Template-Directed Copying of RNA by Non-enzymatic Ligation

The non-enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA-based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template-directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3′-amino group at the 3′-end of each oligonucleotide, in combination with an N-alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.     

The Mechanism of Nonenzymatic Template Copying with Imidazole‐Activated Nucleotides

The emergence of the replication of RNA oligonucleotides was a critical step in the origin of life. An important model for the study of nonenzymatic template copying, which would be a key part of any such pathway, involves the reaction of ribonucleoside‐5′‐phosphorimidazolides with an RNA primer/template complex. The mechanism by which the primer becomes extended by one nucleotide was assumed to be a classical in‐line nucleophilic‐substitution reaction in which the 3′‐hydroxyl of the primer attacks the phosphate of the incoming activated monomer with displacement of the imidazole leaving group. Surprisingly, this simple model has turned out to be incorrect, and the dominant pathway has now been shown to involve the reaction of two activated nucleotides with each other to form a 5′–5′‐imidazolium bridged dinucleotide intermediate. Here we review the discovery of this unexpected intermediate, and the chemical, kinetic, and structural evidence for its role in template copying chemistry.     

DNA with 3′‐5′‐Disulfide Links—Rapid Chemical Ligation through Isosteric Replacement

Efforts to chemically ligate oligonucleotides, without resorting to biochemical enzymes, have led to a multitude of synthetic analogues, and have extended oligomer ligation to reactions of novel oligonucleotides, peptides, and hybrids such as PNA. 1 Key requirements for potential diagnostic tools not based on PCR include a fast templated chemical DNA ligation method that exhibits high pairing selectivity, and a sensitive detection method. Here we report on a solid‐phase synthesis of oligonucleotides containing 5′‐ or 3′‐mercapto‐dideoxynucleotides and their chemical ligations, yielding 3′‐5′‐disulfide bonds as a replacement for 3′‐5′‐phosphodiester units. Employing a system designed for fluorescence monitoring, we demonstrate one of the fastest ligation reactions with half‐lives on the order of seconds. The nontemplated ligation reaction is efficiently suppressed by the choice of DNA modification and the 3′‐5′ orientation of the activation site. The influence of temperature on the templated reaction is shown.     

Enzyme‐Free Replication with Two or Four Bases

All known forms of life encode their genetic information in a sequence of bases of a genetic polymer and produce copies through replication. How this process started before polymerase enzymes had evolved is unclear. Enzyme‐free copying of short stretches of DNA or RNA has been demonstrated using activated nucleotides, but not replication. We have developed a method for enzyme‐free replication. It involves extension with reversible termination, enzyme‐free ligation, and strand capture. We monitored nucleotide incorporation for a full helical turn of DNA, during both a first and a second round of copying, by using mass spectrometry. With all four bases (A/C/G/T), an “error catastrophe” occurred, with the correct sequence being “overwhelmed” by incorrect ones. When only C and G were used, approximately half of the daughter strands had the mass of the correct sequence after 20 copying steps. We conclude that enzyme‐free replication is more likely to be successful with just the two strongly pairing bases than with all four bases of the genetic alphabet.     

Circular DNA by “Bis‐Click” Ligation: Template‐Independent Intramolecular Circularization of Oligonucleotides with Terminal Alkynyl Groups Utilizing Bifunctional Azides

A highly effective and convenient “bis‐click” strategy was developed for the template‐independent circularization of single‐stranded oligonucleotides by employing copper(I)‐assisted azide–alkyne cycloaddition. Terminal triple bonds were incorporated at both ends of linear oligonucleotides. Alkynylated 7‐deaza‐2′‐deoxyadenosine and 2′‐deoxyuridine residues with different side chains were used in solid‐phase synthesis with phosphoramidite chemistry. The bis‐click ligation of linear 9‐ to 36‐mer oligonucleotides with 1,4‐bis(azidomethyl)benzene afforded circular DNA in a simple and selective way; azido modification of the oligonucleotide was not necessary. Short ethynyl side chains were compatible with the circularization of longer oligonucleotides, whereas octadiynyl residues were used for short 9‐mers. Compared with linear duplexes, circular bis‐click constructs exhibit a significantly increased duplex stability over their linear counterparts. The intramolecular bis‐click ligation protocol is not limited to DNA, but may also be suitable for the construction of other macrocycles, such as circular RNAs, peptides, or polysaccharides.     

Semisynthesis of 3′(2′)‐O‐(Aminoacyl)‐tRNA Derivatives as Ribosomal Substrate

An efficient synthesis of (3′‐terminally) 3′(2′)‐O‐aminoacylated pCpA derivatives is described, which could lead to the production of (aminoacyl)‐tRNAs following T4 RNA ligase mediated ligation. The tetrahydrofuranyl (thf) group was used as a permanent protective group for the 2′‐OH of the cytidine moiety which can be removed during the purification of the 3′(2′)‐O‐aminoacylated‐pCpA. This approach allowed for a general synthesis of (3′‐terminally) 3′(2′)‐O‐aminoacylated oligonucleotides. The fully protected pCpA 14 was synthesized by phosphoramidite chemistry and treated with NH₃ solution to remove the 2‐cyanoethyl and benzoyl groups (→ 15 ; Schemes 1 and 2). The 2′‐O‐thf‐protected‐pCpA 15 was coupled with α‐amino acid cyanomethyl esters, and the products 20a – c were deprotected and purified with AcOH buffer to afford 3′(2′)‐O‐aminoacylated pCpA 21a – c in high yields. The 3′(2′)‐O‐aminoacylated pCpA were efficiently ligated with tRNA(? CA) to yield (aminoacyl)‐tRNA which was an active substrate for the ribosome.     

A Fluorescent G‐Quadruplex Sensor for Chemical RNA Copying

Non‐enzymatic RNA replication may have been one of the processes involved in the appearance of life on Earth. Attempts to recreate this process in a laboratory setting have not been successful thus far, highlighting a critical need for finding prebiotic conditions that increase the rate and the yield. Now a highly parallel assay for template directed RNA synthesis is presented that relies on the intrinsic fluorescence of a 2‐aminopurine modified G‐quadruplex. The application of the assay to examine the combined influence of multiple variables including pH, divalent metal concentrations and ribonucleotide concentrations on the copying of RNA sequences is demonstrated. The assay enables a direct survey of physical and chemical conditions, potentially prebiotic, which could enable the chemical replication of RNA.     

Efficient Automated Solid‐Phase Synthesis of DNA and RNA 5′‐Triphosphates

A fast, high‐yielding and reliable method for the synthesis of DNA‐ and RNA 5′‐triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5‐chloro‐saligenyl‐N,N‐diisopropylphosphoramidite was coupled to the 5′‐end. Oxidation of the formed 5′‐phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5′‐cycloSal‐oligonucleotides. Reaction of the support‐bonded 5′‐cycloSal‐oligonucleotide with pyrophosphate yielded the corresponding 5′‐triphosphates. The 5′‐triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer.     

2′‐Ethynyl‐DNA: Synthesis and Pairing Properties

2‐Ethynyl‐DNA was developed as a potential DNA‐selective oligonucleotide analog. The synthesis of 2′‐arabino‐ethynyl‐modified nucleosides was achieved starting from properly protected 2′‐ketonucleosides by addition of lithium (trimethylsilyl)acetylide followed by reduction of the tertiary alcohol. After a series of protecting‐group manipulations, phosphoramidite building blocks suitable for solid‐phase synthesis were obtained. The synthesis of oligonucleotides from these building blocks was successful when a fast deprotection scheme was used. The pairing properties of 2′‐arabino‐ethynyl‐modified oligonucleotides can be summarized as follows: 1) The 2′‐arabino‐ethynyl modification of pyrimidine nucleosides leads to a strong destabilization in duplexes with DNA as well as with RNA. The likely reason is that the ethynyl group sterically influences the torsional preferences around the glycosidic bond leading to a conformation not suitable for duplex formation. 2) If the modification is introduced in purine nucleosides, no such influence is observed. The pairing properties are not or only slightly changed, and, in some cases (deoxyadenosine homo‐polymers), the desired stabilization of the pairing with a DNA complementary strand and destabilization with an RNA complement is observed. 3) In oligonucleotides of alternating deoxycytidine‐deoxyguanosine sequence, the incorporation of 2′‐arabino‐ethynyl deoxyguanosine surprisingly leads to the formation of a left‐handed double helix, irrespective of salt concentration. The rationalization for this behavior is that the ethynyl group locks such duplexes in a left‐handed conformation through steric blockade.     

Nucleotides,Part LXV,Synthesis of 2′‐Deoxyribonucleoside 5′‐Phosphoramidites: New Building Blocks for the Inverse (5′‐3′)‐Oligonucleotide Approach

The syntheses of the 3′‐O‐(4,4′‐dimethoxytrityl)‐protected 5′‐phosphoramidites 25 – 28 and 5′‐(hydrogen succinates) 29 – 32 , which can be used as monomeric building blocks for the inverse (5′‐3′)‐oligodeoxyribonucleotide synthesis are described (Scheme). These activated nucleosides and nucleotides were obtained by two slightly different four‐step syntheses starting with the base‐protected nucleosides 13 – 20 . For the protection of the aglycon residues, the well‐established 2‐(4‐nitrophenyl)ethyl (npe) and [2‐(4‐nitrophenyl)ethoxy]carbonyl (npeoc) groups were used. The assembly of the oligonucleotides required a slightly increased coupling time of 3 min in application of the common protocol (see Table 1). The use of pyridinium hydrochloride as an activator (instead of 1H‐tetrazole) resulted in an extremely shorter activation time of 30 seconds. We established the efficiency of this inverse strategy by the synthesis of the oligonucleotide 3′‐conjugates 33 and 34 which carry lipophilic caps derived from cholesterol and vitamin E, respectively, as well as by the formation of (3′‐3′)‐ and (5′‐5′)‐internucleotide linkages (see Table 2).     

Molecular‐Dynamics Studies of Single‐Stranded Hexitol,Altritol, Mannitol,and Ribose Nucleic Acids (HNA,MNA, ANA,and RNA,Resp.) and of the Stability of HNA⋅RNA,ANA⋅RNA,and MNA⋅RNA Duplexes

The influence of the orientation of a 3′‐OH group on the conformation and stability of hexitol oligonucleotides in complexes with RNA and as single strands in aqueous solution was investigated by molecular‐dynamics (MD) simulations with AMBER 4.1. The particle mesh Ewald (PME) method was used for the treatment of long‐range electrostatic interactions. An equatorial orientation of the 3′‐OH group in the single‐stranded D ‐mannitol nucleic acid (MNA) m(GCGTAGCG) and in the complex with the RNA r(CGCAUCGC) has an unfavorable influence on the helical stability. Frequent H‐bonds between the 3′‐OH group and the O−C(6′) of the phosphate backbone of the following nucleotide explain the distorted conformation of the MNA⋅RNA complex as well as that of the single MNA strand. This is consistent with experimental results that show lowered hybridization potentials for MNA⋅RNA complexes. An axial orientation of the 3′‐OH group in the D ‐altritol nucleic acid (ANA) a(GCGTAGCG) leads to a stable complex with the complementary RNA r(CGCAUCGC), as well as to a more highly preorganized single‐stranded ANA chain. The averaged conformation of the ANA⋅RNA complex is similar to that of A‐RNA, with only minor changes in groove width, helical curvature, and H‐bonding pattern. The relative stabilities of ANA⋅RNA vs. HNA⋅RNA (HNA=D ‐hexitol nucleic acid without 3′‐OH group) can be explained by differences in restricted movements, H‐bonds, and solvation effects.     

Covalent Chemical 5′‐Functionalization of RNA with Diazo Reagents

Functionalization of RNA at the 5′‐terminus is important for analytical and therapeutic purposes. Currently, these RNAs are synthesized de novo starting with a chemically functionalized 5′‐nucleotide, which is incorporated into RNA using chemical synthesis or biochemical techniques. Methods for direct chemical modification of native RNA would provide an attractive alternative but are currently underexplored. Herein, we report that diazo compounds can be used to selectively alkylate the 5′‐phosphate of ribo(oligo)nucleotides to give RNA labelled through a native phosphate ester bond. We applied this method to functionalize oligonucleotides with biotin and an orthosteric inhibitor of the eukaryotic initiation factor 4E (eIF4E), an enzyme involved in mRNA recognition. The modified RNA binds to eIF4E, demonstrating the utility of this labelling technique to modulate biological activity of RNA. This method complements existing techniques and may be used to chemically introduce a broad range of functional handles at the 5′‐end of RNA.     

Targeting of Single‐Stranded Oligonucleotides through Metal‐Induced Cyclization of Short Complementary Strands

A new strategy to cyclize short synthetic oligonucleotides on DNA or RNA target strands is described. The approach is based on metal‐templated cyclization of short synthetic oligonucleotides conjugated with two chelating 2,2′ : 6′,2′′‐terpyridine (Tpy) moieties at their 3′‐ and 5′‐ends. Cyclization after metal addition (Zn²⁺, Fe²⁺) was demonstrated by means of thermal‐denaturation experiments, MALDI‐Q‐TOF‐MS, and gel electrophoresis (PAGE). 1D‐ and 2D‐NMR Experiments were performed to analyze the association of complementary strands after metal‐mediated cyclization. Our protocol allows the efficient circularization of synthetic oligonucleotides. Thereby, the hybridization on a complementary strand was more efficient with an RNA target strand and a 2′‐O‐methylated circularized oligomer.     

Pyranosyl-RNA: chiroselective self-assembly of base sequences by ligative oligomerization of tetra nucleotide-2′,3′-cyclophosphates (with a commentary concerning the origin of biomolecular homochirality)

Background: Why did Nature choose furanosyl-RNA and not pyranosyl-RNA as her molecular genetic system? An experimental approach to this problem is the systematic comparison of the two isomeric oligonucleotide systems with respect to the chemical properties that are fundamental to the biological role of RNA, such as base pairing and nonenzymic replication. Pyranosyl-RNA has been found to be not only a stronger, but also a more selective pairing system than natural RNA; both form hairpin structures with comparable ease. Base sequences of pyranosyl-RNA can be copied by template-controlled replicatioe ligation of short activated oligomers (e.g. tetramer-2′,3′-cyclophosphates) under mild and potentially natural conditions. The copying proceeds with high regio-selectivity as well as chiroselectivity: homochiral template sequences mediate the formation of the correct (4′→2′)-phosphodiester junction between homochiral tetramer units provided they have the same sense of chirality as the template. How could homochiral template sequences assemble themselves in the first place?Results: Higher oligomers of pyranosyl-RNA can self-assemble in dilute solutions under mild conditions by ligative oligomerization of tetramer-2′,3′-cyclophosphates containing hemi self-complementary base sequences. The only side reaction that effectively competes with ligation is hydrolytic deactivation of 2′,3′-cyclophosphate end groups. The ligation reaction is highly chiroselective; it is slower by at least two orders of magnitude when one of the (d)-ribopyranosyl units of a homochiral (d)-tetramer-2′,3′-cyclophosphate is replaced by a corresponding (l)-unit, except when the (l)-unit is at the 4′ end of the tetramer and carries a purine, when the oligomerization rate can be ∼ 10% of that shown for a homochiral isomer. The oligomerization of homochiral tetramers is not, or only weakly, inhibited by the presence of the non-oligomerizing diastereomers.Conclusions: Available data on the chiroselective self-directed oligomerization of tetramer-2′,3′-cyclophosphates allow us to extrapolate that sets of tetramers with different but mutually fitting base sequences can be expected to co-oligomerize stochastically and generate sequence libraries consisting of predominantly homochiral (d)- and (l)-oligomers, starting from the racemic mixture of tetramers containing all possible diastereomers. Such a capability of an oligonucleotide system deserves special attention in the context of the problem of the origin of biomolecular homochirality: breaking molecular mirror symmetry by de-racemization is an intrinsic property of such a system whenever the constitutional complexity of the products of co-oligomerization exceeds a critical level.     

Liquid‐Phase Synthesis of 2′‐Methyl‐RNA on a Homostar Support through Organic‐Solvent Nanofiltration

Due to the discovery of RNAi, oligonucleotides (oligos) have re‐emerged as a major pharmaceutical target that may soon be required in ton quantities. However, it is questionable whether solid‐phase oligo synthesis (SPOS) methods can provide a scalable synthesis. Liquid‐phase oligo synthesis (LPOS) is intrinsically scalable and amenable to standard industrial batch synthesis techniques. However, most reported LPOS strategies rely upon at least one precipitation per chain extension cycle to separate the growing oligonucleotide from reaction debris. Precipitation can be difficult to develop and control on an industrial scale and, because many precipitations would be required to prepare a therapeutic oligonucleotide, we contend that this approach is not viable for large‐scale industrial preparation. We are developing an LPOS synthetic strategy for 2′‐methyl RNA phosphorothioate that is more amenable to standard batch production techniques, using organic solvent nanofiltration (OSN) as the critical scalable separation technology. We report the first LPOS‐OSN preparation of a 2′‐Me RNA phosphorothioate 9‐mer, using commercial phosphoramidite monomers, and monitoring all reactions by HPLC, ³¹P NMR spectroscopy and MS.     

Efficient Solid‐Phase Synthesis of pppRNA by Using Product‐Specific Labeling

A novel solid‐phase synthesis and purification strategy for 5′‐triphosphate oligonucleotides by using lipophilic tagging of the triphosphate moiety is reported. This is based on triphosphate synthesis with 5′‐O‐cyclotriphosphate intermediates, whereby a lipophilic tag, such as decylamine, is introduced during the ring‐opening reaction to give a linear gamma‐phosphate‐tagged species. This method enables the highly efficient synthesis of 5′‐triphosphorylated RNA derivatives and their gamma‐phosphate‐substituted analogues and will especially facilitate the advancement of therapeutic approaches that make use of 5′‐triphosphate oligonucleotides as potent activators of the cytosolic immune sensor RIG‐I.     

Periodic Melting of Oligonucleotides by Oscillating Salt Concentrations Triggered by Microscale Water Cycles Inside Heated Rock Pores

To understand the emergence of life, a better understanding of the physical chemistry of primordial non‐equilibrium conditions is essential. Significant salt concentrations are required for the catalytic function of RNA. The separation of oligonucleotides into single strands is a difficult problem as the hydrolysis of RNA becomes a limiting factor at high temperatures. Salt concentrations modulate the melting of DNA or RNA, and its periodic modulation would enable melting and annealing cycles at low temperatures. In our experiments, a moderate temperature difference created a miniaturized water cycle, resulting in fluctuations in salt concentration, leading to melting of oligonucleotides at temperatures 20 °C below the melting temperature. This would enable the reshuffling of duplex oligonucleotides, necessary for ligation chain replication. The findings suggest an autonomous route to overcome the strand‐separation problem of non‐enzymatic replication in early evolution.