N6-Isopentenyladenosine in RNA Determines the Cleavage Site of Endonuclease Deoxyribozymes

Article abstract of DOI:10.1002/anie.202006218

RNA-cleaving deoxyribozymes can serve as selective sensors and catalysts to examine the modification state of RNA. However, site-specific endonuclease deoxyribozymes that selectively cleave post-transcriptionally modified RNA are extremely rare and their

Full text of DOI:10.1002/anie.202006218

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Accepted Article
Title: N 6-isopentenyladenosine in RNA determines the cleavage site
of endonuclease deoxyribozymes
Authors: Anam Liaqat, Carina Stiller, Manuela Michel, Maksim V
Sednev, and Claudia Höbartner
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202006218
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10.1002/anie.202006218
Angewandte Chemie International Edition
RESEARCH ARTICLE
N6-isopentenyladenosine in RNA determines the cleavage site
of endonuclease deoxyribozymes
Anam Liaqat,[a] Carina Stiller,[a] Manuela Michel,[a] Maksim V. Sednev[a] and Claudia Höbartner*[a]
Abstract: RNA-cleaving deoxyribozymes can serve as selective
sensors and catalysts to examine the modification state of RNA.
However, site-specific endonuclease deoxyribozymes that selectively
cleave posttranscriptionally modified RNA are extremely rare and their
specificity over unmodified RNA is low. In this study, we report that
the native tRNA modification N6-isopentenyladenosine (i6A) strongly
enhances the specificity and has the power to reconfigure the active
site of an RNA-cleaving deoxyribozyme. Using in vitro selection, we
identified a DNA enzyme that cleaves i6A-modified RNA at least 2500-
fold faster than unmodified RNA. Another deoxyribozyme shows
unique and unprecedented behavior by shifting its cleavage site in the
presence of the i6A RNA modification. Together with deoxyribozymes
that are strongly inhibited by i6A, these results highlight intricate ways
of modulating the catalytic activity of DNA by posttranscriptional RNA
modifications.
recognize m6A and modulate the cleavage response is currently
unknown. Best discrimination was observed by the VMC10 DNA
enzyme, which was inhibited by m6A and cleaved unmodified
RNA up to 150-fold faster. In contrast, DNA enzymes that
preferred cleavage of m6A-RNA were less specific, and reached
only 5-10-fold faster cleavage rates for modified versus
unmodified RNA. From these observations, we concluded that a
modified nucleotide like m6A can easily interfere with formation of
a catalytically competent DNA structure, but the opposite direction
is more difficult, i.e. evolving catalytic DNA that is strictly
dependent on the presence of a small chemical modification in
the RNA target is challenging. Along these lines, one may expect
that larger RNA modifications could establish energetically
favourable interactions, and result in deoxyribozymes that more
effectively discriminate modified from unmodified RNA. To
explore this hypothesis, we chose to evolve deoxyribozymes that
selectively cleave N6-isopentenyladenosine (i6A)-modified RNA
and compared their activity to m6A-sensitive endonucleases.
Introduction
N6-isopentenyladenosine (i6A) is a structural analogue of m6A
that contains the bulkier dimethylallyl group as N6-substituent.
The i6A modification is conserved in certain tRNAs in bacteria and
eukaryotes.[7] Located at position 37 next to the anticodon, i6A is
suggested to enhance translation efficiency and fidelity by
stabilizing cognate codon-anticodon interactions,[8] and may
facilitate RNA localization by associating with membranes.[9] In
some cases, i6A is further modified, for example by thio-
methylation to ms2i6A in mitochondrial tRNAs[7] and potentially in
other (nuclear) RNAs,[10] or by oxidation/hydroxylation to ms2io6A,
which is found e.g. in salmonella tRNAs.[7, 11] Interestingly, the i6A
modification is necessary for the expression of human seleno-
proteins,[12] and it has recently been shown as an essential
determinant for the installation of additional tRNA anticodon
modifications, including m3C at positon 32, and Um at position
34.[13] Given the importance of i6A in many natural contexts, i6A-
sensitive deoxyribozymes could become diagnostically useful
tools for the analysis of tRNA modification states.
Cellular RNAs contain a variety of chemically diverse post-
transcriptional modifications with important cellular functions.[1]
Besides sophisticated analytical methods that generate
transcriptome-wide modification maps, simple tools are required
to validate predictions and to examine the modification state of
distinct sites.[2] Focusing on N6-methyladenosine (m6A) as one of
the most abundant mRNA modifications, we have recently
reported m6A-sensitive RNA-cleaving (endonuclease) deoxy-
ribozymes for the site-specific interrogation of m6A modification
levels.[3] Deoxyribozymes with endonuclease activity bind to the
target RNA via Watson-Crick base-pairing and catalyze site-
specific RNA cleavage by promoting the attack of a specific 2'-
hydroxyl group onto the adjacent phosphodiester linkage,
resulting in the formation of 2',3'-cyclic phosphate and 5'-hydroxyl
termini in the fragments of the target RNA.[4] Inhibition of this
mechanisms by 2'-O-methylated nucleotides was used to analyze
ribose methylation in rRNA.[5] The m6A-sensitive DNAzymes were
generally applicable to analyze the presence of m6A in DGACH
sequence motifs, as shown for lncRNAs and a set of C/D box
snoRNAs (m6A),[3] and these analyses were recently extended to
other RNAs.[6] The detailed mechanism, how DNA enzymes
Here we report the discovery and characterization of three
classes of RNA-cleaving deoxyribozymes with distinct responses
to the natural tRNA modification i6A (Figure 1). First, we found that
the activity of three AA deoxyribozymes is abolished by i6A.
Second, the AB08 deoxyribozyme is strongly activated by i6A and
shows unprecedented selectivity in cleaving only i6A-modified
RNA next to the site of modification. Third, we found the
deoxyribozyme AC17 for which the modified nucleotide i6A
causes a distinct shift of the cleavage site by one nucleotide.
[a]
A. Liaqat, C. Stiller, M. Michel, Dr. M.V. Sednev,
Prof. Dr. C. Höbartner
Institute of Organic Chemistry, University of Würzburg
Am Hubland, 97074 Würzburg (Germany)
E-mail: claudia.hoebartner@uni-wuerzburg.de
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.202006218
Angewandte Chemie International Edition
RESEARCH ARTICLE
Figure 1. a) N6-isopentenyladenosine (red) in comparison to unmodified
adenosine (blue, only nucleobases shown). b) RNA-cleaving deoxyribozymes
catalyze the cleavage of the phosphodiester backbone by transesterification. c-
e) Schematic depiction of three different classes of DNAzymes identified in this
study: c) AA DNAzymes are inhibited by i6A. d) The AB08 DNAzyme cleaves
only i6A-modified RNA. e) The cleavage site of the AC17 DNAzyme is influenced
by the modification state of the RNA, i.e. modified and unmodified RNA are
cleaved at different positions.
Figure 2. a) Sequences and predicted secondary structures of three AA
DNAzymes, AA07, AA14, and AA17. b) PAGE analysis of cleavage reaction
with unmodified RNA (R1, labeled with blue '–') and i6A-modified RNA (R2,
labeled with red '+') shows selective cleavage of unmodified RNA, and identifies
the cleavage site: AA07 and AA14 cleave at G15; AA17 cleaves at G16.
Experiments used 3’-fluorescein-labeled RNA. c) Single-turnover kinetics for
AA17. Graphs for others and gel images are shown in Figure S3. d) HR-ESI-
MS analyses of cleaved products. The longer 15/16-nt products contain a 2’,3’-
cyclic phosphate, the shorter 12/13-nt fragments contain 5'-OH and 3'-C6-NH2-
termini.
Results and Discussion
In vitro selection of RNA-cleaving deoxyribozymes
A synthetic DNA library with 20 random nucleotides was used
for the in vitro selection of RNA-cleaving deoxyribozymes,
following established procedures (Figure S1).[3] The 28-nt long
RNA substrates were prepared by solid-phase synthesis (Table
S1), and covalently ligated to the DNA library (Table S2). The
modified nucleotide i6A was incorporated using a suitable i6A
phosphoramidite building block, which was obtained by
regioselective N6-alkylation of an N6-acetylated intermediate.[14]
further upstream at G15. Interestingly, the i6A at position 17 in the
substrate RNA strongly inhibited the catalytic activity of all three
DNA enzymes, independent of their cleavage site. Previously,
m6A was shown to inhibit the cleavage activity of DNAzymes
when located directly next to the cleavage site (e.g. in VMC10),[3]
but such a strong effect of a natural RNA modification at a more
remote location, as is the case of i6A on AA07 and AA14 activities,
has not been observed before. The larger size of the isopentenyl
group in i6A compared to the methyl group in m6A is likely
responsible for this long-range effect. Indeed, when the AA
DNAzymes were tested on m6A-modified RNA (R3, same
sequence as R2, with i6A replaced by m6A), AA17 was strongly
inhibited by m6A, while AA07 and AA14 retained substantial
activity (Figure S3).
During 15 rounds of separation and amplification, catalytically
active DNA sequences were trained to discriminate modified and
unmodified RNA. To achieve the desired selectivity, two separate
selection experiments were carried out, in which positive and
negative selections were altered to favor unmodified RNA (AA
selection) or i6A-modified RNA (AB selection), respectively.
AA DNAzymes are inhibited by i6A
After cloning of the enriched AA selection library, three DNA-
enzymes (AA07, AA14 and AA17) were identified that catalyzed
site-specific cleavage of unmodified RNA (R1) (Figure 2). RNA
cleavage products were only observed with R1 as substrate
(Figure 2b,c), but not with i6A-containing RNA (R2). The cleavage
sites were assigned by comparison to alkaline hydrolysis and
RNase T1 digestion ladders, and confirmed by HR-ESI-MS of
both cleavage products (Figure 2d). AA17 cleaved R1 at position
G16, while the other two DNA enzymes cleaved one nucleotide
AB08 DNAzyme cleaves only i6A RNA
The AB selection revealed the deoxyribozyme AB08, which
showed high activity for cleaving i6A-RNA, but it did not cut
unmodified RNA to any significant extent (Figure 3). This notable
finding reports the first DNA enzyme that specifically cleaves a
post-transcriptionally modified RNA. The AB08 DNA enzyme
strictly requires the modified nucleotide in the substrate RNA and
yields 80% cleaved product after 6 h at pH 7.5 with 20 mM MgCl2.
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10.1002/anie.202006218
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RESEARCH ARTICLE
The cleavage site of AB08 was located directly next to the
modified nucleotide i6A, as confirmed by high-resolution mass
spectrometry of the 12-nt 5'-OH-terminated i6A-containing
fragment (Figure 3d). These findings raised the question if AB08
could be activated by m6A rather than i6A in the RNA substrate.
Using the analogous m6A-modified RNA R3, no cleavage product
was observed upon incubation with AB08 (Figure S4). Thus, the
AB08 DNA enzyme is indeed specific for i6A-modified RNA.
in vitro selection experiments.[4c] The A9G10Y triloop (position 10-
12 in Figure 3e) and a conserved C16G17 dinucleotide in the 3'-
bulge (position 17-18 in the sequence logo) may be involved in a
pseudoknot motif related to the one observed in the crystal
structure of an 8-17 variant.[18] The highly invariable guanosine
triple in the 5'-bulge is a distinctive feature of AB08 and is likely
responsible for the astonishing selectivity for cleaving only i6A
RNA. In the absence of any structural insights into the
organization of the catalytic core, the detailed mechanism of i6A
recognition remains unknown.
i6A shifts the cleavage site of AC17 DNAzyme
The AB selection surfaced an additional highly interesting
DNA enzyme, which was named AC17.[19] Upon screening the
catalytic activity, it was observed that AC17 yielded cleavage
products with both i6A-modified and unmodified RNAs (R1 and
R2), and that these products exhibited different migration on the
analysis gel (Figure 4). Comparison of the migration behavior with
the already assigned cleavage products of AA14 and AB08
revealed that AC17 cuts R1 at G16, but R2 at G15 (Figure 4b).
Inspection of the sequence and secondary structure prediction of
AC17 revealed that it is highly related to the previously identified
VMC10 DNA enzyme that showed strong inhibition by m6A.[3]
A
single point mutation (C12T) converts AC17 into VMC10.
Therefore, a more detailed characterization of the AC17 DNA
enzyme in comparison to VMC10 was undertaken. First, AC17
was tested on m6A-RNA and was found to yield only 10% cleaved
RNA, and this cleavage occurred at G16 (Figure 4c, S7). Thus,
m6A inhibits the ability of AC17 to cut at G16, but at the same time
does not activate it to cut at G15 either. This confirms that the
unrivalled shift of the cleavage site is specific to i6A. On the other
hand, we also checked the activity of VMC10 to cut i6A RNA, and
found about 25% cleavage at G15 (Figure 4d, S7). Thus, both
AC17 and VMC10 cut i6A RNA at G15, but a cytidine at position
12 (C12) of the catalytic core enabled higher rate and yield
compared to thymidine (T12).
Figure 3. a) Secondary structure prediction of AB08 and indicated cleavage site
of i6A RNA. b) Gel image and kinetic graph demonstrating selectivity for i6A-
modified RNA. time points are: 0, 10 min, 0.5, 1, 2, 3, 6, 22 h. d-e) HR-ESI MS
confirming the cleavage site of R2. c) Top: measured spectrum of 12-nt i6A-
modified fragment (i6ACUUCCGUAACU), bottom: simulated spectrum for
C117H149N40O83P11 with exact mass of 3782.58 amu. d) Sequence logo for
Intrigued by the activity of AC17 to cut both RNAs, we
analyzed the enriched DNA library for AC17 abundance and
potential relatives. The round 15 AB pool was separated into
active and inactive fractions, now using unmodified RNA. This
corresponds to a “negative” selection round 16. Both fractions
were amplified with the corresponding NGS primers, and 1.4
million reads were analyzed (Table S3). In accordance with the
faster cleavage rate of R1, a larger number of AC17 variants (with
Levenshtein distance ≤ 4) were found in the cleaved fraction
(1177 variants versus 426 variants in the uncleaved fraction; the
numbers were significantly reduced when filtered for more than
50 reads: 45 and 7 variants, respectively). The sequence logo for
the alignment of 45 AC17 variants is shown in Figure 4f. The 3'-
terminal nucleotides of the catalytic core showed highest
variability. The importance of this region was investigated using
synthetic variants of AC17. Deleting T19 resulted in a DNA
enzyme that cleaved both R1 and R2 with comparable rates, and
the ratio of the cleavage products can be directly translated into
modification levels (Figure S8). Mutation at position 20 resulted in
a series of DNA enzymes with very different behavior: the activity
of the A20G mutant was highly comparable to the parent
deoxyribozyme, while the A20C variant showed strongly preferred
alignment of 54 sequences (within a Levenshtein distance of 4 from AB08),
represented in the active fraction of the enriched DNA library.
To learn more about the abundance of AB08 and potential
related sequences, the enriched selection library was analyzed by
next generation sequencing (NGS). The round 15 library was
separated into active and inactive fractions for cleavage of i6A-
RNA; this corresponds to one additional round of selection, i.e.
round 16. Round 15 and both fractions of round 16 were amplified
with the corresponding Illumina NGS primers (Figure S2), and 1.3
million reads were analyzed (Table S3).[15] AB08 and its relatives
with Levenshtein distance ≤ 4 represented 6.4% of reads in round
15. This fraction increased to 10.9% in round 16. Interestingly, the
number of variants decreased from 73 to 54.[16] Comparison of the
most enriched AB08 variants (Figure S5) and the sequence
logo[17] plotted for the alignment of 54 relatives of AB08 shown in
Figure 3e identified the nucleotides with highest variability at
position 4 and at the 3'-end. The catalytic activities of several
synthetic AB08 variants confirmed the conserved motif (Figure
S6, Table S4), and may suggest a distant relationship of AB08 to
the 8-17 DNA enzyme family, which has been identified in several
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10.1002/anie.202006218
Angewandte Chemie International Edition
RESEARCH ARTICLE
cleavage of unmodified RNA with a 35-fold faster rate (Table S4,
Figure S8). On the other hand, the deletion of A20 completely
changed the activity of the deoxyribozyme:
logos in Figure 3e and 4f, and alignments in the SI). The relative
location of these motifs seems preserved, but the position of the
intervening GG dinucleotide involved in the formation of the base-
paired region is shifted by one nucleotide. In other words, the AGY
motif is located in a predicted triloop in AB08 and a tetraloop in
AC17. It remains to be seen if these motifs are directly involved in
the catalytic mechanism, and how the DNA nucleotides interact
with the RNA bases near the cleavage site to distinctly modulate
the activation of a specific 2'-OH group in response to the
modification state of the adenosine under investigation. Future
crystal structure analyses may also reveal the nucleotides
contacting the cleavage site, enabling rational engineering in
analogy to the structure-based design of RNA-ligating deoxy-
ribozymes.[20]
Conclusion
In summary, this study reported three new classes of RNA-
cleaving DNA enzymes that respond in distinct ways to the
presence of the natural modified nucleotide i6A in the target RNA.
The AB08 DNA enzyme absolutely requires the presence of i6A in
the target RNA for catalytic activity. The finding that i6A can fine-
tune the active site of the DNA enzyme AC17 for cleavage at
distinct positions is exceptional and may be further explored for
analytical applications, for example as a direct readout of
chemically modulated adenosine isopentenylation states.[21] In
the future, it will be interesting to investigate how other tRNA
modifications, including hydroxylated or thiomethylated i6A
analogues are recognized and how they influence the catalytic
activities of AB08, AC17 and future evolved variants for the study
of native tRNAs.[22]
The presented data demonstrate the surprising plasticity of
DNA’s catalytic ability. The reported modes of action are distinct
from previously observed responses of DNA enzymes to m6A in
RNA, and no other nucleobase modification-sensitive DNAzymes
have been reported. The direct comparison how i6A and m6A
affect the catalytic activity supports the conclusion that the larger
and more hydrophobic isopentenyl group permits better
discrimination due to larger energetic and kinetic differences. This
observation correlates with the larger thermodynamically
stabilizing effect of i6A compared to m6A in RNA hairpin
structures.[23] Structural and mechanistic investigations will follow
to allow deeper insights into the architecture of these new DNA
catalysts and their modes of i6A recognition.
Figure 4. a) Predicted secondary structure of AC17 shown to cut unmodified
RNA at G16 and i6A-RNA at G15. Sites of important mutations are indicated. b)
PAGE analysis of cleavage sites of AC17 in comparison to AA14, AA17 and
AB08. Note that this analysis was performed with 5'-32P-labeled RNA (in
contrast to the 3’-fluorescein-labeled RNAs used in Figures 2b and 3b). c) PAGE
analysis with 3’-fluorescein-labeled RNAs and kinetic graphs for cleavage of R1
(A), R2 (i6A) and R3 (m6A) RNA). d-e) kinetic graphs for cleavage of R1 (A), R2
(i6A) andR3 (m6A) RNA with VMC10 (d) and AC17-A20 (e). f) Sequence logo
for alignment of 45 AC17 variants with >50 reads and maximal 4 mutations.
Surprisingly, the AC17-A20 variant showed highly accelerated
cleavage of i6A-RNA, while unmodified RNA R1 was cleaved only
to a minor extent (Figure 4e, S7). In other words, AC17-A20 is
the second i6A-RNA-selective DNA enzyme discovered in this
study, with comparable kinetic activity to AB08, but cleaving one
nucleotide further upstream. Consistent with this result, the
number of reads for AC17-A20 (and its relatives with up to
maximal four mutations) was almost twice as high in the active
fraction of the i6A NGS library compared to the uncleaved fraction
(Table S2). Interestingly, AC17 contains conserved features
reminiscent of the AGY/CG motif and the guanosine triple at the
5'-bulge, which were also found in AB08 (compare sequence
Acknowledgements
This work was supported by the Deutsche Forschungs-
gemeinschaft (DFG, SPP1784 Chemical Biology of native nucleic
acid modifications) and by the European Research Council (ERC
No. 682586). AL acknowledges funding by a PhD scholarship
from the German Academic Exchange Service (DAAD). MVS
thanks the Graduate School for Life Sciences at the University of
Würzburg for a Postdoc Plus fellowship. We thank Michaela
Schraut and Tim Rieseberg for contributions to i6A synthesis, and
Sebastian Mayer for mass spectrometry. Illumina sequencing was
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RESEARCH ARTICLE
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Entry for the Table of Contents
RESEARCH ARTICLE
Probing the presence of prenylation.
The natural tRNA modification i6A
carries an isopentenyl (prenyl) group
at N6 of adenine. This modified
nucleotide fine-tunes the active site of
RNA-cleaving deoxyribozymes
resulting in a distinct shift of the
cleavage site. In addition, this study
reports the first DNA catalysts that
exclusively cut post-transcriptionally
modified RNA.
A. Liaqat, C. Stiller, M. Michel,
M.V. Sednev, C. Höbartner*
Page No. – Page No.
N6-isopentenyladenosine in RNA
determines the cleavage site of
endonuclease deoxyribozymes
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