Abiotic synthesis of purine and pyrimidine ribonucleosides in aqueous microdroplets

Article abstract of DOI:10.1073/pnas.1718559115

Aqueous microdroplets (<1.3 μm in diameter on average) containing 15 mM D-ribose, 15 mM phosphoric acid, and 5 mM of a nucleobase (uracil, adenine, cytosine, or hypoxanthine) are electrosprayed from a capillary at +5 kV into a mass spectrometer at room temperature and 1 atm pressure with 3 mM divalent magnesium ion (Mg²⁺) as a catalyst. Mass spectra show the formation of ribonucleosides that comprise a four-letter alphabet of RNA with a yield of 2.5% of uridine (U), 2.5% of adenosine (A), 0.7% of cytidine (C), and 1.7% of inosine (I) during the flight time of ～50 μs. In the case of uridine, no catalyst is required. An aqueous solution containing guanine cannot be generated under the same conditions given the extreme insolubility of guanine in water. However, inosine can base pair with cytidine and thus substitute for guanosine. Thus, a full set of ribonucleosides to generate the purine–pyrimidine base pairs A-U and I-C are spontaneously generated in aqueous microdroplets under similar mild conditions.

Full text of DOI:10.1073/pnas.1718559115

Abiotic synthesis of purine and pyrimidine
ribonucleosides in aqueous microdroplets
Inho Nama,b, Hong Gil Nama,c,1, and Richard N. Zareb,1
aCenter for Plant Aging Research, Institute for Basic Science, Daegu 42988, Republic of Korea; bDepartment of Chemistry, Stanford University, Stanford, CA
94305; and cDepartment of New Biology, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, Republic of Korea
Contributed by Richard N. Zare, November 27, 2017 (sent for review October 24, 2017; reviewed by Bengt J. F. Nordén and Veronica Vaida)
Aqueous microdroplets (<1.3 μm in diameter on average) containing
15 mM D-ribose, 15 mM phosphoric acid, and 5 mM of a nucleobase
(uracil, adenine, cytosine, or hypoxanthine) are electrosprayed from a
capillary at +5 kV into a mass spectrometer at room temperature and
1 atm pressure with 3 mM divalent magnesium ion (Mg2+) as a cat-
alyst. Mass spectra show the formation of ribonucleosides that com-
prise a four-letter alphabet of RNA with a yield of 2.5% of uridine (U),
2.5% of adenosine (A), 0.7% of cytidine (C), and 1.7% of inosine (I)
during the flight time of ∼50 μs. In the case of uridine, no catalyst is
required. An aqueous solution containing guanine cannot be gener-
ated under the same conditions given the extreme insolubility of
guanine in water. However, inosine can base pair with cytidine and
thus substitute for guanosine. Thus, a full set of ribonucleosides
to generate the purine–pyrimidine base pairs A-U and I-C are
spontaneously generated in aqueous microdroplets under similar
mild conditions.
In a recent study, we showed a synthetic pathway for the
formation of Rib-1-P using aqueous, high–surface-area micro-
droplets. This surface or near-surface reaction circumvents the
fundamental thermodynamic problem of the condensation re-
action (12). It has been suggested that the air–water interface
provides a favorable environment for the prebiotic synthesis of
biomolecules (12–17). Using the Rib-1-P made in the above
abiotic manner and the salvage pathway shown in Fig. 1, we
successfully generated uridine (U) (12), one of the pyrimidine
ribonucleosides, in aqueous microdroplets. However, we did not
investigate whether this process represents a general abiotic
pathway for the generation of both purine and pyrimidine ribo-
nucleosides. Here, we show the abiotic generation of the ribo-
nucleosides adenosine (A), cytidine (C), and inosine (I) by
surmounting the thermodynamic barrier for these reactions at or
near the surface of room-temperature water microdroplets.
Guanine is practically insoluble in water, which precludes the
generation of aqueous microdroplets containing guanine (18).
Nevertheless, inosine (I) can base pair with cytidine (C), and thus
the full base pairs of ribonucleosides can be synthesized and
survive under the same prebiotic conditions. In the micro-
droplets, a small concentration of divalent magnesium ion (Mg2+),
one of the main elements of Earth’s crust (19), serves to catalyze
the process. Because mists, clouds, and sprays are thought to be
common on early Earth, these findings provide a possible missing
link as to how the RNA world came about.
microdroplet chemistry origin of life prebiotic chemistry purine
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ribonucleosides pyrimidine ribonucleosides
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ne hypothesis concerning the origin of life postulates an
RNA world that existed on Earth before modern cells arose
O
(1). According to this hypothesis, RNA stored genetic in-
formation and catalyzed chemical reactions in primitive cells (2,
3). However, a major missing link has been how to construct
from simpler molecules the ribonucleosides, which are the
building blocks of RNA (1–3). This challenge seems quite
daunting because the making of ribonucleosides in a water so-
lution from ribose and some suitable nucleobase, such as the
purines adenine and guanine, and the pyrimidines uracil and
cytosine, is thermodynamically uphill (4, 5). Recently, possible
routes for the prebiotic synthesis of purine and pyrimidine ri-
bonucleosides were studied by bypassing the biological synthesis
mechanism (5–8). These routes demand different environments,
although it might be more favorable if purine and pyrimidine
ribonucleosides could be condensed in the same environment for
the fabrication of RNA. Moreover, these mechanisms require
complex, sequential reactions with reaction times in the range of
days, which may make them less relevant for creating a prebiotic
chemistry in which RNA could develop. As described below, we
present an alternative route for the synthesis of ribonucleosides
that seems to be sufficiently mild and general that it might have
played a role in turning simpler molecules into biomolecules.
In cells, the synthesis of ribonucleosides takes two paths: nu-
cleotide metabolism and the salvage pathway (9–11). In nucle-
otide metabolism, 5-phosphoribosyl-1-pyrophosphate (PRPP)
formed from D-ribose-5-phosphate (Rib-5-P) and nucleobases
are changed into a ribonucleotide and two phosphates (9). As
part of the salvage pathway, Rib-1-P and a nucleobase are
transformed into a ribonucleoside and a phosphate (9–11), as
illustrated in Fig. 1. The salvage pathway in vivo is a reaction in
which ribonucleosides are synthesized from organic intermedi-
ates. Specifically, Rib-1-P is added to a free nucleobase by a
phosphorylase enzyme to form a ribonucleoside. The most im-
portant part of both reactions is that a carbon atom at the first
position (C1) in ribose links to the nucleobase.
Significance
Discovery of an improved prebiotic method for the synthesis of
ribonucleosides provides support to theories that posit a cen-
tral role for RNA in the origin of life. It has been assumed that
ribonucleosides arose through an abiotic process in which ri-
bose and nucleobases became conjoined, but the direct con-
densation of nucleobases with ribose to give ribonucleosides in
bulk solution is thermodynamically uphill. We show a general
synthetic path for ribonucleosides, both purine and pyrimidine
bases, using an abiotic salvage pathway in a microdroplet en-
vironment with divalent magnesium ion (Mg2+) as a catalyst.
Purine and pyrimidine ribonucleosides are formed simulta-
neously under the same conditions, which suggests a possible
scenario for the spontaneous production of random ribonu-
cleosides necessary to generate various types of primitive RNA.
Author contributions: I.N., H.G.N., and R.N.Z. designed research; I.N. performed research;
I.N., H.G.N., and R.N.Z. analyzed data; H.G.N. and R.N.Z. directed and supervised the
project; and I.N., H.G.N., and R.N.Z. wrote the paper.
Reviewers: B.J.F.N., Chalmers University of Technology; and V.V., University of Colorado.
The authors declare no conflict of interest.
This open access article is distributed under Creative Commons Attribution-NonCommercial-
NoDerivatives License 4.0 (CC BY-NC-ND).
1To whom correspondence may be addressed. Email: nam@dgist.ac.kr or zare@stanford.
edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1718559115/-/DCSupplemental.
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120 psi using an electrospray ionization (ESI) source placed in
front of a high-resolution mass spectrometer, as shown in Fig. S1
(20). The microdroplets traveled 5 mm at room temperature and
atmospheric pressure before the reaction was stopped upon
entering the heated inlet of a mass spectrometer (21). Under
these conditions, a corresponding reaction time was estimated to be
∼50 μs on the basis of the droplet speed (∼100 m/s) (22, 23). The
droplets were positively charged by external application of a po-
tential of +5 kV to the ESI source. When a high electric field is
applied to droplets, the electric charge makes an electrical force
inside the droplets, which generates a droplet explosion into very
fine microdroplets (Coulomb explosion) (24). Therefore, the ex-
ternal charge mainly affects the size of droplets, and the size of
droplets controls the yield of ribonucleosides in aqueous micro-
droplets. Here, the sizes of droplets were observed to be, on
average, <1.3 μm in diameter (Fig. S2), as determined optically
(25). Without application of the external potential, the average
diameter of droplets is 12.5 μm, which means the volume of
droplets is at least three orders of magnitude larger than that with
the external voltage. The significantly smaller surface-to-volume
ratio is one of the main reasons that production of ribonucleo-
sides was reduced in uncharged microdroplets (Fig. S2). We also
checked the mass spectra of the products in microdroplets by using
the above experimental scheme without Mg2+. We found peaks for
the mass of precursors D-ribose, phosphoric acid, and a nucleobase,
such as m/z = 136.061 for protonated adenine, m/z = 112.050 for
protonated cytosine, m/z = 137.046 for protonated hypoxanthine,
and m/z = 231.026 for Rib-1-P, as shown in Fig. S3. However, the
ribonucleosides adenosine, cytidine, and inosine were not detected
in microdroplets under these conditions.
Fig. 1. Reaction mechanism for the prebiotic synthesis of ribonucleosides by
an abiotic salvage pathway using Rib-1-P as the intermediate.
Results
Generation of Aqueous Microdroplets Containing Precursors. We
performed reactions to generate ribonucleosides by the abiotic
salvage pathway in aqueous microdroplets (Fig. 1). The micro-
droplets contained 15 mM D-ribose; 15 mM phosphoric acid; and
5 mM adenine, cytosine, or hypoxanthine, which were all in
deionized water. Studies were carried out in the presence of the
divalent magnesium ion (3 mM Mg2+). Microdroplets were
generated from bulk solution with a nebulizing gas (dry N2) at
Thermodynamic Considerations Concerning Ribonucleoside Generation
in Aqueous Microdroplets. To determine the reaction barrier, we
present the free energy diagrams for the abiotic salvage pathways
of adenosine, cytidine, inosine, and uridine showing the changes of
A
B
C
D
Fig. 2. Free energy diagrams for the abiotic synthesis of the ribonucleosides (A) adenosine, (B) cytidine, (C) inosine, and (D) uridine. The condensation occurs
between ribose and phosphoric acid to generate Rib-1-P. The salvage reaction occurs between Rib-1-P and nucleobase to generate the corresponding ri-
bonucleoside. The ΔG values in bulk solution (blue lines) are positive, whereas in microdroplets (red lines) they are negative.
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1.5
1
With Mg2+
Without Mg2+
1.2
1
D-ribose + Phosphoric acid (P) + Adenine + Mg2+
A
B
100
80
60
40
20
0
0.8
0.6
0.4
0.2
0
0.5
0
100
150
200
m/z
250
300
267
269
m/z
271
267
269
m/z
271
With Mg2+
Without Mg2+
0.5
0.4
0.3
0.2
0.1
0
0.2
0.15
0.1
D-ribose + P+ Cytosine + Mg2+
100
80
60
40
20
0
0.05
0
100
150
200
250
300
243.5 244 244.5 245 245.5
243.5 244 244.5 245 245.5
m/z
m/z
m/z
2
With Mg2+
Without Mg2+
2
D-ribose + P + Hypoxanthine + Mg2+
C
100
80
60
40
20
0
1.5
1
1.5
1
0.5
0
0.5
0
268
270
272
274
100
150
200
m/z
250
300
268
270
272
274
m/z
m/z
Fig. 3. Mass spectra of reactions in microdroplets containing 15 mM D-ribose, 15 mM phosphoric acid, and 5 mM of the nucleobase (A) adenine, (B) cytosine,
and (C) hypoxanthine, with and without 3 mM Mg2+. The red boxes show the detected ion signals of ribonucleosides with the Mg2+ catalyst, and the red
letters denote the detected m/z peaks of (A) adenosine, (B) cytidine, and (C) inosine.
Gibbs free energy, ΔG, in which the data are based on previous shown that the synthesis of Rib-1-P from ribose and phosphoric
studies (12, 26, 27) (Fig. 2). For cytidine, the ΔG value of the acid in microdroplets has a ΔG value of −1.1 kcal/mol (12). We
salvage reaction is calculated by density functional theory (DFT) previously demonstrated that the phosphorylation of ribose in
using the projector-augmented wave method. The DFT calcula- microdroplets displays a negligible change of entropy (ΔS = 7 ×
10−4 kcal/mol·K). Therefore, microdroplets alter the kinetics and
tion considers unsolvated reactants and products; however, we
already observed that the calculated value is very close to the real
value for the reaction in aqueous microdroplets (12). In bulk
aqueous solution, a condensation reaction between ribose and
phosphate has a large negative value of TΔS (−6.3 kcal/mol) or
ΔS = −2.1 × 10−2 kcal/mol·K, which results in a positive Gibbs free
energy change (ΔG = +5.4 kcal/mol) (12, 27). However, in bulk
solution reactions between phosphate in Rib-1-P and adenine,
cytosine, or hypoxanthine following a salvage pathway, the values
of ΔG are negative: −3.1, −1.94, and −1.88 kcal/mol, respectively
(26, 27). It appears that the salvage reaction between Rib-1-P and
nucleobase, both purine and pyrimidine, can occur spontaneously
once Rib-1-P is formed as an intermediate. This is in good
agreement with uridine fabrication by a salvage pathway, which
also has a negative ΔG value, −2.13 kcal/mol (28). Combining this
information, we find that syntheses of adenosine, cytidine, inosine,
and uridine all have positive ΔG values, +2.30, +3.46, +3.52 and
+3.27 kcal/mol, respectively (blue lines in Fig. 2), in bulk aqueous
solution.
thermodynamics of ribose phosphorylation. The decreased en-
tropic change for this chemical reaction in microdroplets com-
pared with bulk solution could be attributed to the molecular
organization and alignment of reactants at the air–water in-
terface of microdroplet surfaces (29). Consequently, the ΔG
values for the synthesis of the ribonucleosides adenosine, cyti-
dine, inosine, and uridine become negative in microdroplets:
−4.12, −2.96, −2.90, and −3.15 kcal/mol, respectively (red lines in
Fig. 2). These results indicate that the reaction to generate both
purine and pyrimidine ribonucleosides becomes thermodynami-
cally allowed in microdroplets based on the salvage pathway. Of
course, this condition is necessary but not sufficient for the for-
mation of the purine and pyrimidine ribonucleosides. We must also
examine the kinetics of these reactions.
Mg2+ Catalytic Effect for Ribonucleoside Generation. The thermo-
dynamic feasibility of the abiotic salvage reaction suggests that
the lack of ribonucleoside production for adenosine, cytidine,
and inosine in microdroplets (Fig. S4) is the consequence of
poor kinetics. To accelerate the kinetics of the ribonucleoside
In sharp contrast to the thermodynamic inhibition of ribose
phosphorylation in bulk solution (ΔG = +5.4 kcal/mol), we have
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A
B
C
D
Fig. 4. The synthetic path of adenosine in microdroplets with Mg2+ catalyst. (A) Mass spectra for the products of reaction between adenine and Rib-1-P and
(B) the mechanism of salvage reaction between adenine and Rib-1-P to give adenosine. Red letters denote the detected m/z peak of the adenosine from an
abiotic salvage reaction. (C) Mass spectrum for the products of reaction between D-ribose and adenine in the absence of phosphate and (D) the previously
assumed mechanism for the direct ribosylation of adenine. C6H13N and P denote a fragment of adenine (cyclohexylamine) and phosphoric acid.
generation, we investigated the use of different metal ion cata-
lysts, including Mg2+, Ca2+, and Fe2+. We found that 3 mM Mg2+
in microdroplets worked well. In previous research, Mg2+ was also
needed with inorganic polyphosphates for making adenosine using
heating and drying (7). In addition, the microdroplet environment
can support the acceleration of chemical reactions, in contrast to
what happens in bulk solution. We and other groups have shown
the acceleration property of microdroplets (103- to 106-fold higher
than bulk) for various chemical reactions, such as redox reactions
(20, 30), protein unfolding (21), chlorophyll demetallation
(31), addition/condensation reactions (32), carbon-carbon bond-
forming reactions (33), and elimination/substitution reactions
(34). With Mg2+, the abiotic salvage reaction in microdroplets for
generation of ribonucleosides was dramatically changed. In each
mass spectrum of microdroplets containing Mg2+ we observed ad-
ditional peaks (Fig. 3) that were not present in the reactant solutions
without Mg2+. In particular, peaks at m/z = 268.104 for adenosine,
m/z = 244.089 for cytosine, and m/z = 269.088 for hypoxanthine were
identified as protonated adenosine [Adenosine +H+]+, protonated
cytidine [Cytidine +H+]+, and protonated inosine [Inosine +H+]+,
respectively. That means that ribonucleosides, both purine and py-
rimidine bases, proceed spontaneously to form under these condi-
tions in tens of microseconds at room temperature and atmospheric
pressure. By ionization efficiency calibrations, the product yields
for adenosine, cytosine, and inosine were determined to be ∼2.5%,
0.7%, and 1.7%, respectively (Fig. S4).
in the presence of Mg2+. First, we tested the solution containing
15 mM Rib-1-P and 5 mM adenine (Fig. 4 A and B). After gener-
ation of droplets, the adenosine was still synthesized (yield of ∼4%),
which means that the exchange reaction occurs between Rib-1-P and
adenine in the same manner as a salvage reaction. However, from a
solution containing D-ribose and adenine, there was no observation
of adenosine, as shown in Fig. 4 C and D. Thus, a direct condensation
reaction between ribose and adenine is negligible compared with the
abiotic salvage synthesis. Understandably, Rib-1-P was readily gen-
erated from the solution, including ribose and phosphate, as shown in
our previous research (Fig. S6).
Role of Mg2+ Catalyst. The abiotic generation of ribonucleosides
occurs on the order of microseconds with the Mg2+ catalyst.
Mg2+ plays important roles in biology—for example, stabilizing
proteins, nucleic acids, and cell membranes, as well as serving as
a cofactor for enzymes (18). We also speculate that Mg2+ could
contribute to transition state stabilization, because Mg2+appears to
change the kinetics of reactions. An example of Mg2+ stabilizing
the transition states in biochemical reactions has been reported in
self-cleaving ribozymes (35). It is notable that synthesis of uridine
does not require Mg2+, which may provide a mechanistic insight on
the role of Mg2+
.
Conclusions
We have provided what we believe to be a feasible prebiotic
pathway to synthesize ribonucleosides, both purine and pyrimi-
dine bases, under the same conditions of aqueous microdroplets
containing small amounts of Mg2+. In contrast to bulk solution,
microdroplets can overcome the thermodynamic barrier for
these condensation reactions. The thermodynamic change for
chemical reactions in microdroplets could be attributed, in part,
to the molecular organization and alignment of reactants at the
air–water interface of microdroplet surfaces (36, 37). The air–
water interface possesses a strong electric field, which could in-
fluence the organization of reactant molecules, as well as the
kinetics and thermodynamics of chemical reactions (29). With
We performed tandem MS using collision-induced dissocia-
tion (CID) to confirm the structure of generated molecules in
the microdroplets. The fragmentation pattern for each reacted
microdroplet includes a nucleobase peak, [Adenine +H+]+,
[Cytosine +H+]+, or [Hypoxanthine +H+]+, which exactly
matched that of the respective ribonucleoside (Fig. S5).
Discussion
Plausible Mechanism for Ribonucleoside Generation in Aqueous
Microdroplets. To determine the plausible mechanism of ribonucle-
oside generation, we tested multiple-step reactions in microdroplets
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the unique properties of Mg2+ in biology, the present study
suggests that the inorganic chemistry of Mg2+ might have a key
role in the origin of life with microdroplet chemistry. It is of
special importance that both purine and pyrimidine ribonucle-
osides are made in the same environment, which suggests the
simultaneous production of large pools of random ribonucleo-
sides in the same microdroplets that might lead (38) to the
production of self-replicating RNA.
chemicals were purchased from Sigma-Aldrich. HPLC-grade solvents were
purchased from Fisher Scientific.
Quantitative Analysis. The quantitative analysis for the estimate of percent-
age yield of the above reactions was performed by an ionization efficiency
calibration. Calibration plots were made by electric spraying the mixture of
the reactant and the corresponding authentic product in known concen-
tration ratios. As the ion signal intensities of the reactant (IR) and the product
(IP) both depend on their concentrations and ionization efficiencies, we
calculated the ratio IP:IR and plotted it against the product-to-reactant
concentration ratio ([P]:[R]). We estimated the yield of the reaction from
this calibration plot.
Materials and Methods
Generation of Aqueous Microdroplets Containing Precursors. The aqueous
solution of 15 mM D-ribose, 15 mM phosphoric acid, and 5 mM nucleobases
was injected with 3 mM magnesium sulfate by a mechanical syringe pump
through a hypodermic needle to the fused silica capillary directing toward a
mass spectrometer inlet. A coaxial sheath gas (dry N2 at 120 psi) flow around
the capillary results in nebulization and also helps to direct the spray
emerging from the capillary tip toward the mass spectrometer inlet (the
flow rate of 5 μL/min through silica tubing). A positive voltage, +5 kV, was
applied to the hypodermic needle. At a mass spectrometer inlet, the reac-
tants, intermediates, and products are released from droplets by Coulomb
fission and enter into the mass spectrometer through a heated capillary. The
capillary temperature was maintained at 275 °C and the capillary voltage at
44 V. To confirm the identities of synthesized ribonucleosides, tandem MS
was conducted by CID. For all mass spectrometric analyses, the spray distance
(the distance from spray tip to the entrance of the heated capillary) was kept
at 5 mm. Mass spectra were detected by high-resolution MS (Thermo Sci-
entific LTQ Orbitrap XL Hybrid Ion Trap-Orbitrap). All of the necessary
DFT Calculations. First-principles calculations were carried out on the basis of
periodic DFT using a generalized gradient approximation within the Perdew-
Burke-Ernzerhof exchange correction functional (39, 40). We used the
projector-augmented wave method for describing ionic cores as imple-
mented in the Vienna ab initio simulation package (VASP) (41). The wave
functions were constructed from the expansion of plane waves with an
energy cutoff of 520 eV. A 6 × 6 × 6 k-point mesh Monkhorst-Pack method
was used to sample the Brillioun zone. The electronic optimization steps
were self-consistently converged over 10−4 eV per formula unit.
ACKNOWLEDGMENTS. We thank Yin-Hung Lai for helping us measure
microdroplet size as a function of applied voltage to the ESI source. We also
thank Eric Kool, who suggested using hypoxanthine. This work was
supported by Institute for Basic Science Grant IBS-R013-D1 and the Air Force
Office of Scientific Research through Basic Research Initiative Grant
AFOSR FA9550-12-1-0400.
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