Enhanced nonenzymatic RNA copying with 2-aminoimidazole activated nucleotides

Article abstract of DOI:10.1021/jacs.6b13148

Achieving efficient nonenzymatic replication of RNA is an important step toward the synthesis of self-replicating protocells that may mimic early forms of life. Despite recent progress, the nonenzymatic copying of templates containing mixed sequences remains slow and inefficient. Here we demonstrate that activating nucleotides with 2-aminoimidazole results in superior reaction kinetics and improved yields of primer extension reaction products. This new leaving group significantly accelerates monomer addition as well as trimer-assisted RNA primer extension, allowing efficient copying of a variety of short RNA templates with mixed sequences.

Full text of DOI:10.1021/jacs.6b13148

Communication
pubs.acs.org/JACS
Enhanced Nonenzymatic RNA Copying with 2‑Aminoimidazole
Activated Nucleotides
Li Li, Noam Prywes,† Chun Pong Tam, Derek K. O’Flaherty, Victor S. Lelyveld, Enver Cagri Izgu,
Ayan Pal, and Jack W. Szostak*
Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology,
Massachusetts General Hospital, Boston, Massachusetts 02114, United States
S
* Supporting Information
reaction by 10−100 times, depending on whether the
downstream nucleotide is an activated monomer6 or an
ABSTRACT: Achieving efficient nonenzymatic replica-
activated oligonucleotide.7 We have recently suggested that
tion of RNA is an important step toward the synthesis of
self-replicating protocells that may mimic early forms of
life. Despite recent progress, the nonenzymatic copying of
templates containing mixed sequences remains slow and
inefficient. Here we demonstrate that activating nucleo-
tides with 2-aminoimidazole results in superior reaction
kinetics and improved yields of primer extension reaction
products. This new leaving group significantly accelerates
monomer addition as well as trimer-assisted RNA primer
extension, allowing efficient copying of a variety of short
RNA templates with mixed sequences.
the physical origin of this rate enhancement is due to the
covalent reaction of 2-MI-activated monomers with each other
to form imidazolium bridged dinucleotides, which are highly
reactive intermediates in the copying of RNA templates by
primer extension.8
In an effort parallel to the above mechanistic investiga-
tions,5−8 we sought to develop a leaving group with enhanced
catalytic activity when used to activate nucleotides in positions
downstream of the reaction site from a screen of leaving groups.
We measured primer extension rates on a template that
encodes the product sequence -CGGG-3′. Reaction mixtures
contained C monomers activated by 2-MI (2-MeImpC), which
could only bind the initial +1 position, and G monomers
eplication of the genetic information is a key chemical
Rprocess in living organisms. Whereas modern cells copy activated by various leaving groups, which could only bind
downstream (1a−1h, Figure 1). The results revealed two
general trends. First, a larger substituent at the 2-position slows
down the reaction (1a−1d). Second, the reaction rate decreases
as the pKa of the leaving group decreases (1a, 1e−1h).
Replacing 2-MI on the downstream G monomers by 2-
methylpyrrole (1i) abolishes the catalytic effect, indicating that
neither pi-stacking between neighboring leaving groups, nor the
NH moiety alone, which may serve as a hydrogen-bond donor,
are sufficient to catalyze primer extension; the lack of a catalytic
effect is consistent with primer extension proceeding through
an imidazolium-bridged intermediate.
Guided by these two empirical rules, we reasoned that 2-
aminoimidazole (2-AI, 1j), which has a small substituent on the
2-position and a higher pKa than 2-MI,9 might serve as a better
catalyst of primer extension with 2-MeImpC. Indeed, 2-AI
activated pG accelerated 2-MeImpC addition by 7 times
compared to its 2-MI activated counterpart. A plausible
explanation for the enhanced reactivity of 2-AI-activated pG
is that 2-AI accelerates the formation of the 2-amino-
imidazolium bridged dinucleotide intermediate due to the
greater nucleophilicity of its imidazole N3. The larger 2-
methylaminoimidazole leaving group (1k) strongly reduced the
rate despite its high pKa. Although the poor reactivity of 1k-
activated pG can be attributed in part to steric hindrance by the
bulkier 2-methylamino substituent, the magnitude of the effect
is surprising and is the subject of ongoing investigation.
their genomes using sophisticated protein enzymes, primordial
cells had to rely on primitive mechanisms without access to an
elaborate translation system. One such mechanism is non-
enzymatic RNA replication,1 in which chemically activated
nucleotides, such as phosphoroimidazolides, spontaneously
polymerize when guided by a template sequence. Its simplicity
has made it an attractive approach in laboratory attempts to
develop self-replicating protocells that mimic early forms of
life.2 However, a major limitation of this approach is its poor
yield and slow rate, particularly when copying templates with
mixed sequences.3 Here we report the development of a
superior leaving group, 2-aminoimidazole, which enables
efficient copying of short RNA templates with mixed
sequences.
Since the discovery in 1981 that 2-methylimidazole (2-MI)
activated nucleotides show both good reactivity and the ability
to direct the formation of natural 3′-5′ linkages, 2-MI has been
the most widely used leaving group for studies of nonenzymatic
RNA template copying by primer extension.4 Interestingly, 2-
MI activated monomers are more reactive in primer extension
than the corresponding imidazole and 4-methylimidazole
derivatives,4 both of which contain better leaving groups than
2-MI as judged by their hydrolysis rates.5 The higher reactivity
of 2-MI activated nucleotides has been attributed to an
interaction between leaving groups of neighboring monomers,
such that the leaving group on the downstream nucleotide
exhibits a catalytic effect that increases the rate of reaction
between the upstream nucleotide and the adjacent primer.6
This leaving-group interaction accelerates the primer extension
Received: December 22, 2016
© XXXX American Chemical Society
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DOI: 10.1021/jacs.6b13148
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Journal of the American Chemical Society
Communication
short templates, including those directing the incorporation of
A, U, and 2-thiouridine (s2U) monomers, 2-AI activated
monomers react from 6 to 100 times faster than their 2-MI
activated counterparts (Table 1). The rate enhancement of s2U
Table 1. Reaction Rates of Nonenzymatic Primer Extension
Measured by Primer Consumption
a
reaction rates (h−1
)
product sequence
2-AI leaving group
2-MI leaving group
-CCC-3′
-GGG-3′
-CGG-3′
-GCC-3′
-AGG-3′
-UGG-3′
-s2UGG-3′
6.7 0.1
4.92 0.09
7.5 0.1
1.11 0.03
0.61 0.01
0.31 0.02
0.090 0.003
0.026 0.003
4.5 0.2
2.81 0.03
0.62 0.01
1.4 0.3
b
n.d.
0.035 0.002
a
Reaction conditions as described in Figure 1. Results are given as the
mean s.e.m. of triplicate experiments. Too slow to be measured.
b
over U is consistent with our previous studies using the 2-MI
leaving group.10 Collectively, these results establish that
monomers activated by 2-AI are superior substrates for
nonenzymatic RNA primer extension.
Figure 1. Development of 2-aminoimidazole as a superior leaving
group for nonenzymatic primer extension. Top row: the template
(cyan)-dependent reaction of 2-MI activated C monomers (red) with
the primer (green) was used to probe the effect of adjacent leaving
groups (LG, black) on the reaction rate. Curved arrow indicates the
site of reaction. Varying the leaving group (1a−1k) influenced the
rates by up to 200 times, with 2-aminoimidazole (1j) giving the fastest
rate. Reaction conditions: 1.5 μM primer, 5 μM template, 200 mM
HEPES pH 8.0, 50 mM MgCl2, and 20 mM monomers. All
experiments were performed in triplicate. Blue: mean observed rate
in h−1; standard error of the mean (s.e.m.) in the last digit is shown in
parentheses. Magenta: pKa of the leaving group.
Encouraged by the superior performance of 2-AI activated
monomers, we next asked if these monomers can copy
templates containing mixed sequences with the assistance of
downstream activated “helper” trinucleotides (trimers).7,11 To
copy a template that encodes the product sequence -UGAC-3′,
four 2-AI activated trimers (GAC, ACG, CGG, and GGU,
where the underlined nucleotides have a 5′-phosphoro-(2-
aminoimidazole) moiety) were mixed with 2-AI activated A, C,
G, and s2U monomers. The reaction time course (Figure 3a)
revealed +1, + 2, and +3 extension intermediates, indicating
that most primers extended via addition of monomers rather
than ligation of trimers. After 1 day, 95% of the primer had
been converted to +4 products, some of which had then further
reacted to form +5 to +7 products.
We confirmed the identities of the primer extension products
by liquid chromatography−mass spectrometry (Figure 3b). To
enhance the chromatographic separation, we used unlabeled
primers, and templates that contained five-nucleotide 3′-
overhangs. An exhaustive mass-based search revealed peaks
corresponding to the correct +1 to +7 products including a
major peak of the +4 full-length product and only one minor
peak that is consistent with a product having misincorporated
s2U in place of A at the +3 position, likely due to an s2U:U
mismatch.12 Our analysis did not reveal any misincorporated +4
products, indicating that such a mismatch slows down further
primer extension13 even in the presence of “helper” trimers. In
addition, we did not observe products containing misincorpo-
rations caused by G-U wobble pairing, confirming that 2-
thiolation effectively suppresses G-U mismatch formation.10
Compared with the 2-MI leaving group, the new 2-AI leaving
group significantly enhances the efficiency of trimer-assisted
template copying. Until now, an AA dinucleotide in the
template has been a significant roadblock for primer
extension.3,14 Even with the assistance of trimers activated by
2-MI, the yield of +2 products is only 10% and 50% for U and
s2U monomers, respectively.7 With 2-AI as a leaving group, the
yield increased to 92% for U monomers (Figure 4a) and 98%
for s2U monomers (Figure 4b), respectively. As such, the
nonenzymatic copying of an AA dinucleotide, a reaction long
In addition to their superior catalytic effect, monomers
activated by 2-AI are also better substrates at the +1 site than
those activated by 2-MI (Figure S1), an observation consistent
with their faster hydrolysis rates in a solution mimicking the
primer-extension condition (Figure S2). As a result, the
phosphoroimidazolide 1j-pG extends the primer far more
efficiently than 1a-pG on a template that encodes the product
sequence -GGGG-3′ (Figure 2). This motivated us to try using
2-AI activated monomers in both the upstream and down-
stream positions in primer extension reactions. For various
Figure 2. Efficient nonenzymatic RNA copying using phosphoroimi-
dazolides with 2-AI leaving group. The time course of primer
extension reaction using phosphoroimidazolides 1j-pG and 1a-pG
were assayed using polyacrylamide gel electrophoresis (PAGE).
Positions of primer and +1 to +4 products are indicated. Reaction
conditions as described in Figure 1.
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Figure 4. Efficient one-pot nonenzymatic copying of various short
RNA templates. (a) Copying AA dinucleotide templates using 2-AI
activated U monomers and trimers. (b) Copying AA dinucleotide
templates using 2-AI activated s2U monomers and trimers. (c)
Extending primers by UAGC using 2-AI activated A, C, G, and U
monomers and trimers. (d) Extending primers by the sequence
AUGAGGC using 2-AI activated A, C, G, and U monomers and
trimers. The extension products were assayed by PAGE. Positions of
primers and extended products are indicated. Reaction conditions as
described in Figure 3.
efficient nonenzymatic copying of various short RNA templates
with mixed sequences. Notably, 2-AI is also related to 2-
aminooxazole, a key intermediate in the prebiotic synthesis of
the pyrimidine ribonucleotides.17 Further optimization of the
primer-extension reaction may reveal a prebiotically plausible
pathway to the efficient copying of RNAs that are long enough
to encode important biological functions such as ribozyme
activity.
Figure 3. Efficient one-pot extension of primers by four nucleotides.
(a) Electrophoretogram of extension products after different reaction
times, as assayed by PAGE. Positions of primer and +1 to +7 products
are indicated. Reaction conditions: 1.2 μM primer (green), 1.5 μM
template (cyan), 200 mM HEPES pH 8.0, 50 mM MgCl2, 0.5 mM
trimer (black), and 10 mM monomer (red). (b) Overlay of extracted
ion chromatograms of residual primer (P) and extension products
after 1 day (+1 to +7) observed by LC-MS. Observed ions for the peak
labeled +3* are consistent with a product having misincorporated s2U
in place of A at the +3 position. Observed ions for the peak +7† are
consistent with either the correct +7 product or an alternative double
mutant product (see Table S1).
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b13148.
Figures S1−S2, Tables S1−S3, experimental procedures
and analytical data (1H and 13C NMR, HRMS) for all
new compounds (PDF)
considered to be impossible under normal reaction conditions,
has now been achieved by combining the 2-thiolation-induced
preorganization of monomers in the reactive C3′-endo
conformation,10,15 the catalytic effect of helper trimers,7 and
the superior reactivity of the 2-AI leaving group.
To test the generality of this method, we synthesized two
additional mixed-sequence templates containing AU and UA
sequences that are also considered to be challenging for
nonenzymatic RNA copying.3 Satisfyingly, both templates can
be copied with overall yields higher than 85% in just 1 day with
unmodified A, C, G, and U monomers (Figure 4c,d). The
average single-nucleotide extension yield is 97−98%. We are
currently engaged in a systematic exploration of the sequence
space available to nonenzymatic RNA copying using the 2-AI
leaving group.
The development of the 2-aminoimidazole leaving group is a
significant advance in nonenzymatic RNA copying. 2-AI
imparts higher reactivity to phosphoroimidazolides, signifi-
cantly increasing both the rate and the yield of primer
extension. The improvement in leaving-group reactivity also
synergizes with 2-thiolation of uridine and the use of catalytic
trimers, both of which are prebiotically plausible,7,16 enabling
AUTHOR INFORMATION
Corresponding Author
*szostak@molbio.mgh.harvard.edu
ORCID
Chun Pong Tam: 0000-0001-6381-9011
Victor S. Lelyveld: 0000-0002-3890-0288
Enver Cagri Izgu: 0000-0001-6673-3635
Jack W. Szostak: 0000-0003-4131-1203
■
Present Address
†Department of Plant and Environmental Sciences, The
Weizmann Institute of Science, P.O. Box 26, Rehovot 76100,
Israel
Funding
J.W.S. is an Investigator of the Howard Hughes Medical
Institute. L.L. is a Life Sciences Research Foundation Fellow.
D.K.O. is a recipient of a Postdoctoral Research Scholarship
from the FRQNT, Quebec, Canada. This work was supported
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Journal of the American Chemical Society
Communication
in part by a grant (290363) from the Simons Foundation to
J.W.S. and by a grant from the NSF (CHE-1607034) to J.W.S.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Szostak lab members Dr. L. Zhou for
assistance with oligonucleotide synthesis and C. Giurgiu for
helpful discussions.
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