Prebiotically Plausible RNA Activation Compatible with Ribozyme-Catalyzed Ligation

Article abstract of DOI:10.1002/anie.202010918

RNA-catalyzed RNA ligation is widely believed to be a key reaction for primordial biology. However, since typical chemical routes towards activating RNA substrates are incompatible with ribozyme catalysis, it remains unclear how prebiotic systems generated and sustained pools of activated building blocks needed to form increasingly larger and complex RNA. Herein, we demonstrate in situ activation of RNA substrates under reaction conditions amenable to catalysis by the hairpin ribozyme. We found that diamidophosphate (DAP) and imidazole drive the formation of 2′,3′-cyclic phosphate RNA mono- and oligonucleotides from monophosphorylated precursors in frozen water-ice. This long-lived activation enables iterative enzymatic assembly of long RNAs. Our results provide a plausible scenario for the generation of higher-energy substrates required to fuel ribozyme-catalyzed RNA synthesis in the absence of a highly evolved metabolism.

Full text of DOI:10.1002/anie.202010918

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Accepted Article
Title: Prebiotically plausible RNA activation compatible with ribozyme-
catalyzed ligation
Authors: Emilie Yeonwha Song, Eddy Ivanhoe Jiménez, Huacan Lin,
Kristian Le Vay, Ramanarayanan Krishnamurthy, and Hannes
Mutschler
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Prebiotically plausible RNA activation compatible with ribozyme-
catalyzed ligation
E. Y. Song, E. I. Jiménez, H. Lin,[b]+ K. Le Vay, R. Krishnamurthy * and H. Mutschler[a],[c]*
[
a]
[b]+
[a]
[b]
[
a]
E. Y. Song, Dr. K. Le Vay, Prof. Dr. H. Mutschler
Max Planck Institute of Biochemistry
Am Klopferspitz 18, 82152 Martinsried (Germany)
E-mail: mutschler@biochem.mpg.de
[
b]
Dr. E. I. Jiménez, Dr. H. Lin, Prof. Dr. R. Krishnamurthy
Department of Chemistry
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
E-mail: rkrishna@scripps.edu
[
[
c]
Prof. Dr. H. Mutschler
Technical University Dortmund
Otto-Hahn-Str. 4a, 44227 Dortmund (Germany)
These authors contributed equally to this work.
+]
Supporting information for this article is given via a link at the end of the document.
Abstract: RNA-catalyzed RNA ligation is widely believed to be a key
reaction for primordial biology. However, since typical chemical routes
towards activating RNA substrates are incompatible with ribozyme
catalysis, it remains unclear how prebiotic systems generated and
sustained pools of activated building blocks needed to form
increasingly larger and complex RNA. Herein, we demonstrate in situ
activation of RNA substrates under reaction conditions amenable to
catalysis by the hairpin ribozyme. We found that diamidophosphate
In the laboratory, RNA synthesis by ribozymes is achieved
through the ligation of pre-activated RNA substrates. For example,
some artificial ribozymes obtained by in vitro selection utilize 5’-
[
2–
triphosphate activation chemistry for phosphodiester formation.
4]
Their substrates are typically obtained from in vitro transcription
reactions involving RNA polymerase proteins and retain the 5'-
triphosphate of the first nucleotide. In vitro selection experiments
also yielded ribozymes that can triphosphorylate specific RNA
substrates using trimetaphosphate.[ However, credible non-
5]
(
DAP) and imidazole drive the formation of 2’,3’-cyclic phosphate RNA
mono- and oligonucleotides from monophosphorylated precursors in
frozen water-ice. This long-lived activation enables iterative
enzymatic assembly of long RNAs. Our results provide a plausible
scenario for the generation of higher-energy substrates required to
fuel ribozyme-catalyzed RNA synthesis in the absence of a highly
evolved metabolism.
enzymatic pathways for high-yielding 5’-triphosphate activation of
RNA are currently missing.[
6,7]
Other ligase ribozymes utilize
short-lived 5’-phosphoramidate-activated RNA. These substrates
are obtained through dedicated pre-activation of 5’-phosphorylated
RNA with reagents such as imidazole derivatives,[8–10] and either
labile and nonspecific carbodiimides or prebiotically implausible
2
,2-dipyridyl disulfide and triphenyl phosphine.[11] Thus, robust
chemical pathways that yield activated RNA under conditions that
also enable ribozyme activity remain elusive.
O
P
O
H
2
N
NH
2
R
R
Diamidophosphate (DAP) was recently identified as a
promising water-stable and prebiotically plausible reagent to
phosphorylate biological building blocks such as nucleic acids,
amino acids, and lipid precursors.[12,13] Of particular interest for the
activation of RNA is the ability of DAP to produce nucleoside 2’,3’-
cyclic monophosphates (>P), which can subsequently polymerize
into short RNA oligonucleotides without the need for additional
activating reagents. Enthalpically, >P-dependent formation of
RNA phosphodiester bonds is favored due to the small amount of
HO
O
HO
(
DAP)
O
O
P
imidazole
pH 6, ice
O
OH
O
O
O
P
HO
R
OH
OH
HO
O
O
O
O
OH
H
2
N
P
P
O
OH
O
energy stored in the strained 2',3'-cyclic phosphate, but is
Scheme 1. Diamidophosphate-mediated formation of 2’,3’-cyclic phosphate
ribonucleotides under conditions compatible with ribozyme catalysis.
disfavored entropically.[
14,15]
However, stable substrate binding
and/or low temperatures can compensate for the entropic costs
and lead to a strong shift in reaction equilibrium towards
ligation.[16,17] Consequently, >P-activated mono- and oligo-
Modern cells have evolved elaborate metabolic networks in mild
aqueous conditions to ensure their self-preservation and to
sustain their pool of activated building blocks. In contrast, it
remains unclear how analogous (re-)activation of building blocks
in primitive precursor systems could have been possible without
access to sophisticated protein enzymes. Given a likely central
role of RNA catalysts (ribozymes) during the Origin of Life,[
robust prebiotic processes must have provided pools of activated
mono- and oligonucleotides e.g. for RNA self-replication.
nucleotides may act as potent building blocks for primitive
ribozyme-catalyzed[17–20] and even spontaneous RNA ligation
reactions that increase the diversity and length distribution of RNA
polymer chains.[21,22] The mild conditions required for DAP-
dependent phosphorylation provide an attractive approach to
activate free 3'-termini of RNA for ligation reactions in the context
of primitive metabolism. Under solution conditions and high
millimolar concentrations of DAP, nucleoside, and metal chloride,
1]
[12]
quantitative >P formation is observed within days to weeks.
1
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COMMUNICATION
Figure 1. (a) Comparison of activator performance in the formation of N>Ps from their respective 3’-NMPs (1 mM) after 28 days at pH = 6 and -20 °C in the presence
of 1 mM DAP, 5 mM MgCl and 5 mM of either imidazole, 2-methylimidazole (2MI) or 2-aminoimidazole (2AI); similar trends were observed at pH 5, 7 and 8 (Figures
S13-S16). (b) Percentage conversion to >P as a function of time with imidazole as activator at pH = 6.
2
2
+
>P-dependent in-ice ligation of RNA substrates.[18] Thus, we
probed whether in-ice activation of RNA substrates by DAP could
enable direct downstream HPz-dependent ligation reactions.
To monitor the coupled activation-ligation reactions, we
developed a reporter electrophoretic mobility shift assay (EMSA)
based on a cis-ligating version of the HPz (cHPz, Figures 2, S17).
We confirmed the self-ligation activity of cHPz using >P RNA
(sub>P) generated with ethyl-3-(3’-dimethylaminopropyl)-
carbodiimide (EDC), with which ~88% self-ligation was observed
via urea-PAGE in reaction conditions used in previous HPz in-ice
studies (Figure S18).[18] No band shift was observed in the
presence of sub-P, the non-activated 3’-monophosphate RNA.
Following the implementation of the reporter EMSA, we
sought to test the efficacy of the coupled DAP activation-ligation
reactions under various conditions. Specifically, we tested a
However, high levels of metal ions such as Mg , which are
required for efficient >P formation under aqueous conditions, are
incompatible with half-lives of ribozymes in water.[23]
Here, we demonstrate RNA activation by DAP under
conditions that are amenable to the catalytic activity of >P-
dependent ribozymes. In particular, we found that the
concentrating and preserving environment of water-ice systems
enables efficient formation of >P-activated RNA. The activated
RNA can be used in situ for RNA ligation by derivatives of the
naturally occurring hairpin ribozyme (HPz), which serves as a
versatile model system for prebiotic RNA ligation.[17–19]
To explore the potential of water-ice in combination with
DAP to enable >P RNA formation, we incubated individual
canonical nucleoside 3’-monophosphates (3’-NMPs) with DAP
and imidazole (or its derivatives 2-amino- and 2-methylimidazole)
at -20 °C and monitored the reactions using ion-exchange liquid
chromatography (Figures S25-72). In solution, DAP reacts with
phosphates to form the corresponding amidopyrophosphate. This
intermediate forms the 2’,3’-cyclophosphate in the presence of a
’-OH group, with the amidophosphate as the leaving group
Scheme 1).[12] Intriguingly, even in ice at -20 °C, we observed
moderate to good yields for the U, C, A and G >Ps after 28 days
Figures 1a, S1-S12) confirmed by 31P-NMR (Figures S73-74).
Among the three activators tested in this study, the reactions with
imidazole was relatively faster (Figures S1-S12) and yielded the
highest amounts of >Ps (Figures 1a, S13-S16) under the four pH
conditions tested. We also note remarkable differences in the
efficiency of >P formation depending on the nucleobase (Figure
2+
range of concentrations of DAP, Mg , and imidazole for their
potential to enable coupled activation-ligation reactions. We
2+
identified the inclusion of 5 mM DAP, 5 mM Mg , and 5 mM
imidazole as optimal conditions thus far for the combined
activation and ligation of the RNA substrate sub-P (Figure S19),
and used these conditions in all further experiments. We also
noted an increased ligation yield at 50 mM DAP in the absence of
imidazole. We presumed that the increased concentration of DAP,
especially in eutectic ice, compensates for the absence of
imidazole. However, increasing both DAP and imidazole
concentrations to 50 mM resulted in a lower ligation yield
compared to with 5 mM (Figure S20). This result may be due to
the high DAP and imidazole concentrations that reduce the
concentration-by-freezing efficiency for the RNA components.
The higher initial concentration of the two components reduces
the amount of water-ice necessary to reach the molal equilibrium
concentrations of the unfrozen phase.[ Thus, the resulting cHPz
and substrate concentrations in the aqueous phase may be lower
than with decreased DAP and imidazole concentrations, leading
to less efficient activation and/or ligation.
2
(
(
1
b). After 7 days at pH = 6 in the presence of imidazole, ~50% of
both pyrimidine nucleotides 3’-UMP and 3’-CMP were converted
into their respective >Ps. In contrast, conversion of the purine
nucleotides under the same conditions was much slower with
33% for 3’-AMP (33%), and only <3% for 3’-GMP. After 28 days,
the conversion yields for 3’-UMP, 3’-CMP and 3’-AMP reached
2-88% while it approached 30% for 3’-GMP. See below for
24]
~
7
further mechanistic explanations for the observed influence of the
nucleobase moieties.
We then explored the pH dependency of the activation-
ligation reaction. We speculated that a highly acidic environment
may decrease the availability of the DAP-activating agent
Having shown that DAP can be used as an efficient
activation agent for N>P formation in frozen water-ice systems,
we sought to explore whether DAP can also activate
oligonucleotides to fuel ribozyme-catalyzed ligation reactions.
Previous studies have shown that HPzs are capable of efficient
imidazole (pK
a
= 6.95[25]), whose nucleophilicity decreases upon
protonation. Moreover, the activity of HPz decreases below pH
5[26] while the stability of RNA is optimal at pH 4–5. Indeed, we did
2
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Figure 2. A reporter electrophoretic band shift assay based on a modified
hairpin ribozyme (cHPz) which accepts a 2’,3’-cyclic phosphate substrate
(
sub>P) for its catalytic activity. (a) A simplified schematic of the reaction during
which the substrate (sub-P) is activated to yield the 2’,3’-cyclic phosphate RNA
oligomer. LP = ligation product. (b) Denaturing EMSA of the species shown
in (a). sub-P is 5’ tagged with a fluorescein derivative to aid in analysis. (c) A
detailed secondary structure of the substrate-ribozyme complex prior to ligation.
The location of the 2’,3’-cyclic phosphate and site of ligation are indicated in red.
not detect significant RNA degradation during the experiment
(Figure S21). Consistent with these considerations and with our
results with monomeric ribonucleotides, we observed the highest
ligation yields and reaction rates at pH 5–6 (Figures 3 and S22).
We postulate that a nucleophilic pool of unprotonated imidazole
is maintained at this mildly acidic pH while preserving the activity
of cHPz. The decrease in yields at pH above 6 likely reflects that
>
~
P formation requires protonation of the -NH
2 a
group of DAP (pK
5.5).[27] This assumption is supported by the pH dependence of
Figure 3. DAP-mediated formation of 2’,3’-cyclic phosphate RNA and its
subsequent ligation in the presence of imidazole occur under various conditions
compatible with ribozyme catalysis and is optimal at pH 5-6. (a) Time course
analysis of in situ activation-ligation reactions at different pH values. Reaction
mixtures containing 10 mM of glycine∙HCl buffer (pH 3), MES buffer (pH 5-7),
Tris∙HCl buffer (pH 9), or carbonate/bicarbonate buffers (pH 11) were analysed
via TBE-urea PAGE. Data points were fitted assuming a simplified 2-step
reaction mechanism to extrapolate maximum ligation yields. (b) pH dependency
of the maximum ligation yield using the best-fit parameters from (a). Error bars
indicate the 95% confidence intervals. (c) Dependency of coupled activation-
ligation reactions on the 3’-terminal base of sub-P. Reactions were performed
using four 2’/3’-monophosphorylated variants and analysed via TBE-urea PAGE.
the activation of 3'-NMPs (Figures S1-S12). While we note that it
is typically difficult to estimate the pH of the interstitial brine in
water-ice accurately, we expect that the pH in our buffered
samples is increased by only ~0.5 pH units at -9 °C.[28]
We initially observed a considerable influence of the base
moiety on DAP-mediated phosphate activation for the four
mononucleotides (Figure 1b). Therefore, we wondered whether a
similar underlying nucleobase dependency might govern the DAP
activation of RNA oligonucleotides in the EMSA analysis. Indeed,
we observed increased ligation yields for 3’-terminal pyrimidines
over purines (Figure 3c). The difference was most obvious
between substrates with a 3'-terminal C and G: After 84 days, the
the nucleobases A and C are protonated at pH 5–6 and become
more electron-withdrawing, thereby increasing the acidity of the
2’-OH group.[31] For U, the 2-keto-oxygen may coordinate with the
2’-OH group, thus facilitating its deprotonation.[
3
'-terminal pyrimidine resulted in 68% ligation yield compared to
the purine at 43%. The yield differences are unlikely to be the
result of a preference of the cHPz for different 3' ends, because
the influence of the 3' base identity was only marginal in control
experiments with pre-activated substrates (Figure S23). While the
32]
To further test the generality of the reaction, we investigated
the influence of the co-activator imidazole on substrate activation.
2-methylimidazole (2MI) and 2-aminoimidazole (2AI) are close
analogues of imidazole (IMI) that are considered as superior 5’-
leaving groups for non-enzymatic copying of RNA templates.[33,34]
We repeated our EMSA assay with 5 mM 2MI or 2AI instead of
IMI (Figure S25) and found that ligation yields were considerably
lower (~25% after 84 days) in the presence of 2AI or 2MI
solubilities of monophosphates (CMP, 16.3 g/L; UMP, 12 g/L;
AMP, 8 g/L; GMP, 8 g/L)[
29]
may be a factor for the different
activation yields at the monomer level, this is unlikely with
oligonucleotides. pK values of the 2’-OH group, between 13.22
a
and 13.47,[30] would predict minimal differences in the
nucleophilicity of the 2’-OH groups of the four nucleotides for the
formation of >Ps. However, it is possible that a larger fraction of
3
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COMMUNICATION
phosphates back to >Ps. Furthermore, EDC modifies nucleic acid
base moieties at rates comparable to those of RNA-catalyzed
ligation[37–39]. In contrast, we observed no indication of similar
undesirable side reactions with DAP.
Finally, we investigated whether DAP activation also allows
multiple ligations to produce long RNAs, which is a prerequisite
for the formation of more complex RNA molecules. To this end,
we probed our one-pot activation/ligation scheme for
concatenation reactions of RNA oligomers (Figures 4bc and S26),
including the assembly of an RNA polymerase ribozyme from
seven ≤30 nt fragments (RPR7, Figure S26) catalyzed by
fragmented hairpin ribozymes.[ Strikingly, for both reactions we
observed six or more successive ligation events after 21 days of
in-ice incubation.
19]
The data herein present DAP-mediated formation of
activated RNA substrates under conditions compatible with
ribozyme catalysis. DAP is an attractive candidate reagent for the
prebiotic activation of RNA due to its long half-life and reactivity in
aqueous environments.[40] Moreover, its potential in the primordial
activation of lipids, peptides, and 5’ ends of nucleic acids – and
therefore as a key reagent in the origin of all life on Earth – has
been documented.[
12,13]
Furthermore, the compatibility of the
reaction with frozen water-ice matrices that upconcentrate solutes
allows for low initial DAP and imidazole concentrations to achieve
high-yielding activation of mono- and oligonucleotides. DAP is
therefore
a powerful reagent to mitigate quasi-irreversible
hydrolysis of 2',3'-cyclic phosphates that occurs over time in the
2+
[41]
presence of M ions, and to sustain a pool of activated >P RNA
molecules that can serve as starting material for both enzymatic
and non-enzymatic ligation reactions.
>P RNA can also be formed by enzymatic or non-enzymatic
RNA cleavage. In combination with strand dissociation,
Figure 4. (a) Comparison of DAP and EDC under in situ activation-ligation
conditions. Reaction mixtures differing only in the inclusion of either EDC or
DAP were analysed via TBE-urea PAGE. The data points were fitted assuming
a simplified irreversible 2-step reaction mechanism. (b) Simplified schematic of
1
6 nt RNA oligomer concatenation. SBS = substrate-binding strand. (c) DAP-
consecutive cleavage-ligation reactions may therefore lead to the
mediated RNA concatenation. Time course analysis via SYBR Gold-stained
TBE-urea PAGE of reaction mixtures containing a fragmented ribozyme and 16
nt 3’-monophosphorylated substrates. We note that the RNA ladder displays
lower electrophoretic mobility, presumably due to different salt concentrations
in the loading buffer and / or conjugated fluorophores.
formation of longer products.[
19,22,42–44]
While offering an intriguing
alternative for RNA synthesis in the absence of direct chemical
activation, such recombination reactions have several
disadvantages. The extra cleavage and dissociation steps slow
product formation and decrease final yields due to a higher
number of reversible steps required for product formation.[
Moreover, ribozyme-catalysed recombination requires the
presence of additional sequence elements on substrates which
reduces the pool of compatible oligonucleotides in randomized
libraries. Finally, recombination does not regenerate hydrolysed
19,22]
compared to IMI (~55%). The lower pK
8
a
of IMI (6.95 compared to
.46 for 2AI[35] and 7.86 for 2MI[36]) – and therefore its larger
unprotonated nucleophilic pool – coupled with less steric
hindrance may amplify the formation of the activated imidazole-
amidophosphate that is responsible for phosphorylation.
Nevertheless, our results show that different imidazole derivatives
can serve as activators for the DAP-dependent generation of
cyclic phosphates.
Next, we examined the performance of DAP compared to
the prebiotically implausible carbodiimide EDC, which is typically
used to generate >Ps. Intriguingly, the long-term kinetics of RNA
activation under in situ ribozyme catalysis-compatible conditions
showed higher ligation yields with DAP compared to EDC (Figure
>
Ps. Thus, while both direct ligation and recombination are
capable of exploring a large sequence space through repeated
ligation reactions[ , both reactions can ultimately benefit from the
mild re-activating properties of DAP.
22]
The resulting high-diversity products could provide the
starting material for evolutionary processes in the form of nascent
ribozymes.[
22]
Additionally, continuous activation of RNA
substrates provides the possibility of maintaining cross-catalytic
[
19]
reaction networks consisting of several ribozyme components
4a). While one-pot in situ activation with EDC caused higher initial
and may enable new strategies for continuous RNA evolution
under prebiotically plausible conditions. In conclusion, our work
underlines the importance of identifying ribozyme catalysis-
compatible RNA activation reagents in enabling one-pot
processes, which has implications for abiotic molecular evolution
of nucleic acids.
yields during the first 10 days of incubation, likely due to higher
reactivity of the carbodiimide moiety, the yields in the DAP-based
reactions significantly exceeded those incubated with EDC during
long-term incubation, reaching >50% more RNA ligation after 28
days. A plausible explanation for this observation is the overall
low stability of EDC due to hydrolysis, which hampers reactivation
of hydrolyzed cyclic phosphates. In contrast, the high stability of
DAP may allow repeated reactivation of hydrolyzed 3’-terminal
Acknowledgements
4
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HM and EYS acknowledge funding by the Deutsche
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Angewandte Chemie International Edition
10.1002/anie.202010918
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Easy one-pot recipe: a plausible scenario for the prebiotic generation of activated RNA substrates under mild aqueous conditions.
Using water-soluble diamidophosphate, we demonstrate in situ production of 2’,3’-cyclic phosphate-activated oligoribonucleotides
and their subsequent ligation by a ribozyme.
Institute and/or researcher Twitter usernames: @MPI_Biochem, @MutschlerLab
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This article is protected by copyright. All rights reserved.