Noncanonical RNA Nucleosides as Molecular Fossils of an Early Earth—Generation by Prebiotic Methylations and Carbamoylations

Article abstract of DOI:10.1002/anie.201801919

The RNA-world hypothesis assumes that life on Earth started with small RNA molecules that catalyzed their own formation. Vital to this hypothesis is the need for prebiotic routes towards RNA. Contemporary RNA, however, is not only constructed from the four canonical nucleobases (A, C, G, and U), it also contains many chemically modified (noncanonical) bases. A still open question is whether these noncanonical bases were formed in parallel to the canonical bases (chemical origin) or later, when life demanded higher functional diversity (biological origin). Here we show that isocyanates in combination with sodium nitrite establish methylating and carbamoylating reactivity compatible with early Earth conditions. These reactions lead to the formation of methylated and amino acid modified nucleosides that are still extant. Our data provide a plausible scenario for the chemical origin of certain noncanonical bases, which suggests that they are fossils of an early Earth.

Full text of DOI:10.1002/anie.201801919

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Accepted Article
Title: Prebiotic methylations and carbamoylations generate non-
canonical RNA nucleosides as molecular fossils of an early Earth
Authors: Christina Schneider, Sidney Becker, Hidenori Okamura,
Antony Crisp, Tynchtyk Amatov, Michael Stadlmeier, and
Thomas Carell
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201801919
Angew. Chem. 10.1002/ange.201801919
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Prebiotic methylations and carbamoylations generate non-
canonical RNA nucleosides as molecular fossils of an early Earth
Christina Schneider, Sidney Becker, Hidenori Okamura, Antony Crisp, Tynchtyk Amatov, Michael
Stadlmeier and Thomas Carell*
The RNA world hypothesis assumes that life on earth started with
small RNA molecules that catalyzed their own formation. Vital to this
hypothesis is the need for prebiotic routes towards RNA.
Contemporary RNA, however, is not only constructed from the four
canonical nucleobases (A, C, G and U), but it contains in addition
many chemically modified (non-canonical) bases. A yet open
question is if these non-canonical bases were formed in parallel to
the canonical bases (chemical origin), or whether they were created
later, when life demanded higher functional diversity (biological
origin). Here we show that isocyanates in combination with sodium
nitrite establish methylating and carbamoylating reactivity compatible
with early Earth conditions. This chemistry leads to the formation of
methylated and amino acid modified nucleosides that are still extant.
Our data provide a plausible scenario for the chemical origin of
certain non-canonical bases, which suggests that they are fossils of
an early Earth.
polypeptides[15, 16] and simple anabolic pathways[17, 18] may have
supported an early RNA based metabolism.
This hypothesis requires the presence of the key building blocks
of life such as nucleosides and amino acids or of primitive
anabolic processes that led to their formation.[19] This raises the
question if life began with only the four canonical bases (A, C, G
21]
and U)[20,
or if an early preRNA was chemically more
diverse,[22] containing non-canonical nucleosides.[22] Those non-
canonical bases that are until today found in RNA, may be
considered fossils of this early phase of chemical evolution.[23-25]
Finding evidence for this idea requires simple chemistry
compatible with early Earth geochemical models that generate
these non-canonical bases. Here we show that the majority of
methylated nucleosides, which play important roles in RNAs of
all three domains of life, can be prebiotically generated upon
reaction of canonical nucleosides with N-methyl-N-nitrosourea,
which is readily formed from N-methylurea and sodium nitrite[26]
(Scheme 1).
More than 120 modified bases were identified in RNA, which are
important for the correct folding into complex three-dimensional
structures and for the fine tuning of RNA/RNA and RNA/protein
interactions.[1-3] Modified nucleosides are for example found in
close proximity to the anticodon stem loop in tRNA, where they
are involved in translation of the genetic code.[4, 5] Methylated
nucleosides such as m6A are involved in regulating mRNA
8]
stability,[6] splicing,[7,
translation[9-11] and
X chromosome
inactivation.[12] Another methylated nucleosides, m7G, is part of
the 5’-cap structure of eukaryotic mRNA.[13]
The RNA world hypothesis postulates that life started with self-
replicating RNA molecules that were amenable to process of
chemical evolution involving replication, randomization and
selection.[14] Since RNA is able to store genetic information and
perform catalytic processes, the hypothesis further posits that an
early replicating cell could proliferate and maintain a primitive
metabolism in the absence of coded proteins. Non-coded
Scheme 1. Chemistry that leads to the formation of methylated derivatives of
canonical nucleobases that are today found in RNA in all three domains of life.
Methylurea functions as a storage for reactive isocyanic acid.

NO2 was potentially available on the early Earth from NO and
NO2,[27] which are formed during lightning in an N2
atmosphere.[28] Alternatively, NO can form by reaction of N2 with
CO2 in hot impact plumes.[29]
Next to the methylated RNA bases, we also find amino acid
modified nucleosides among the many contemporary non-
31]
canonical RNA bases.[30,
They are directly involved in
decoding the genetic information.[32, 33] We show that our NO2 -
based chemistry provides these modified bases as well, which
suggests an early intimate contact between nucleobases and
amino acids that might have been the basis for the co-evolution
of RNA and proteins and the establishment of primitive proto-
metabolic pathways.

[a]
Christina Schneider, Sidney Becker, Dr. Hidenori Okamura, Antony
Crisp, Dr. Tynchtyk Amatov, Michael Stadlmeier and Prof. Dr. T.
Carell
Center for Integrated Protein Science (CiPSM) at the Department of
Chemistry, LMU München
Butenandtstr. 5-13, 81377 München
Fax: (+) 49 2180 77756
E-mail: Thomas.Carell@lmu.de
Homepage: www.carellgroup.de
The chemistry starts with methyl urea 1, which is one of the
molecules that was likely present on the early Earth[34]. Methyl
urea (1) is for example formed by reaction of ammonia with
Supporting information for this article is given via a link at the end of
the document.
methyl isocyanate which
was detected on comet
36]
67P/Churyumov-Gerasimenko.[35,
Methyl urea was also
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shown to form directly in the Urey-Miller experiment[37] and it is
available with high yields by the reaction of methylamine 2 with
HNCO (3) in water (90%).[38] HNCO in turn was detected in
interstellar gases,[39] and likewise on comet 67P/Churyumov-
Gerasimenko.[35, 40] Urea itself is also known to decompose into
ammonium isocyanate.[41, 42] Despite the potential formation of 3,
however, it is difficult to conceive the accumulation of HNCO (3)
because of its high reactivity. If, however, a little methyl urea (1)
is present, it can readily react with NO(Scheme 1). Methyl urea
(3) is easily nitrosylated, which gives N-methyl-N-nitrosourea
(4)[26] with a yield of 62%. This compound can physically
separate as a foam from the aqueous phase, which allows 4 to
potentially accumulate, so that it may have been locally available
at high concentrations. Under slightly basic conditions, for
example in the presence of borax (reported to be important for
ribose forming reactions),[43] 4 quickly decomposes to furnish 1-
hydroxy-2-methyldiazene (5) under liberation of HNCO (3). As
such, only small amounts of HNCO are required to help
converting MeNH2 and NaNO2 into 1-hydroxy-2-methyldiazene
(5). 1-Hydroxy-2-methyldiazene (5) in turn eliminates water and
decomposes to diazomethane (6), which is
a common
methylating agent.[44] Since all starting materials are likely
components of the organic matter on the early Earth, it is
therefore plausible that diazomethane was an accessible
component. The controlled release of 6 from the stable
precursor methyl urea (1) could have made it available for
chemical transformations despite its high reactivity and
consequently short half-life time on the early Earth.
When we performed this base-catalyzed diazomethane (6)
formation in the presence of the canonical nucleobases, we
obtained a large set of methylated compounds (Fig. 1). For the
experiment we dissolved the nucleosides in a 1:1 mixture of
borate buffer and formamide. Formamide is accessible under
early Earth conditions through the reaction of HCN and H2O.[45]
N-methyl-N-nitrosourea (4) was then added to the nucleoside
mixture in one portion. After one hour at 70°C, samples were
taken and analysed by LC-MS and tandem mass spectrometry.
The observed results are depicted in Fig. 1. In order to correctly
assign the resulting methylated nucleosides, co-injections with
synthetic reference compounds were performed (SI). The
products were further elucidated by analysis of the
fragmentation patterns in LC-MS2 experiments. When we
performed the reaction in the presence of adenosine, we
obtained m1A, Am and m6A, together with the 3’ and 5’
methylated derivatives (marked as mxA, SI). When guanosine
was methylated under the same conditions, we detected m7G
(7%) as well as Gm, m1G and m2G, all of which are known non-
canonical bases. In the presence of cytidine, the bases m3C and
Cm were generated. Furthermore, the reaction of uridine
furnished the methylated compounds Um and m3U. m3U is
formed in high yield of 11%. We also investigated the
Figure 1. HPLC traces of the reaction mixtures obtained in the reaction of N-
methyl-N-nitrosourea (1) in the presence of the canonical nucleobases A, G, C
and U. The modified nucleosides are shown in blue, the canonical ones in red.
Peaks labeled with “mx” were identified as sugar modified modifications based
on data from fragmentation studies (SI).
methylation reaction with inosine (I) as the hydrolysis product of
47]
A.[46,
When I was subjected to the same conditions, we
detected formation of Im and m1I (see SI). Importantly, nearly all
of the methylated nucleosides we observed are today found in
RNAs of all three domains of life.[2, 48]
We next asked the question of whether the simple chemistry can
be used to enable the attachment of larger chemical moieties
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genetic analysis shows surprising consensus (Fig. 3). Most of
the simple modifications that were present in LUCA could also
be formed by the chemistry presented here.
such as amino acids to the canonical nucleobases to give RNA
modifications such as t6A and g6A.
Figure 2. A: Plausible reaction scheme for the prebiotic access to t6A and g6A.
B: MS-chromatograms of the reactions of N-methyl urea derivatives 9 with the
canonical nucleoside A under formation of isocyanates of the corresponding
amino acids. The chromatogram in blue shows the reaction without the
Figure 3 Non-canonical nucleosides generated in this study (yellow area) and
non-canonical nucleosides that were found based on phylogenetic analysis to
be likely early nucleobases bases in the biosphere (blue area).[50] Modified
nucleosides that were found in both studies are shown in the green area.
ms2t6A, m22G and m62A were deposited at the border since they could be
generated as well using the here described chemistry starting from ms2A, m2G
and m6A.
addition of Ni2+-salts and the ones in black represent the reaction in the
presence of Ni2+-salts. Co-injections with synthetic standards are shown in
red. The additional peaks arise from the reaction of the sugars with the amino
acid isocyanate. Selectivity can be increased by the addition of [Ni(ClO4)2].
In summary, we report a simple cascade reaction that starts with
isocyanic acid, methylisocyanate, methylamine, ammonia and
sodium nitrite. In this cascade the unstable molecule isocyanic
acid (3) is captured by methylamine and stored in form of methyl
urea. It can be released under basic conditions from N-methyl-
N-nitrosourea (4) which is produced by nitrosylation of
methylurea (1). The chemistry allows us to convert the canonical
pyrimidine and purine bases, for which prebiotically plausible
formation processes were recently described[20, 21, 51, 52] into non-
canonical nucleosides. As such, the reported results provide
chemical evidence that the canonical and many non-canonical
ribonucleosides can form spontaneously under plausible
prebiotic conditions. The here described chemistry can be linked
to the nitrosylation chemistry that was recently reported to
enable the parallel formation of canonical and non-canonical
bases.[22] The non-canonical bases, particularly the amino acid
modified purines, potentially increase the chemical diversity of
RNA in order to broaden its folding and catalytic capabilities.
This complements ideas that non-canonical base pairs might
have existed in pre-RNA.[22, 53]
This was indeed possible (Fig. 2), when we replaced HNCO by
methyl isocyanate CH3NCO (7). 7 can be generated under
prebiotic conditions via UV-irradiation of CH4 and HNCO. In an
aqueous environment, we observed that 7 reacts rapidly with
amino acids such as glycine (8a) and threonine (8b) to give the
corresponding methyl urea derivatives 9 (Fig. 2) in nearly
quantitative yields. The compounds 9a and
b can be
nitrosylated[49] under the same conditions as methyl urea 1 to
form the nitroso compounds 10a and 10b in high yields of 82-
95%. A pH switch to slightly basic conditions with either
phosphate or borate buffer converts the intermediate nitroso
compounds 10a,b into the isocyanates of the corresponding
amino acids 11a,b. Upon treatment with adenosine, these
intermediates react to give the corresponding N6-derivatives g6A
and t6A. Since the reaction takes place under basic conditions,
not only N6 but also the 2’-, 3’-, and 5’-hydroxyl groups can react
with the isocyanate-derivative of the amino acids (see Fig. 2).
Interestingly, the selectivity of the reaction can be controlled to
favour the N6 position by the addition of Ni2+, which is generated
during prebiotic nucleoside formation.[22] At the same time CH2N2
(6) is formed which can facilitate subsequent methylations (see
SI).
Acknowledgements
Interestingly, the amino acid modified nucleosides that are
formed as described here, are present today in all three domains
of life.[2, 48] Recently, a comparative phylogenetic analysis[50] has
suggested that non-canonical bases were likely already present
in the ancient parent of all life on Earth, known conventionally as
LUCA, the last universal common ancestor. An overlay of the
nucleosides accessed in this study with those derived from the
We thank the Deutsche Forschungsgemeinschaft for financial
support via SFB1032 (TP-A5), SPP1784, CA275/11-1 and the
Excellence Cluster CiPSM. This project has received funding
from the European Research Council (ERC) under the European
Union's Horizon 2020 research and innovation programme
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