S5). Alongside the lack of catalytic activity from the
selection using the Ala-Phe-Ala substrate 3, these results
indicate that the mere presence of an aromatic ring in the
substrate is insufficient to promote DNA-catalyzed hydrolysis.
Instead, the identity of the leaving group as an aniline rather
than an aliphatic amide enabled the reaction.
4) and 0.21 0.03 h−1 (n = 5), respectively, and hydrolysis
yields up to 80% (Figure 4). These kobs values correspond to
rate enhancements of >3600 and >5000 (background
hydrolysis undetected, <0.4% in 96 h). Each deoxyribozyme
had maximal activity at pH 7.5, with generally little activity at
pH 7.2 or 7.8 (Figure S9). All five deoxyribozymes required
both Zn2+ and Mn2+; in two cases including 8ZC9, Mg2+ was
additionally required for optimal activity (Figure S10). When
No DNA catalysts emerged from direct selections using 6
with N20−N60 random regions (data not shown). We also made
several attempts to evolve ester- and anilide-hydrolyzing
deoxyribozymes into DNA catalysts for hydrolysis of aliphatic
amide 6, considering that both ester and amide hydrolysis
should proceed through addition−elimination mechanisms.
Toward this goal, numerous ester-hydrolyzing 10ZA deoxy-
Figure 4. Activities of aromatic amide-hydrolyzing deoxyribozymes
identified from the selection under conditions A. kobs values (top to
bottom in legend): 0.13, 0.18, 0.028, 0.19, 0.18 h−1; kbkgd <0.00004
h−1. Only the anilide bond of substrate 2 is hydrolyzed (Figure S5).
See Figures S9 and S10 for detailed pH and metal ion requirements.
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ribozymes and anilide-hydrolyzing 8ZC deoxyribozymes were
partially randomized (25% per nucleotide) and reselected for
cleavage of 6. In all cases, however, no activity was observed
(data not shown).
ASSOCIATED CONTENT
* Supporting Information
Experimental details and additional data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
To investigate the mechanistic basis for the observed DNA-
catalyzed hydrolysis of aromatic amide 2, linear free energy
relationships (LFERs) were obtained. Substituents were placed
individually onto the aromatic ring of 2 in the position ortho to
the anilide nitrogen atom, noting that the para position is
already occupied by the benzamide carbonyl group. The
hydrolysis rate constants kobs for all five 8ZC deoxyribozymes
(N40) were determined for several electron-donating sub-
stituents [σp < 0: (CH3)2NH, CH3O and CH3] as well as
electron-withdrawing substituents [σp > 0: Cl and CF3]. Each
plot of log(kX/kH) versus σp was linear with slope ρ ≈ 0
(Figures 5 and S12). These LFER data are consistent with an
AUTHOR INFORMATION
Corresponding Author
scott@scs.illinois.edu
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by grants to S.K.S. from the
National Institutes of Health (R01GM065966), the Defense
Threat Reduction Agency (HDTRA1-09-1-0011), and the
National Science Foundation (CHE0842534). B.M.B. was
partially supported by NIH T32 GM070421.
REFERENCES
■
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Figure 5. LFER data for the 8ZC9 and 8ZC30 deoxyribozymes. See
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addition−elimination mechanistic model in which aromatic
amide hydrolysis proceeds with rate-determining general acid-
catalyzed elimination involving nitrogen protonation (see
Figure S13 for a full explanation of this conclusion). We also
note that other mechanistic explanations are possible, e.g.,
involving a rate-determining conformational change. When the
phenol analogue of 2 was tested with the five 8ZC
deoxyribozymes, substantial activity (kobs 5- to 33-fold above
kbkgd = 0.4 h−1) was observed for all but 8ZC4 (Figure S14).
This finding indicates that at least two distinguishable modes of
interaction are possible between an anilide-hydrolyzing DNA
catalyst and its substrate.
(c) Ameta, S.; Jaschke, A. Chem. Sci. 2013, 4, 957.
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In summary, we have demonstrated that DNA can catalyze
hydrolysis of esters and aromatic amides (anilides). Linear free
energy relationship analysis of the anilide-hydrolyzing deoxy-
ribozymes suggests that the rate-determining mechanistic step
involves concomitant nitrogen protonation and C−N bond
cleavage. What explains the absence of deoxyribozymes for
hydrolysis of aliphatic amides such as substrate 6? A reasonable
hypothesis is that the decreased electrophilicity of the carbonyl
carbon of 6 relative to 2 shifts the rate-determining step for
potential deoxyribozymes to carbonyl addition rather than
elimination. If correct, then identifying these deoxyribozymes
requires accelerating the addition step, e.g., by use of a stronger
nucleophile such as an amine rather than water. Our current
efforts are evaluating this hypothesis. In the longer term,
deoxyribozymes for amide cleavage will be developed for free
peptide and protein substrates, as we have recently established
for phosphatase activity.15
(13) Each deoxyribozyme was named as (for example) 10ZA8, where
10 is the round number, ZA is the systematic alphabetical designation
for the particular selection, and 8 is the clone number.
(14) Velez, T. E.; Singh, J.; Xiao, Y.; Allen, E. C.; Wong, O.; Chandra,
M.; Kwon, S. C.; Silverman, S. K. ACS Comb. Sci. 2012, 14, 680.
(15) Chandrasekar, J.; Silverman, S. K. Proc. Natl. Acad. Sci. U.S.A.
2013, 110, 5315.
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