Kinetic explanations for the sequence biases observed in the nonenzymatic copying of RNA templates.

The identification of nonenzymatic pathways for nucleic acid replication is a key challenge in understanding the origin of life. We have previously shown that nonenzymatic RNA primer extension using 2-aminoimidazole (2AI) activated nucleotides occurs primarily through an imidazolium-bridged dinucleotide intermediate. The reactive nature and preorganized structure of the intermediate increase the efficiency of primer extension but remain insufficient to drive extensive copying of RNA templates containing all four canonical nucleotides. To understand the factors that limit RNA copying, we synthesized all ten 2AI-bridged dinucleotide intermediates and measured the kinetics of primer extension in a model system. The affinities of the ten dinucleotides for the primer/template/helper complexes vary by over 7,000-fold, consistent with nearest neighbor energetic predictions. Surprisingly, the reaction rates at saturating intermediate concentrations still vary by over 15-fold, with the most weakly binding dinucleotides exhibiting a lower maximal reaction rate. Certain noncanonical nucleotides can decrease sequence dependent differences in affinity and primer extension rate, while monomers bridged to short oligonucleotides exhibit enhanced binding and reaction rates. We suggest that more uniform binding and reactivity of imidazolium-bridged intermediates may lead to the ability to copy arbitrary template sequences under prebiotically plausible conditions.

Chemical Synthesis of Modified RNA.

Ribonucleic acids (RNAs) play a vital role in living organisms. Many of their cellular functions depend critically on chemical modification. Methods to modify RNA in a controlled manner-both in vitro and in vivo-are thus essential to evaluate and understand RNA biology at the molecular and mechanistic levels. The diversity of modifications, combined with the size and uniformity of RNA (made up of only 4 nucleotides) makes its site-specific modification a challenging task that needs to be addressed by complementary approaches. One such approach is solid-phase RNA synthesis. We discuss recent developments in this field, starting with new protection concepts in the ongoing effort to overcome current size limitations. We continue with selected modifications that have posed significant challenges for their incorporation into RNA. These include deazapurine bases required for atomic mutagenesis to elucidate mechanistic aspects of catalytic RNAs, and RNA containing xanthosine, N4-acetylcytidine, 5-hydroxymethylcytidine, 3-methylcytidine, 2'-OCF3, and 2'-N3 ribose modifications. We also discuss the all-chemical synthesis of 5'-capped mRNAs and the enzymatic ligation of chemically synthesized oligoribonucleotides to obtain long RNA with multiple distinct modifications, such as those needed for single-molecule FRET studies. Finally, we highlight promising developments in RNA-catalyzed RNA modification using cofactors that transfer bioorthogonal functionalities.

A continuous reaction network that produces RNA precursors.

Continuous reaction networks, which do not rely on purification or timely additions of reagents, serve as models for chemical evolution and have been demonstrated for compounds thought to have played important roles for the origins of life such as amino acids, hydroxy acids, and sugars. Step-by-step chemical protocols for ribonucleotide synthesis are known, but demonstrating their synthesis in the context of continuous reaction networks remains a major challenge. Herein, compounds proposed to be important for prebiotic RNA synthesis, including glycolaldehyde, cyanamide, 2-aminooxazole, and 2-aminoimidazole, are generated from a continuous reaction network, starting from an aqueous mixture of NaCl, NH4Cl, phosphate, and HCN as the only carbon source. No well-timed addition of any other reagents is required. The reaction network is driven by a combination of γ radiolysis and dry-down. γ Radiolysis results in a complex mixture of organics, including the glycolaldehyde-derived glyceronitrile and cyanamide. This mixture is then dried down, generating free glycolaldehyde that then reacts with cyanamide/NH3 to furnish a combination of 2-aminooxazole and 2-aminoimidazole. This continuous reaction network models how precursors for generating RNA and other classes of compounds may arise spontaneously from a complex mixture that originates from simple reagents.

Regioselective formation of RNA strands in the absence of magnesium ions.

The oligomerization of ribonucleotides can produce short RNA strands in the absence of enzymes. This reaction gives one of two regioisomeric phosphodiester linkages, a 2',5'- or a 3',5'-diester. The former, non-natural linkage is detrimental for duplex stability, and is known to form preferentially in oligomerizations occurring in homogeneous solution with preactivated nucleotides in the presence of magnesium cations. We have studied ribonucleotide oligomerization with in situ activation, using NMR as monitoring technique. Unexpectedly, the known preference for 2',5'-linkages in the oligomerization of AMP was reversed in the absence of magnesium ions at slightly basic pH. Further, oligomerization was surprisingly efficient in the absence of Mg2+ salts, producing oligomers long enough for duplex formation. A quantitative systems chemistry analysis then revealed that the absence of magnesium ions favors the activation of nucleotides, and that the high concentration of active species can compensate for slower coupling. Further, organocatalytic intermediates can help to overcome the unfavorable regioselectivity of the magnesium-catalyzed reactions. Our findings allay concerns that RNA may have been difficult to form in the absence of enzymes. They also show that there is an efficient path to genetic material that does not require mineral surfaces or cations known to catalyze RNA hydrolysis.

Spontaneous advent of genetic diversity in RNA populations through multiple recombination mechanisms.

There are several plausible abiotic synthetic routes from prebiotic chemical materials to ribonucleotides and even short RNA oligomers. However, for refinement of the RNA World hypothesis to help explain the origins of life on the Earth, there needs to be a manner by which such oligomers can increase their length and expand their sequence diversity. Oligomers longer than at least 10-20 nucleotides would be needed for raw material for subsequent natural selection. Here, we explore spontaneous RNA-RNA recombination as a facile means by which such length and diversity enhancement could have been realized. Motivated by the discovery that RNA oligomers stored for long periods of time in the freezer expand their lengths, we systematically investigated RNA-RNA recombination processes. In addition to one known mechanism, we discovered at least three new mechanisms. In these, one RNA oligomer acts as a splint to catalyze the hybridization of two other oligomers and facilitates the attack of a 5'-OH, a 3'-OH, or a 2'-OH nucleophile of one oligomer onto a target atom of another. This leads to the displacement of one RNA fragment and the production of new recombinant oligomers. We show that this process can explain the spontaneous emergence of sequence complexity, both in vitro and in silico.

Synthesis of crosslinked 2'-OMe RNA duplexes and their application for effective inhibition of miRNA function.

The interstrand crosslinking of nucleic acids is one of the strategies to create the stable complex between an oligonucleotide and RNA by covalent bond formation. We previously reported that fully 2'-O-methylated (2'-OMe) RNAs having the 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. In this study, we established a chemical method to efficiently synthesize the crosslinked 2'-OMe RNA duplexes using AVP and prepared the anti-miRNA oligonucleotides (AMOs) containing the antisense targeting miR-21 and crosslinked duplex at the terminal sequences. These AMOs showed a markedly higher anti miRNA activity than that of the commercially-available miR-21 inhibitor which has locked nucleic acid (LNA) residues.

Synthesis and duplex-forming ability of oligonucleotides modified with 4'-C,5'-C-methylene-bridged nucleic acid (4',5'-BNA).

We describe herein the design and synthesis of 4'-C,5'-C-methylene-bridged nucleic acid (4',5'-BNA), a novel artificial nucleic acid with the torsion angle γ in a non-canonical +ac range. The 4',5'-BNA phosphoramidite bearing a thymine nucleobase was synthesized from a commercially available thymidine analog in 11 steps and successfully incorporated into oligonucleotides. The resulting oligonucleotides were evaluated for their duplex-forming ability toward single-stranded DNA and RNA.

Synthesis and applications of RNAs with position-selective labelling and mosaic composition.

Knowledge of the structure and dynamics of RNA molecules is critical to understanding their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be dramatically enhanced by methods that enable incorporation of modified or labelled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. Here we develop a hybrid solid-liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labelling. We demonstrate its use by successfully preparing various isotope- or fluorescently labelled versions of the 71-nucleotide aptamer domain of an adenine riboswitch for nuclear magnetic resonance spectroscopy or single-molecule Förster resonance energy transfer, respectively. Those RNAs include molecules that were selectively isotope-labelled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently labelled in and near kissing loops. These selectively labelled RNAs have the same fold as those transcribed using conventional methods, but they greatly simplify the interpretation of NMR spectra. The single-position isotope- and fluorescently labelled RNA samples reveal multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labelling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection, and disease diagnostics.

Lanthanide Tagging of Oligonucleotides to Nucleobase for Paramagnetic NMR.

Although lanthanide tags, which have large anisotropic magnetic susceptibilities, have already been introduced to enrich NMR parameters by long-range pseudoconact shifts (PCSs) and residual dipolar couplings (RDCs) of proteins, their application to nucleotides has so far been limited to one previous report, due to the high affinities of lanthanides for the phosphodiester backbone of nucleotides and difficult organic synthesis. Herein, we report successful attachment of a lanthanide tag to a chemically synthesized oligonucleotide via a disulfide bond. NMR experiments reveal PCSs of up to 1 ppm and H-H RDCs of up to 8 Hz at 950 MHz. Although weaker magnetic alignment was achieved than with proteins, the paramagnetic data could be fitted to the known structure of the DNA, taking the mobility of the tag into account. While further rigidification of the tag is desirable, this tag could also be used to measure heteronuclear RDCs of 13 C,15 N-labeled chemically synthesized DNA and RNA.

Non-Watson-Crick RNA synthesis suited to origin functions.

A templated RNA synthesis is characterized in which G5'pp5'G accelerates synthesis of A5'pp5'A from pA and chemically activated ImpA precursors. Similar acceleration is not observable in the presence of UppU, CppC, AppG, AppA, or pG alone. Thus, it seems likely that AppA is templated by GppG via a form or forms of G:A base-pairing. AppA also appears, more slowly, via a previously known untemplated second-order chemical route. Such AppA synthesis requires only ordinary near-neutral solutions containing monovalent and divalent salts, and rates are only slightly sensitive to variation in pH. Templated synthesis rates are first order in pA, ImpA, and template GppG; thus third order overall. Therefore, this reaction resembles cross-templating of AppA on poly(U), but is notably slower and less sensitive to temperature. Viewing AppA as a coenzyme analog, GppG templating provides a simpler molecular route, termed para-templating, to encoded chemical functions. Para-templating can also arise from a single, localized nucleobase geosynthetic event which yields purines. It requires only a single backbone-forming chemistry. Thus it may have appeared earlier and served as evolutionary precursor for more complex forms of encoded genetic expression.

An expedient process for reducing the formation of process-related impurities during solid-phase synthesis of potential nucleic acid-based drugs.

With the intent of mitigating the formation of process-related impurities during solid-phase synthesis of DNA or RNA sequences, a hydroxylated controlled-pore glass support conjugated to three, five or seven hexaethylene glycol spacers was prepared and demonstrated to provide a more efficient and robust synthesis process. Indeed, the use of a support conjugated to five hexaethylene glycol spacers led to a 19% up to 42% reduction of process-related impurities contaminating synthetic nucleic acid sequences, when compared to that obtained from the same DNA/RNA sequences synthesized using a commercial long-chain alkylamine controlled-pore glass support under highly similar conditions.

Chemical methods for the modification of RNA.

RNA is often considered as being the vector for the transmission of genetic information from DNA to the protein synthesis machinery. However, besides translation RNA participates in a broad variety of fundamental biological roles such as gene expression and regulation, protein synthesis, and even catalysis of chemical reactions. This variety of function combined with intricate three-dimensional structures and the discovery of over 100 chemical modifications in natural RNAs require chemical methods for the modification of RNAs in order to investigate their mechanism, location, and exact biological roles. In addition, numerous RNA-based tools such as ribozymes, aptamers, or therapeutic oligonucleotides require the presence of additional chemical functionalities to strengthen the nucleosidic backbone against degradation or enhance the desired catalytic or binding properties. Herein, the two main methods for the chemical modification of RNA are presented: solid-phase synthesis using phosphoramidite precursors and the enzymatic polymerization of nucleoside triphosphates. The different synthetic and biochemical steps required for each method are carefully described and recent examples of practical applications based on these two methods are discussed.

Postsynthetic On-Column 2' Functionalization of RNA by Convenient Versatile Method.

We report a universal straightforward strategy for the chemical synthesis of modified oligoribonucleotides containing functional groups of different structures at the 2' position of ribose. The on-column synthetic concept is based on the incorporation of two types of commercial nucleotide phosphoramidites containing orthogonal 2'-O-protecting groups, namely 2'-O-thiomorpholine-carbothioate (TC, as "permanent") and 2'-O-tert-butyl(dimethyl)silyl (tBDMS, as "temporary"), to RNA during solid-phase synthesis. Subsequently, the support-bound RNA undergoes selective deprotection and follows postsynthetic 2' functionalization of the naked hydroxyl group. This convenient method to tailor RNA, utilizing the advantages of solid phase approaches, gives an opportunity to introduce site-specifically a wide range of linkers and functional groups. By this strategy, a series of RNAs containing diverse 2' functionalities were synthesized and studied with respect to their physicochemical properties.

Synthesis of 5'-Thiamine-Capped RNA.

RNA 5'-modifications are known to extend the functional spectrum of ribonucleotides. In recent years, numerous non-canonical 5'-modifications, including adenosine-containing cofactors from the group of B vitamins, have been confirmed in all kingdoms of life. The structural component of thiamine adenosine triphosphate (thiamine-ATP), a vitamin B1 derivative found to accumulate in Escherichia coli and other organisms in response to metabolic stress conditions, suggests an analogous function as a 5'-modification of RNA. Here, we report the synthesis of thiamine adenosine dinucleotides and the preparation of pure 5'-thiamine-capped RNAs based on phosphorimidazolide chemistry. Furthermore, we present the incorporation of thiamine-ATP and thiamine adenosine diphosphate (thiamine-ADP) as 5'-caps of RNA by T7 RNA polymerase. Transcripts containing the thiamine modification were modified specifically with biotin via a combination of thiazole ring opening, nucleophilic substitution and copper-catalyzed azide-alkyne cycloaddition. The highlighted methods provide easy access to 5'-thiamine RNA, which may be applied in the development of thiamine-specific RNA capture protocols as well as the discovery and confirmation of 5'-thiamine-capped RNAs in various organisms.

Large-Scale Photolithographic Synthesis of Chimeric DNA/RNA Hairpin Microarrays To Explore Sequence Specificity Landscapes of RNase HII Cleavage.

Ribonuclease HII (RNase HII) is an essential endoribonuclease that binds to double-stranded DNA with RNA nucleotide incorporations and cleaves 5' of the ribonucleotide at RNA-DNA junctions. Thought to be present in all domains of life, RNase HII protects genomic integrity by initiating excision repair pathways that protect the encoded information from rapid degradation. There is sparse evidence that the enzyme cleaves some substrates better than others, but a large-scale study is missing. Such large-scale studies can be carried out on microarrays, and we employ chemical photolithography to synthesize very large combinatorial libraries of fluorescently labeled DNA/RNA chimeric sequences that self-anneal to form hairpin structures that are substrates for Escherichia coli RNase HII. The relative activity is determined by the loss of fluorescence upon cleavage. Each substrate includes a double-stranded 5 bp variable region with one to five consecutive ribonucleotide substitutions. We also examined the effect of all possible single and double mismatches, for a total of >9500 unique structures. Differences in cleavage efficiency indicate some level of substrate preference, and we identified the 5'-dC/rC-rA-dX-3' motif in well-cleaved substrates. The results significantly extend known patterns of RNase HII sequence specificity and serve as a template using large-scale photolithographic synthesis to comprehensively map landscapes of substrate specificity of nucleic acid-processing enzymes.

Template-Directed Nonenzymatic Primer Extension Using 2-Methylimidazole-Activated Morpholino Derivatives of Guanosine and Cytidine.

Efforts to develop self-replicating nucleic acids have led to insights into the origin of life and have also suggested potential pathways to the design of artificial life forms based on non-natural nucleic acids. The template-directed nonenzymatic polymerization of activated ribonucleotide monomers is generally slow because of the relatively weak nucleophilicity of the primer 3'-hydroxyl. To circumvent this problem, several nucleic acids based on amino-sugar nucleotides have been studied, and as expected, the more-nucleophilic amine generally results in faster primer extension. Extending this logic, we have chosen to study morpholino nucleic acid (MoNA), because the secondary amine of the morpholino-nucleotides is expected to be highly nucleophilic. We describe the synthesis of 2-methylimidazole-activated MoNA monomers from their corresponding ribonucleoside 5'-monophosphates and the synthesis of an RNA primer with a terminal MoNA nucleotide. We show that the activated G and C MoNA monomers enable rapid and efficient extension of the morpholino-terminated primer on homopolymeric and mixed-sequenced RNA templates. Our results show that MoNA is a non-natural informational polymer that is worthy of further study as a candidate self-replicating material.

Bioinspired Design of Lysolytic Triterpenoid-Peptide Conjugates that Kill African Trypanosomes.

Humans have evolved a natural immunity against Trypanosoma brucei infections, which is executed by two serum (lipo)protein complexes known as trypanolytic factors (TLF). The active TLF ingredient is the primate-specific apolipoprotein L1 (APOL1). The protein has a pore-forming activity that kills parasites by lysosomal and mitochondrial membrane fenestration. Of the many trypanosome subspecies, only two are able to counteract the activity of APOL1; this illustrates its evolutionarily optimized design and trypanocidal potency. Herein, we ask whether a synthetic (syn) TLF can be synthesized by using the design principles of the natural TLF complexes but with different chemical building blocks. We demonstrate the stepwise development of triterpenoid-peptide conjugates, in which the triterpenoids act as a cell-binding, uptake and lysosomal-transport modules and the synthetic peptide GALA acts as a pH-sensitive, pore-forming lysolytic toxin. As designed, the conjugate kills infective-stage African trypanosomes through lysosomal lysis thus demonstrating a proof-of-principle for the bioinspired, forward-design of a synTLF.

Introduction of Para-Hydroxy Benzyl Spacer Greatly Expands the Utility of Ortho-Hydroxy-Protected Aryl Sulfate System: Application to Nonphenolic Payloads.

The ortho-hydroxy-protected aryl sulfate (OHPAS) linker is composed of a diaryl sulfate backbone equipped with a latent phenol moiety at the ortho position of one of the aryl units. The Ar-OH released when the ortho phenol undergoes intramolecular cyclization and displaces the second aryl unit can be viewed as a payload. We have shown in the preceding paper that the OHPAS linkers are highly stable chemically and in various plasmas, yet release payloads when exposed to suitable triggering conditions. As an extension of the OHPAS system, we employed a para-hydroxy benzyl (PHB) spacer for coupling to nonphenolic payloads; this tactic again provided a highly stable system capable of smooth release of appended payloads. The PHB modification works beautifully for tertiary amine and N-heterocycle payloads.

Studying sparsely populated conformational states in RNA combining chemical synthesis and solution NMR spectroscopy.

Using chemical synthesis and solution NMR spectroscopy, RNA structural ensembles including a major ground state and minor populated excited states can be studied at atomic resolution. In this work, atom-specific 13C labeled RNA building blocks - a 5-13C-uridine and a 2,8-13C2-adenosine building block - are used to introduce isolated 13C-1H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5-13C-uridine 2'-O-TBDMS-phosphoramidite building block was introduced into a 21 nucleotide (nt) tP5c stem construct of the tP5abc subdomain of the Tetrahymena group I ribozyme. Then, the 2,8-13C2-adenosine 2'-O-TBDMS-phosphoramidite building block was incorporated into a 9 kDa and a 15 kD construct derived from the epsilon (ε) RNA element of the duck Hepatitis B virus. The 2,8-13C2-adenosine resonances of the 9 kDa 28 nt sequence could be mapped to the full-length 53 nt construct. The isolated NMR active nuclei pairs were used to probe for low populated excited states (<10%) via 13C-Carr-Purcell-Meiboom-Gill (CPMG)-relaxation dispersion NMR spectroscopy. The 13C-CPMG relaxation dispersion experiment recapitulated a secondary structure switching event in the P5c hairpin of the group I intron construct previously revealed by 15N relaxation dispersion experiments. In the ε-HBV RNA an unfolding event occurring on the millisecond time scale was found in the upper stem in-line with earlier observations. This unpaired conformational state is presumed to be important for the binding of the epsilon reverse transcriptase (RT) enzyme. Thus, a full description of an RNA's folding landscape helps to obtain a deeper understanding of its function, as these high energy conformational states often represent functionally important intermediates involved in (un)folding or ribozyme catalysis.