DNA-catalyzed glycosylation using aryl glycoside donors

Article abstract of DOI:10.1039/c6cc04329a

We report the identification by in vitro selection of Zn²⁺/Mn²⁺-dependent deoxyribozymes that glycosylate the 3′-OH of a DNA oligonucleotide. Both β and α anomers of aryl glycosides can be used as the glycosyl donors. Individual deox

Full text of DOI:10.1039/c6cc04329a

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DNA-catalyzed glycosylation using aryl glycoside
donors†
Cite this: Chem. Commun., 2016,
52, 9259
Anthony R. Hesser,‡ Benjamin M. Brandsen,‡ Shannon M. Walsh, Puzhou Wang
Received 23rd May 2016,
Accepted 18th June 2016
and Scott K. Silverman*
DOI: 10.1039/c6cc04329a
www.rsc.org/chemcomm
We report the identification by in vitro selection of Zn2+/Mn2+
-
dependent deoxyribozymes that glycosylate the 30-OH of a DNA
oligonucleotide. Both b and a anomers of aryl glycosides can be
used as the glycosyl donors. Individual deoxyribozymes are each
specific for a particular donor anomer.
Deoxyribozymes are particular single-stranded sequences of DNA
that have catalytic activity. They are identified by in vitro selection
starting from entirely random sequence pools.1 DNA catalysts have
been found for many chemical reactions, including RNA cleavage
by transesterification, RNA ligation, and DNA and RNA hydrolysis.2
More recently, deoxyribozymes have been identified for creation or
removal of many common post-translational modifications (PTMs)
of peptides,3 including phosphorylation,4 dephosphorylation,5 and
formation of dehydroalanine.6 Glycosylation is an important PTM
for which de novo catalysts that allow sequence-selective peptide
and protein modification will have broad utility.7 Toward the
longer-term goal of peptide glycosylation deoxyribozymes, we
previously sought DNA-catalyzed glycosylation using the sugar
nucleotide UDP-GlcNAc as the glycosyl donor8 during the key
in vitro selection step of each round, followed by capture via
NaIO4 oxidation of the GlcNAc moiety and reductive amination
with NH2-modified DNA. However, that approach was subverted by
a UDP-GlcNAc-independent DNA-catalyzed DNA deglycosylation
reaction.9 Here we report that both b and a anomers of aryl
glycosides, which have been used as synthetic glycosyl donors by
glycosyltransferase10 and mutant glycoside hydrolase11 protein
enzymes, are effective glycosyl donors with DNA catalysts, using
a DNA oligonucleotide 30-OH group as the glycosyl acceptor.
The aryl glycoside donors used in this study are the derivatives
of D-glucose (D-Glc) shown in Fig. 1, and the in vitro selection
strategy is shown in Fig. 2. In each selection experiment, the
30-OH glycosyl acceptor and the glycosyl donor were presented
Fig. 1 Structures of glycosyl donors. Each is an aryl glycoside of D-glucose
(1, b-D-Glc; 2, a-D-Glc). Compounds 1a, 1b, 1c, and 2b were evaluated in
this study.
Fig. 2 Key step of in vitro selection, in which DNA catalyzes glycosylation
of a 30-OH using an aryl glycoside donor, illustrated with a b-D-Glc donor.
Glycosylation leads to a PAGE shift; the catalytically active DNA sequences
are separated and PCR-amplified to enable the next selection round. The
product is depicted here as the a-anomer, but the configurations for individual
deoxyribozyme products in this study have not been assigned experimentally.
See Fig. S1 and Table S1 (ESI†) for nucleotide details.
Department of Chemistry, University of Illinois at Urbana-Champaign,
600 South Mathews Avenue, Urbana, Illinois 61801, USA.
E-mail: scott@scs.illinois.edu
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cc04329a
‡ A. R. H. and B. M. B. are co-first-authors of this manuscript.
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simultaneously to an N40 DNA pool (40 random nucleotides).
The glycosyl donor was attached to a DNA oligonucleotide,
which binds via Watson–Crick interactions to a fixed sequence
of DNA adjacent to the N40 region. The DNA oligonucleotide
with the 30-OH was connected through a covalent loop to the
N40 pool. In the key selection step, glycosylation of the 30-OH
enables subsequent separation by polyacrylamide gel electro-
phoresis (PAGE) of the DNA sequences that catalyzed the reaction.
The PAGE-shifted DNA sequences are amplified by PCR and ligated
to the 30-OH DNA oligonucleotide to begin a new selection round.
The first selection experiment used the 2-chloro-4-nitrophenyl
b-D-Glc glycosyl donor 1a. 2-Chloro-4-nitrophenyl b-D-glucuronic
acid was prepared in two steps from 1-bromo-2,3,4-tri-O-acetyl-a-D-
glucuronide methyl ester (see ESI†) and conjugated to a 50-NH2-
modified DNA oligonucleotide. This conjugate was stable (o0.5%
degradation in 24 h) in both of our commonly used selection
conditions,3 which are (A) 70 mM HEPES, pH 7.5, 1 mM ZnCl2,
20 mM MnCl2, 40 mM MgCl2, and 150 mM NaCl at 37 1C, and
(B) 50 mM CHES, pH 9.0, 40 mM MgCl2, and 150 mM NaCl at
37 1C. Note that Mg2+ is present in both conditions A and B,
whereas Zn2+ and Mn2+ cannot be used at the higher pH of
conditions B due to precipitation and oxidation, respectively,
although the higher pH could facilitate greater DNA-catalyzed
reactivity. After 11 rounds in conditions A with 14 h incubation
in the selection step of each round, the pool activity was 13%
(see selection progression in Fig. S2A, ESI†). Individual deoxy-
ribozymes were cloned from the round 11 pool, revealing two
distinct sequences (Fig. S3A, ESI†): 11GV112 with rate constant
kobs = 0.11 ꢀ 0.01 hꢁ1 (with Zn2+/Mn2+/Mg2+; n = 4, mean ꢀ sd)
and 55% yield at 48 h (Fig. 3A; the glycosylation product
identity confirmed by MALDI mass spectrometry, Table S2,
ESI†) and 11GV103 with kobs = 0.07 hꢁ1 and 15% yield at 48 h
(data not shown).§ The anomeric configurations of the glyco-
sylation products were not assigned. For calibration regarding the
kobs values of B0.1 hꢁ1, ‘‘speed limits’’ of nucleic acid enzymes
are generally considered to be B1 min ;
ꢁ1 12 on that basis, there is
room for improvement in kobs. Separately, after 8 rounds in
conditions B, aberrantly migrating product bands were observed,
and the selection experiment was discontinued.
Fig. 3 Glycosylation of a 30-OH acceptor using the 11GV112 deoxy-
ribozyme. (A) Metal ion dependence when using the 2-chloro-4-nitrophenyl
b-D-Glc glycosyl donor 1a. In the PAGE image, representative timepoints are
shown at t = 0, 12, and 48 h for the indicated metal ion combinations. The
metal ion combinations not shown, including Mn2+ or Mg2+ alone, had no
activity. S = substrate, P = product. Incubation conditions: 70 mM HEPES,
pH 7.5, 150 mM NaCl, combinations of 1 mM ZnCl2, 20 mM MnCl2, and 40 mM
MgCl2 as indicated, at 37 1C. The Zn2+ concentration was optimized; with 1a,
the yield was lower at either 0.8 or 1.2 mM Zn2+ (data not shown). (B) Glycosyl
donor dependence (in presence of all of Zn2+, Mn2+, and Mg2+).
The 11GV112 deoxyribozyme was partially randomized and
reselected (Fig. S2B, ESI†), providing a phylogeny that revealed
many conserved nucleotides (Fig. S3B, ESI†). However, none of
the reselected sequence variants had improved rate constant or
yield in comparison with the parent sequence (data not shown).
Evaluation of 11GV112 using various incubation conditions
revealed dependence upon both Zn2+ and Mn2+ (kobs with
Zn2+/Mn2+ 0.06 hꢁ1), with trace activity in Zn2+/Mg2+ and no
detectable activity in Zn2+ alone (Fig. 3A). 11GV112 was assayed the glycosyl donor. 4-Nitrophenyl a-D-glucuronic acid was prepared
with the b-series of glycosyl donors 1a, 1b, and 1c as well as the by TEMPO oxidation of 4-nitrophenyl a-D-glucose (see ESI†)
4-nitrophenyl a-D-Glc donor 2b (Fig. 3B). Donor 1b supported and joined to 50-NH2-modified DNA, providing a conjugate that
glycosylation rate (kobs 0.15 hꢁ1) and yield (44% at 48 h) compar- was stable under selection conditions A (conditions B were not
able to that of 1a, whereas neither 1c nor 2b permitted any activity. evaluated). After 16 selection rounds, the pool activity was 13%
In a separate selection experiment, the 4-nitrophenyl a-D-Glc (Fig. S2C, ESI†), and individual deoxyribozymes were cloned,
donor 2b was instead used. The 4-nitrophenyl glycoside (i.e., 2b providing two distinct sequences (Fig. S3C, ESI†). The 16MJ132
rather than 2a) was used for synthetic simplicity; the key considera- deoxyribozyme had kobs = 0.080 ꢀ 0.010 hꢁ1 (with Zn2+/Mn2+
/
tion is that in this experiment, 2b is the a rather than b anomer of Mg2+; n = 4) and 28% yield at 48 h (Fig. 4), and 16MJ101 had
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DNA-anchored UDP-Glc. This finding emphasizes the value of
aryl glycosides for such selection experiments. RNA-catalyzed
nucleotide synthesis by glycosylation of nucleobases with sugar
pyrophosphates has been reported,15 but such glycosyl donors
are similarly anticipated to be rather unstable under common
selection conditions, and their synthesis is less straightforward
than for aryl glycosides.
In summary, we have demonstrated that both b and a anomers
of aryl glycosides can be effective glycosyl donors for DNA catalysts
that glycosylate the 30-OH group of a DNA oligonucleotide sub-
strate. A deoxyribozyme identified with one anomer is inactive with
the other anomer, suggesting but not requiring mechanistic
differences between the two varieties of DNA enzyme. Glycosylated
oligonucleotides (i.e., oligonucleotide–carbohydrate conjugates)
have practical applications;16 with continued development, deoxy-
ribozymes may provide an alternative synthetic route that avoids
various complications associated with carbohydrate synthetic
chemistry. Many challenges remain in order to achieve our long-
term goal of identifying peptide-glycosylating deoxyribozymes.
In particular, the new DNA catalysts reported here function only
with a DNA 30-OH glycosyl acceptor and also require that the aryl
glycoside donor is DNA-anchored. Both reaction partners must be
addressed via further selection efforts, which are ongoing in our
Fig. 4 Glycosylation of a 30-OH acceptor using the 16MJ132 deoxy-
ribozyme. Shown are the metal ion dependence using the 4-nitrophenyl
a-D-Glc glycosyl donor 2b, as well as assay with the 4-nitrophenyl b-D-Glc
glycosyl donor 1b. Incubation conditions: 70 mM HEPES, pH 7.5, 150 mM
NaCl, combinations of 0.4 mM ZnCl2, 20 mM MnCl2, and 40 mM MgCl2 as
indicated, at 37 1C. The Zn2+ concentration was optimized; with 2b, similar
k
obs and yield were observed with 0.2–0.5 mM Zn2+, whereas the yield was
lower at or above 0.6 mM Zn2+ (data not shown). The metal ion combinations
not shown, including Mn2+ or Mg2+ alone, had no activity.
kobs = 0.07 hꢁ1 and 17% yield at 48 h (data not shown). Using laboratory.
the 4-nitrophenyl a-D-Glc donor 2b, 16MJ132 required both
This work was supported by a grant to S. K. S. from the
Zn2+ and Mn2+ (kobs with Zn2+/Mn2+ 0.07 hꢁ1), and no activity National Institutes of Health (R01GM065966). B. M. B. was
was observed with the isomeric b-donor 1b. These patterns of partially supported by NIH T32GM070421. S. M. W. was partially
metal dependence and glycosyl donor anomer dependence are supported by an NIH predoctoral fellowship (F31GM115147). Mass
analogous to those found for 11GV112.
spectrometry was performed at the UIUC School of Chemical
In parallel with the above-described selection experiments Sciences Mass Spectrometry Laboratory on an instrument pur-
in which a DNA 30-OH was the glycosyl acceptor, we performed chased with support from NIH grant S10RR027109A.
a separate selection that used a DNA-anchored CAAYAA hexa-
peptide as the intended glycosyl acceptor (see Fig. S4, ESI,† for
structure) along with the 2-chloro-4-nitrophenyl b-D-Glc donor 1a.
Notes and references
Although two new deoxyribozymes were identified (Fig. S3D, ESI†),
neither DNA catalyst used the Tyr side chain as the acceptor.
Instead, both of the new deoxyribozymes used a DNA nucleobase
functional group within or very near to the 50-end of the initially
random N40 region as the nucleophile to attack the glycosyl donor,
whereas the peptide moiety of the substrate was dispensable
(Fig. S5, ESI†).¶ Similar branched products were observed in a
separate selection with a CAASAA Ser-containing hexapeptide
(data not shown). These observations indicate the need to
improve our selection strategy to disallow the survival of DNA
sequences that catalyze their own glycosylation rather than
modification of the intended peptide substrate. In previous
studies, we have initially found unanticipated DNA-catalyzed
reactivity and then resolved the issue to achieve our initial goal;
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§ Each deoxyribozyme in this study was named as, for example, 11GV112,
where 11 is the round number, GV1 is the systematic alphabetic designation
for the particular selection, and 12 is the clone number.
¶ We speculate that the conditions B selection experiment with the DNA
oligonucleotide acceptor and 1a donor provided analogous branched
products rather than 30-OH glycosylation, although the products were not
investigated in detail.
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