Amino Acid Modified RNA Bases as Building Blocks of an Early Earth RNA‐Peptide World

Abstract Fossils of extinct species allow us to reconstruct the process of Darwinian evolution that led to the species diversity we see on Earth today. The origin of the first functional molecules able to undergo molecular evolution and thus eventually able to create life, are largely unknown. The most prominent idea in the field posits that biology was preceded by an era of molecular evolution, in which RNA molecules encoded information and catalysed their own replication. This RNA world concept stands against other hypotheses, that argue for example that life may have begun with catalytic peptides and primitive metabolic cycles. The question whether RNA or peptides were first is addressed by the RNA‐peptide world concept, which postulates a parallel existence of both molecular species. A plausible experimental model of how such an RNA‐peptide world may have looked like, however, is absent. Here we report the synthesis and physicochemical evaluation of amino acid containing adenosine bases, which are closely related to molecules that are found today in the anticodon stem‐loop of tRNAs from all three kingdoms of life. We show that these adenosines lose their base pairing properties, which allow them to equip RNA with amino acids independent of the sequence context. As such we may consider them to be living molecular fossils of an extinct molecular RNA‐peptide world.

Abstract: Fossils of extinct speciesa llow us to reconstruct the process of Darwinian evolution that led to the species diversity we see on Earth today.T he origin of the first functional molecules able to undergo molecular evolution and thus eventually able to create life, are largely unknown.T he most prominenti dea in the field posits that biologyw as preceded by an era of molecular evolution, in which RNA molecules encoded information and catalysed their own replication.T his RNA worldc oncept stands againsto ther hypotheses, that argue for example that life may have begun with catalytic peptides and primitive metabolic cycles.T he questionw hether RNA or peptides were first is addressed by the RNA-peptidew orld concept, which postulates ap arallel existence of both molecular species. Ap lausible experimental model of how such an RNA-peptide world may have lookedl ike, however, is absent.H ere we reportt he synthesis andp hysicochemical evaluation of amino acid containing adenosine bases, which are closely related to molecules that are found today in the anticodons tem-loop of tRNAs from all three kingdoms of life.W es how that these adenosines lose their base pairing properties, which allow them to equip RNA with amino acids independent of the sequence context. As such we may consider them to be living molecular fossils of an extinct molecular RNA-peptide world.
The RNA-peptide co-evolution hypothesis describes the emergence of self-replicating molecules that contained aminoa cids and RNA. [1] At the macromolecular level,t his tight coexistence of peptides and RNA is established in the ribosome,w here encoding and catalytic RNA is supported by proteins. [2] Although we cannot delineate how such an early RNA-peptide world may have looked like, it seems not too implausible to assume that some of the molecular components may have survived until today as vestiges of this extinct world. [3] tRNAs derived from all three kingdoms of life contain al arge number of modified bases, [4] and some of them are indeed modified with aminoa cids. [3] The most wide spread amino acid modified basesa re adenosine nucleosides,i nw hich the amino acid is linkedv ia urea connector to the N 6 -amino group of the heterocycle as depicted in Figure 1a.P articularly ubiquitousa re adenosinem odificationsc ontaining the aminoa cids threonine (t 6 A) [5][6][7] and glycine( g 6 A), [8] together with hn 6 A. [9,10] Based upon recent phylogenetic analyses andt he fact that t 6 Ai s foundi na ll three kingdoms of life, it has been suggested that such amino acid modified bases were already present in the last universalc ommon ancestor (LUCA), from which all life forms descended. [11][12][13][14] t 6 Ai sf or example today found in nearly all ANN decoding tRNAs. [15] We recently reported ap lausible prebiotic route to some of these amino acidm odified A-bases, which strengthens the idea that they could indeed be living chemicalf ossils of the extinctR NA-peptidew orld. [16] Despite the interesting philosophical genotype-phenotype dualism that characterizes these structures and their contemporary importancef or the faithful decoding of genetici nformation, a general synthesis of aa 6 Am odified bases (Figure 1a)a nd as ystematicstudy of their properties is lacking.
Here we report the synthesis of av ariety of aa 6 An ucleosides with canonical amino acids (aa = Asp, Gly,H is, Phe, Thr, [17] Ser, Val), their incorporation into DNA and RNA and an investigation of how they influence the physicochemical properties of oligonucleotides. We were particularly interested to study how they might affect the stability of RNA and DNA. The computer visualization shows that in A-form RNA (Figure 1b), the amino acid part of the aa 6 Ab ase would need to reside inside the helix, shielded from the outside. In the B-form DNA one could imagine ad ecoration of the major groove with the amino acid side chains as depicted in Figure 1c.
In the Schemes 1a nd 2w es how the synthesis of the different ureal inked amino acidA -derivatives (aa 6 A). We first prepared the amino acid components for the coupling to the Anucleoside (Scheme1). Our starting points for Thr 6 A, Ser 6 Aa nd Asp 6 Aw ere the free amino acids 1-3,i nw hich we first transformed all carboxylic acids into the p-nitrophenylethyl esters (npe, 4-6). [17] The hydroxy groups of the Thr and Ser compounds were finally protected as TBS-ethers to give the final products 7 and 8 (Scheme 1a). For Val, Gly and Phe we started with the Boc-protected amino acids 9-11,w hichw ealso converted into the npe-esters 12-14 using Mitsunobu type chemistry [18] followed by acidic (4 m HCl in dioxane) Boc-deprotection to give the amino acid products 15-17 (Scheme 1b). [19] For His 6 A, we agains tartedw ith the Boc-protected amino acid 18 (Scheme 1c)a nd used HBTU activationt og enerate the npe ester 19.P rotection of the imidazole N t with POM-chloride followed again by Boc-deprotection furnished the ready to couple amino acid 21.
The connection of the aminoa cid with the A-nucleoside via the uream oiety wasn ext carriedo ut as depicted in Scheme 2. We first treated phenylc hloroformate with N-methylimidazole to obtain the 1-N-methyl-3-phenoxycarbonyl-imidazolium chloride (22). [20] Adenosine was converted in parallel into the cyclic 3',5'-silyl protected nucleoside, followed by conversion of the 2'-OH group into the TBS-ether. [21] The reaction of compound 24 with the activated carbonate and the corresponding amino acid, provided in all cases the amino acid coupled products 25-31 in good to excellent yields. Subsequentc leavage of the Scheme1.Synthesis of the amino acid building blocks as needed for the coupling to the nucleoside Atog ive Thr 6 A, Ser 6 A, Asp 6 A, Val 6 A, Gly 6 A, Phe 6 A and His 6 A. cyclic silylether with HF·pyridine complex, [22,23] protection of the 5'-OH group with dimethoxytritylchloride (DMTCl) [24] allowed the final conversion of the compounds into the corresponding phosphoramidites 46-52.S tandard solid phase RNA chemistry [25][26][27][28][29][30][31] was subsequently employed to prepareR NA strandsc ontaining the individual aa 6 An ucleosides stably embedded.T he standard RNA synthesis protocol did not require any adjustment. In all cases we observed fair coupling of the aa 6 Ap hosphoramidites and no decomposition during deprotection. Deprotection required three steps.F irst, with DBU in THF at r.t. for 2h we cleaved the npe-protecting group. Second, we deprotectedt he bases and cleaved from the solid support with aqueous NH 3 /MeNH 2 .F inally,w er emoved the 2'silyl group with HF in NEt 3 .
In order to investigate how aa 6 Ab ases would affect the stability of DNA duplexes we also prepared as ar epresentative molecule t 6 dA as depicted in Scheme3.T ot his end we first acetyl-protected dA 53, [32] performed the coupling of the protected threonine with the activatedc arbonate 22,c leaved the acetyl groups and converted the nucleoside subsequently into the 5'-DMTp rotected phosphoramidite 57.T he purification of compound 57 was quite difficult due to its high polarity.W e neededt ou se rather polar mixture of EtOAc/Hex (2/1) as the mobile phase for the chromatographic separation. This provided the phosphoramidite 57,h owever the materialh ad al ower purity in comparison to the RNA phosphoramidites.N evertheless, solid phase DNA synthesis and deprotection of the DNA strand ODN1 proceededa gain smoothly and in high yields. Figure 2a shows as an example the raw HPL-chromatograms of ON1 (RNA strand with embedded t 6 A) and the corresponding chromatogram after purification (inset) together with the obtained MALDI-TOF mass spectrum (Figure 2b). The chromatogramso ft he raw material show ag ood quality of the obtained RNA material. The analytical chromatogram after purification and the MALDI-TOF data prove the purity of the finally obtained RNA oligonucleotide and the integrity of the t 6 A-containing RNA strand. Figure 2c and 2d show the same data set for the t 6 dA containing DNA oligonucleotide (ODN1), proving again the successfuls ynthesis of t 6 dA containing oligonucleotide. The aa 6 (d)A nucleosides can exist in two different conformations. [33] The first, s-trans,m aintains the Watson-Crick hydrogen bonding capabilities with the urea amino acid oriented towards the imidazole ring system (Figure 3a). Thisa llows formation of a Hoogsteen type 7-membered ring H-bondw ith the N 7 .I nt he corresponding s-cis-conformation, the urea amino acid orients towardst he Watson-Crick sidet hereby establishing at ypically strong intramolecular 6-membered H-bond with N 1 (Figure 3b). In order to investigate if the embeddingo ft he amino acid would enforce s-trans-conformation and hence Watson-Crick H-bonding, we measured meltingp oints of all aa 6 Ac ontaining RNA strandsa nd of the t 6 dA containing DNA strand hybridized to the corresponding counter strands ( Figure 3). In the RNA:RNA situation we noted for all aa 6 As trandst hat we investigated, as ingle clear melting point, showingt hat only one conformer of the aa 6 Ab ase likely exists in the RNA:RNA duplexes. In situation where the aa 6 Ab ase exists in two different stable conformations, one would expect am ore complexm elting behaviour.I na ll cases we saw that the meltingp oint is strongly reduced by 10-15 8C. When we embedded two aa 6 A buildingb locks into as hort RNA strand no duplex formation Scheme3.Synthesis of t 6 dA phosphoramidite and its incorporationi nto DNA. www.chemeurj.org 2020 The Authors. Published by Wiley-VCH GmbH was obtained. Even stronger reduction of the melting point was observed for the DNA duplexc ontaining one t 6 dA. Here, we also saw just one sharpm eltingp oint and ar eduction of the T m by over 20 8C. These data show that the aa 6 Ab ases and among them t 6 Aa nd g 6 Aa re unable to base pair.A lthough we have no direct proof of the structure the data argue for ap referred s-cis-conformation (Figure 3b)i na greement with the literature. [34] This conclusion is also supported by the observation that irrespective of the chirality of the attached amino acid (l-v ersus d-Phe 6 A), we measured the same melting temperature. This would not be expected if the s-trans-conformation and base pairing would be possible. These data suggestt hat aa 6 An ucleosides within RNA position ag iven amino acid outside the A-form helix in an unpaired situation and hence independent from the counterbase. As such, multiple aa 6 Ac ontaining RNA strandsw ould be structures in which the RNA part is decorated by the amino acid side chains. In order to show that RNAstructures containingm ultiple amino acids as representatives of an RNA-peptidew orld can stably form, we prepared two RNA duplexes (Figure 4). In the first (D5), we placed three t 6 A bases as extra bases in an otherwise undisturbed RNA duplex. Indeed,n ow the stability of this duplex was indistinguishable from the same construct containing just canonicalb ases (D6). Finally,w ep repared an RNA duplex D7,i nw hichw ep laced the amino acids Ser-Asp-His directly next to each other to simulate what is known in the peptidew orld as the catalytic triad present in serinep eptidases. [35] Again in this case as table duplex structure forms with the three aa 6 Ab ases creatinga loop. Although we do not show any catalytic activity here, we believe that it is easily imaginable that if these aminoa cids are properly positioned in as tably folded RNA the structure could gain catalytic properties.
The melting data show,t hat aa 6 Ab ases alone are unable to establish base pairing, which hindert hem to encodes equence information.O nt he other side, these bases allow the incorporation of amino acids into RNA structuresi rrespective of the counterbase. Because RNAsa re mostly stably folded structures in which many bases are not involved in any base pairing or establish no Watson-Crick interactions the amino acid adenosine nucleosides allow the stable incorporationo fa mino acid functionality into RNA.
In summary,h ere we investigated the synthesis and properties of aa 6 An ucleoside-amino acidc onjugates,s ome of which (t 6 A, g 6 A, hn 6 A) are today found as key components in the tRNAso fm anys pecies. In these tRNAs the aa 6 An ucleosides reside at the general purine position 37 adjacent to the anticodon loop. They are not involved in base pairing but fine tune the codon-anticodon interaction to enable faithful translation of informationi nto ap eptide sequence. [36] Here we show that these bases are indeed unable to base pair.T hey have to be placed outside the pairing regime that is neededf or RNA folding. As such they functiona sa nchors that allow the connection of amino acid to RNA structures independento ft he counterbase. The side chains are then available to equip RNA with additional functions that might have been beneficial in an early RNA-peptide world. The fact that aa 6 An ucleosides are stable structures and until today broadly found in today's RNA make them prime candidates to develop idea about the chemical constitution of the vanished RNA-peptideworld.