Ribosome-Free Translation up to Pentapeptides via Template Walk on RNA Sequences.

The origin of translation is one of the most difficult problems of molecular evolution. Identifying molecular systems that translate an RNA sequence into a peptide sequence in the absence of ribosomes and enzymes is a challenge. Recently, single-nucleotide translation via coupling of 5' phosphoramidate-linked amino acids to 2'/3'-aminoacyl transfer-NMPs, as directed by the sequence of an RNA template, was demonstrated for three of the four canonical nucleotides. How single-nucleotide translation could be expanded to include all four bases and to produce longer peptides without translocation along the template strand remained unclear. Using transfer strands of increasing length containing any of the four bases that interrogate adjacent positions along the template, we now show that pentapeptides can be produced in coupling reactions and subsequent hydrolytic release in situ. With 2'/3'-aminoacylated mono-, di-, tri- and tetranucleotides we thus show how efficient translation can be without biomacromolecules.

A Fluorinated Chaperone Gives X-ray Crystal Structures of Acyclic Natural Product Derivatives up to 338 Molecular Weight.

Crystallizing molecules with long flexible chains is a challenge, making it difficult to perform X-ray crystallography. Chaperones can assist in the crystallization of compounds that do not crystallize by themselves by producing solvate crystals that contain the analyte in their three-dimensional lattices. Among the most versatile chaperones for liquid analytes are tetraaryladamantanes (TAAs), but the size of the compounds that can be encapsulated is limited, and attempts to surpass this limit with known TAAs were unsuccessful. Here we report that 1,3,5,7-tetrakis(2-fluoro-4-methoxyphenyl)adamantane (TFM) is a crystallization chaperone for acyclic molecules up to the molecular weight of phytyl acetate (338 g/mol). Encapsulation of such a large acyclic compound was achieved when the analyte was esterified and when a two-step temperature protocol was used for crystallization. Exploratory work indicates that a drop to -20 °C allows for encapsulation of squalene (Mr 411 g/mol), albeit with positional disorder of the analyte. Our X-ray crystal structures of solvates with flexible analytes shed light on how crystalline order can be imposed on large acyclic analytes. The new, fluorinated TAA gives access to crystal structures that were inaccessible thus far.

Prolinyl Phosphoramidates of Nucleotides with Increased Reactivity.

Nucleoside monophosphates (NMPs) are the subunits of RNA. They are incorporated into growing complementary strands when sequences are copied in enzyme-free reactions using organic leaving groups at the phosphates. Amino acids are rarely considered as leaving groups, but proline can act as a leaving group when N-linked to NMPs, so that prolinyl NMPs hydrolyze in aqueous buffer at 37 °C, with half-life times as short as 2.4 h, and they act as monomers in enzyme-free primer extension. Still, their level of reactivity is insufficient for practical purposes, requiring months for some extensions. Herein we report the synthesis of eight substituted prolinyl AMPs together with seven related compounds and the results of a study of their reactivity. A δ-carboxy prolinyl NMP was found to be converted with a half-life time of just 11 min in magnesium-free buffer, and a δ-isopropyl prolinyl NMP was shown to react sevenfold faster than its prolinyl counterpart in enzyme-free genetic copying of RNA. Our results indicate that both anchimeric and steric effects can be employed to increase the reactivity of aminoacidyl nucleotides, i.e. compounds that combine two fundamental classes of biomolecules in one functional entity.

A ProTide of AZT Shows Activity Against Human Papillomaviruses.

Infection by human papillomaviruses (HPV) can cause warts and tumors. So far, no small molecule antiviral has been approved for the treatment of infections with this DNA virus, although preclinical studies show activity for nucleosidic compounds, such as 9-(2-phosphonylmethoxy)ethylguanine (PMEG) or cidofovir. This prompted us to test new prodrug versions of the nucleoside analog 3'-azido-2',3'-dideoxythymidine (AZT), known to be active against reverse transcriptases and approved for the treatment of HIV. Here we report the synthesis of an ethylbutyl alaninyl ester phosphosphoramidate prodrug of AZT, dubbed AZAEB, and its activity against HPV, a target not known to be sensitive to AZT. A methyl ester derivative was found to be inactive against this and three other DNA viruses, while the phosphoramidate prodrug AZAEB showed a modest inhibitory effect against HPV types 6, 11, 18 and 31. Our results open up new avenues of study for the treatment of diseases caused by members of the papillomaviridae family.

Prolinyl Nucleotides Drive Enzyme-Free Genetic Copying of RNA.

Proline is one of the proteinogenic amino acids. It is found in all kingdoms of life. It also has remarkable activity as an organocatalyst and is of structural importance in many folded polypeptides. Here, we show that prolinyl nucleotides with a phosphoramidate linkage are active building blocks in enzyme- and ribozyme-free copying of RNA in the presence of monosubstituted imidazoles as organocatalysts. Both dinucleotides and mononucleotides are incorporated at the terminus of RNA primers in aqueous buffer, as instructed by the template sequence, in up to eight consecutive extension steps. Our results show that condensation products of amino acids and ribonucleotides can act like nucleoside triphosphates in media devoid of enzymes or ribozymes. Prolinyl nucleotides are metastable building blocks, readily activated by catalysts, helping to explain why the combination of α-amino acids and nucleic acids was selected in molecular evolution.

Caging of a Strongly Pairing Fluorescent Thymidine Analog with Soft Nucleophiles.

Controlling the pairing strength of nucleobases in DNA through reactions with compounds found inside the cell is a formidable challenge. Here we report how a thiazolyl substituent turns a strongly pairing ethynylpyridone C-nucleoside into a reactive residue in oligonucleotides. The thiazolyl-bearing pyridone reacts with soft nucleophiles, such as glutathione, but not with hard nucleophiles like hydroxide or carbonate. The addition products pair much more weakly with adenine in a complementary strand than the starting material, and also change their fluorescence. This makes oligonucleotides containing the new deoxynucleoside interesting for controlled release. Due to its reactivity toward N, P, S, and Se-nucleophiles, and the visual signal accompanying chemical conversion, the fluorescent nucleotide reported here may also have applications in chemical biology, sensing and diagnostics.

Synthesis of a Peptidoyl RNA Hairpin via a Combination of Solid-Phase and Template-Directed Chain Assembly.

Peptidoyl RNAs are the products of ribosome-free, single-nucleotide translation. They contain a peptide in the backbone of the oligoribonucleotide and are interesting from a synthetic and a bioorganic point of view. A synthesis of a stabilized version of peptidoyl RNA, with an amide bond between the C-terminus of a peptide and a 3'-amino-2',3'-dideoxynucleoside in the RNA chain was developed. The preferred synthetic route used an N-Teoc-protected aminonucleoside support and involved a solution-phase coupling of the amino-terminal oligonucleotide to a dipeptido dinucleotide. Exploratory UV-melting and NMR analysis of the hairpin 5'-UUGGCGAAAGCdC-LeuLeu-AA-3' indicated that the peptide-linked RNA segments do not fold in a cooperative fashion. The synthetic access to doubly RNA-linked peptides on a scale sufficient for structural biology opens the door to the exploration of their structural and biochemical properties.

Enzyme-Free Copying of 12 Bases of RNA with Dinucleotides.

The synthesis of complementary strands is the reaction underlying the replication of genetic information. It is likely that the earliest self-replicating systems used RNA as genetic material. How RNA was copied in the absence of enzymes and what sequences were most likely to have supported replication is not clear. Here we show that mixtures of dinucleotides with C and G as bases copy an RNA sequence of up to 12 nucleotides in dilute aqueous solution. Successful enzyme-free copying occurred with in situ activation at 4 °C and pH 6.0. Dimers were incorporated in favor of monomers when both competed as reactants, and little misincorporation was detectable in mass spectra. Simulations using experimental rate constants confirmed that mixed C/G sequences are good candidates for successful replication with dimers. Because dimers are intermediates in the synthesis of longer strands, our results support evolutionary scenarios encompassing formation and copying of RNA strands in enzyme-free fashion.

Efficient Oligomerization of Aromatic Amino Acids Induced by Gaps in Four-Helix Bundles of DNA or RNA.

The formation of peptides from amino acids is one of the processes associated with life. Because of the dominant role of translation in extant biology, peptide-forming processes that are RNA induced are of particular interest. We have previously reported the formation of phosphoramidate-linked peptido RNAs as the products of spontaneous condensation reactions between ribonucleotides and free amino acids in aqueous solution. We now asked whether four-helix bundle (4HB) DNA or RNA folding motifs with a single- or double-nucleotide gap next to a 5'-phosphate can act as reaction sites for phosphoramidate formation. For glycine, this was found to be the case, whereas phenylalanine and tryptophan showed accelerated formation of peptides without a covalent link to the nucleic acid. Free peptides with up to 11 tryptophan or phenylalanine residues were found in precipitates forming in the presence of gap-containing DNA or RNA 4HBs. Control experiments using motifs with just a nick or primer alone did not have the same effect. Because folded structures with a gap in a double helix are likely products of hybridization of strands formed in statistically controlled oligomerization reactions, our results are interesting in the context of prebiotic scenarios. Independent of a putative role in evolution, our findings suggest that for some aromatic amino acids an RNA-induced pathway for oligomerization exists that does not have a discernable link to translation.

High Fidelity Enzyme-Free Primer Extension with an Ethynylpyridone Thymidine Analog.

High fidelity base pairing is important for the transmission of genetic information. Weak base pairs can lower fidelity, complicating sequencing, amplification and replication of DNA. Thymidine 5'-monophosphate (TMP) is the most weakly pairing nucleotide among the canonical deoxynucleotides, causing high errors rates in enzyme-free primer extension. Here we report the synthesis of an ethynylpyridone C-nucleoside analog of 3'-amino-2',3'-dideoxythymidine monophosphate and its incorporation in a growing strand by enzyme-free primer extension. The ethynylpyridone C-nucleotide accelerates extension more than five-fold, reduces misincorporation and readily displaces TMP in competition experiments. The results bode well for the use of the C-nucleoside as replacements for thymidine in practical applications.

The Effect of Pooling on the Detection of the Nucleocapsid Protein of SARS-CoV-2 with Rapid Antigen Tests.

The COVID-19 pandemic puts significant stress on the viral testing capabilities of many countries. Rapid point-of-care (PoC) antigen tests are valuable tools but implementing frequent large scale testing is costly. We have developed an inexpensive device for pooling swabs, extracting specimens, and detecting viral antigens with a commercial lateral flow test for the nucleocapsid protein of SARS-CoV-2 as antigen. The holder of the device can be produced locally through 3D printing. The extraction and the elution can be performed with the entire set-up encapsulated in a transparent bag, minimizing the risk of infection for the operator. With 0.35 mL extraction buffer and six swabs, including a positive control swab, 43 ± 6% (n = 8) of the signal for an individual extraction of a positive control standard was obtained. Image analysis still showed a signal-to-noise ratio of approximately 2:1 at 32-fold dilution of the extract from a single positive control swab. The relative signal from the test line versus the control line was found to scale linearly upon dilution (R2 = 0.98), indicating that other pooling regimes are conceivable. A pilot project involving 14 participants and 18 pooled tests in a laboratory course at our university did not give any false positives, and an individual case study confirmed the ability to detect a SARS-CoV-2 infection with five-fold or six-fold pooling, including one swab from a PCR-confirmed COVID patient. These findings suggest that pooling can make frequent testing more affordable for schools, universities, and similar institutions, without decreasing sensitivity to an unacceptable level.

Single nucleotide translation without ribosomes.

The translation of messenger RNA sequences into polypeptide sequences according to the genetic code is central to life. How this process, which relies on the ribosomal machinery, arose from much simpler precursors is unclear. Here, we demonstrate that single nucleotides charged with an amino acid couple with amino acids linked to the 5'-terminus of an RNA primer in reactions directed by the nucleotides of an RNA template in dilute aqueous solution at 0 °C. When a mixture of U-Val, A-Gly and G-Leu competed for coupling to Gly-RNA, base pairing dictated which dipeptide sequence formed preferentially. The resulting doubly anchored dipeptides can retain their link to the primer for further extension or can be fully released under mild acidic conditions. These results show that a single-nucleotide-based form of translation exists that requires no more than oligoribonucleotides and anchored amino acids.

Co-crystallization of an organic solid and a tetraaryladamantane at room temperature.

Tetraaryladamantanes have proven useful as chaperones for the co-crystallization of small molecules that do not readily crystallize by themselves. The co-crystals are often useful for structure elucidation. Usually, the small molecules are encapsulated in the crystal lattice of the aryladamantane that forms during rapid thermal crystallization. Thus far, co-crystallization has been limited to liquids as guest molecules. Here we report the co-crystal structures of phenol, which is solid at room temperature, with both 1,3,5,7-tetrakis(2,4-dimethoxyphenyl)adamantane (TDA) and 1,3,5,7-tetrakis(2,4-diethoxyphenyl)adamantane (TEO). The co-crystals were obtained from solutions in dichloromethane by slow evaporation or diffusion. The implications for generating other co-crystals of two solids are briefly discussed.

Determining the Diastereoselectivity of the Formation of Dipeptidonucleotides by NMR Spectroscopy.

Proteins are composed of l-amino acids, but nucleic acids and most oligosaccharides contain d-sugars as building blocks. It is interesting to ask whether this is a coincidence or a consequence of the functional interplay of these biomolecules. One reaction that provides an opportunity to study this interplay is the formation of phosphoramidate-linked peptido RNA from amino acids and ribonucleotides in aqueous condensation buffer. Here we report how the diastereoselectivity of the first peptide coupling of the peptido RNA pathway can be determined in situ by NMR spectroscopy. When a racemic mixture of an amino acid ester was allowed to react with an 5'-aminoacidyl nucleotide, diastereomeric ratios of up to 72 : 28 of the resulting dipeptido nucleotides were found by integration of 31 P- or 1 H-NMR peaks. The highest diastereomeric excess was found for the homochiral coupling product d-Ser-d-Trp, phosphoramidate-linked to adenosine 5'-monophosphate with its d-ribose ring. When control reactions with an N-acetyl amino acid and valine methyl ester were run in organic solvent, the diastereoselectivity was found to be lower, with diastereomeric ratios≤62 : 38. The results from the exploratory study thus indicate that the ribonucleotide residue not only facilitates the coupling of lipophilic amino acids in aqueous medium but also the formation of a homochiral dipeptide. The methodology described here may be used to search for other stereoselective reactions that shed light on the origin of homochirality.

Hybridization Networks of mRNA and Branched RNA Hybrids.

Messenger RNA (mRNA) is emerging as an attractive biopolymer for therapy and vaccination. To become suitable for vaccination, mRNA is usually converted to a biomaterial, using cationic peptides, polymers or lipids. An alternative form of converting mRNA into a material is demonstrated that uses branched oligoribonucleotide hybrids with the ability to hybridize with one or more regions of the mRNA sequence. Two such hybrids with hexamer arms and adamantane tetraol as branching element were prepared by solution-phase synthesis. When a rabies mRNA was treated with the branched hybrids at 1 M NaCl concentration, biomaterials formed that contained both of the nucleic acids. These results show that branched oligoribonucleotides are an alternative to the often toxic reagents commonly used to formulate mRNA for medical applications.

An AZT Analog with Strongly Pairing Ethynylpyridone Nucleobase and Its Antiviral Activity against HSV1.

Challenges resulting from novel viruses or new strains of known viruses call for new antiviral agents. Nucleoside analogs that act as inhibitors of viral polymerases are an attractive class of antivirals. For nucleosides containing thymine, base pairing is weak, making it desirable to identify nucleobase analogs that pair more strongly with adenine, in order to compete successfully with the natural substrate. We have recently described a new class of strongly binding thymidine analogs that contain an ethynylmethylpyridone as base and a C-nucleosidic linkage to the deoxyribose. Here we report the synthesis of the 3'-azido-2',3'-deoxyribose derivative of this compound, dubbed AZW, both as free nucleoside and as ProTide phosphoramidate. As a proof of principle, we studied the activity against Herpes simplex virus type 1 (HSV1). Whereas the ProTide phosphoramidate suffered from low solubility, the free nucleoside showed a stronger inhibitory effect than that of AZT in a plaque reduction assay. This suggests that strongly pairing C-nucleoside analogs of pyrimidines have the potential to become active pharmaceutical ingredients with antiviral activity.

The Enzyme-Free Release of Nucleotides from Phosphoramidates Depends Strongly on the Amino Acid.

Phosphoramidates composed of an amino acid and a nucleotide analogue are critical metabolites of prodrugs, such as remdesivir. Hydrolysis of the phosphoramidate liberates the nucleotide, which can then be phosphorylated to become the pharmacologically active triphosphate. Enzymatic hydrolysis has been demonstrated, but a spontaneous chemical process may also occur. We measured the rate of enzyme-free hydrolysis for 17 phosphoramidates of ribonucleotides with amino acids or related compounds at pH 7.5. Phosphoramidates of proline hydrolyzed fast, with a half-life time as short as 2.4 h for Pro-AMP in ethylimidazole-containing buffer at 37 °C; 45-fold faster than Ala-AMP and 120-fold faster than Phe-AMP. Crystal structures of Gly-AMP, Pro-AMP, ßPro-AMP and Phe-AMP bound to RNase A as crystallization chaperone showed how well the carboxylate is poised to attack the phosphoramidate, helping to explain this reactivity. Our results are significant for the design of new antiviral prodrugs.

Absolute Configuration of Small Molecules by Co-Crystallization.

The most reliable method to determine the absolute configuration of chiral molecules is X-ray crystallography, but small molecules can be difficult to crystallize. We report rapid co-crystallization of tetraaryladamantanes with small molecules as different as n-decane to nicotine to produce crystals for X-ray analysis and the assignment of absolute configuration when the molecules are chiral. A screen of 52 diverse compounds gave inclusion in co-crystals for 88 % of all cases and a high-resolution structure in 77 % of cases. Furthermore, starting from three milligrams of analyte, a combination of NMR spectroscopy and X-ray crystallography produced a full structure in less than three days using an adamantane crystallization chaperone that encapsulates the analyte at room temperature.

Thoughts on What Chemists Can Contribute to Fighting SARS-CoV-2 - A Short Note on Hand Sanitizers, Drug Candidates and Outreach.

The SARS-CoV-2 outbreak causing the respiratory disease COVID-19 has left many chemists in academia without an obvious option to contribute to fighting the pandemic. Some of our recent experiences indicate that there are ways to overcome this dilemma. A three-pronged approach is proposed.

2'/3' Regioselectivity of Enzyme-Free Copying of RNA Detected by NMR.

The RNA-templated extension of oligoribonucleotides by nucleotides produces either a 3',5' or a 2',5'-phosphodiester. Nature controls the regioselectivity during RNA chain growth with polymerases, but enzyme-free versions of genetic copying have modest specificity. Thus far, enzymatic degradation of products, combined with chromatography or electrophoresis, has been the preferred mode of detecting 2',5'-diesters produced in enzyme-free reactions. This approach hinges on the substrate specificity of nucleases, and is not suitable for in situ monitoring. Here we report how 1 H NMR spectroscopy can be used to detect the extension of self-templating RNA hairpins and that this reveals the regioisomeric nature of the newly formed phosphodiesters. We studied several modes of activating nucleotides, including imidazolides, a pyridinium phosphate, an active ester, and in situ activation with carbodiimide and organocatalyst. Conversion into the desired extension product ranged from 20 to 90 %, depending on the leaving group. Integration of the resonances of H1' protons of riboses and H5 protons of pyrimidines gave regioselectivities ranging from 40:60 to 85:15 (3',5' to 2',5' diester), but no simple correlation between 3',5' selectivity and yield. Our results show how monitoring with a high-resolution technique sheds a new light on a process that may have played an important role during the emergence of life.