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.

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.

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.

Amino Acid-Specific, Ribonucleotide-Promoted Peptide Formation in the Absence of Enzymes.

Nucleic acids and polypeptides are at the heart of life. It is interesting to ask whether the monomers of these biopolymers possess intrinsic reactivity that favors oligomerization in the absence of enzymes. We have recently observed that covalently linked peptido RNA chains form when mixtures of monomers react in salt-rich condensation buffer. Here, we report the results of a screen of the 20 proteinogenic amino acids and four ribonucleotides. None of the amino acids prevent phosphodiester formation, so all of them are compatible with genetic encoding through RNA chain growth. A reactivity landscape was found, in which peptide formation strongly depends on the structure of the amino acid, but less on the nucleobase. For example, proline gives ribonucleotide-bound peptides most readily, tyrosine favors pyrophosphate and phosphodiester formation, and histidine gives phosphorimidazolides as dominant products. When proline and aspartic acid were allowed to compete for incorporation, only proline was found at the N-terminus of peptido chains. The reactivity described here links two fundamental classes of biomolecules through reactions that occur without enzymes, but with amino acid specificity.

Ribonucleotides and RNA Promote Peptide Chain Growth.

All known forms of life use RNA-mediated polypeptide synthesis to produce the proteins encoded in their genes. Because the principal parts of the translational machinery consist of RNA, it is likely that peptide synthesis was achieved early in the prebiotic evolution of an RNA-dominated molecular world. How RNA attracted amino acids and then induced peptide formation in the absence of enzymes has been unclear. Herein, we show that covalent capture of an amino acid as a phosphoramidate favors peptide formation. Peptide coupling is a robust process that occurs with different condensation agents. Kinetics show that covalent capture can accelerate chain growth over oligomerization of the free amino acid by at least one order of magnitude, so that there is no need for enzymatic catalysis for peptide synthesis to begin. Peptide chain growth was also observed on phosphate-terminated RNA strands. Peptide coupling promoted by ribonucleotides or ribonucleotide residues may have been an important transitional form of peptide synthesis that set in when amino acids were first captured by RNA.

The effect of leaving groups on binding and reactivity in enzyme-free copying of DNA and RNA.

The template-directed incorporation of nucleotides at the terminus of a growing primer is the basis of the transmission of genetic information. Nature uses polymerases-catalyzed reactions, but enzyme-free versions exist that employ nucleotides with organic leaving groups. The leaving group affects yields, but it was not clear whether inefficient extensions are due to poor binding, low reactivity toward the primer, or rapid hydrolysis. We have measured the binding of a total of 15 different activated nucleotides to DNA or RNA sequences. Further, we determined rate constants for the chemical step of primer extension involving methylimidazolides or oxyazabenzotriazolides of deoxynucleotides or ribonucleotides. Binding constants range from 10 to >500 mM and rate constants from 0.1 to 370 M(-1) h(-1) For aminoterminal primers, a fast covalent step and slow hydrolysis are the main factors leading to high yields. For monomers with weakly pairing bases, the leaving group can improve binding significantly. A detailed mechanistic picture emerges that explains why some enzyme-free primer extensions occur in high yield, while others remain recalcitrant to copying without enzymatic catalysis. A combination of tight binding and rapid extension, coupled with slow hydrolysis induces efficient enzyme-free copying.

The strength of the template effect attracting nucleotides to naked DNA.

The transmission of genetic information relies on Watson-Crick base pairing between nucleoside phosphates and template bases in template-primer complexes. Enzyme-free primer extension is the purest form of the transmission process, without any chaperon-like effect of polymerases. This simple form of copying of sequences is intimately linked to the origin of life and provides new opportunities for reading genetic information. Here, we report the dissociation constants for complexes between (deoxy)nucleotides and template-primer complexes, as determined by nuclear magnetic resonance and the inhibitory effect of unactivated nucleotides on enzyme-free primer extension. Depending on the sequence context, Kd's range from 280 mM for thymidine monophosphate binding to a terminal adenine of a hairpin to 2 mM for a deoxyguanosine monophosphate binding in the interior of a sequence with a neighboring strand. Combined with rate constants for the chemical step of extension and hydrolytic inactivation, our quantitative theory explains why some enzyme-free copying reactions are incomplete while others are not. For example, for GMP binding to ribonucleic acid, inhibition is a significant factor in low-yielding reactions, whereas for amino-terminal DNA hydrolysis of monomers is critical. Our results thus provide a quantitative basis for enzyme-free copying.

Cascade of reduced speed and accuracy after errors in enzyme-free copying of nucleic acid sequences.

Nonenzymatic, template-directed synthesis of nucleic acids is a paradigm for self-replicating systems. The evolutionary dynamics of such systems depend on several factors, including the mutation rates, relative replication rates, and sequence characteristics of mutant sequences. We measured the kinetics of correct and incorrect monomer insertion downstream of a primer-template mismatch (mutation), using a range of backbone structures (RNA, DNA, and LNA templates and RNA and DNA primers) and two types of 5'-activated nucleotides (oxyazabenzotriazolides and imidazolides, i.e., nucleoside 5'-phosphorimidazolides). Our study indicated that for all systems studied, an initial mismatch was likely to be followed by another error (54-75% of the time), and extension after a single mismatch was generally 10-100 times slower than extension without errors. If the mismatch was followed by a matched base pair, the extension rate recovered to nearly normal levels. On the basis of these data, we simulated nucleic acid replication in silico, which indicated that a primer suffering an initial error would lag behind properly extended counterparts due to a cascade of subsequent errors and kinetic stalling, with the typical mutational event consisting of several consecutive errors. Our study also included different sequence contexts, which suggest the presence of cooperativity among monomers affecting both absolute rate (by up to 2 orders of magnitude) and fidelity. The results suggest that molecular evolution in enzyme-free replication systems would be characterized by large "leaps" through sequence space rather than isolated point mutations, perhaps enabling rapid exploration of diverse sequences. The findings may also be useful for designing self-replicating systems combining high fidelity with evolvability.

Templating efficiency of naked DNA.

Template-directed synthesis of complementary strands is pivotal for life. Nature employs polymerases for this reaction, leaving the ability of DNA itself to direct the incorporation of individual nucleotides at the end of a growing primer difficult to assess. Using 64 sequences, we now find that any of the four nucleobases, in combination with any neighboring residue, support enzyme-free primer extension when primer and mononucleotide are sufficiently reactive, with >or=93% primer extension for all sequences. Between the 64 possible base triplets, the rate of extension for the poorest template, CAG, with A as templating base, and the most efficient template, TCT, with C as templating base, differs by less than two orders of magnitude. Further, primer extension with a balanced mixture of monomers shows >or=72% of the correct extension product in all cases, and >or=90% incorporation of the correct base for 46 out of 64 triplets in the presence of a downstream-binding strand. A mechanism is proposed with a binding equilibrium for the monomer, deprotonation of the primer, and two chemical steps, the first of which is most strongly modulated by the sequence. Overall, rates show a surprisingly smooth reactivity landscape, with similar incorporation on strongly and weakly templating sequences. These results help to clarify the substrate contribution to copying, as found in polymerase-catalyzed replication, and show an important feature of DNA as genetic material.

Chemical primer extension: individual steps of spontaneous replication.

The replication of genetic information, as we know it from today's biology, relies on template-directed, polymerase-catalyzed extension of primers. It is known that short stretches of complementary RNA can form on templates in the absence of enzymes. This account summarizes recent work on efficient enzyme-free primer extension, both with 3'-amino-terminal deoxyribonucleotide primers and with primers made of unmodified RNA. Near-quantitative primer extension with half-life times on the order of hours has been demonstrated by using azaoxybenzotriazolides of nucleotides and downstream-binding oligomers. Further, small non-nucleosidic substituents placed on the terminus of the template or the downstream-binding oligomer have been shown to increase the rate and fidelity of primer-extension reactions. Since all four templating bases (A, C, G, T/U) direct sequence-selective primer-extension steps, we feel that there is renewed hope that full, nonenzymatic replication from monomers may eventually be achieved.

Structural and functional comparison of HemN to other radical SAM enzymes.

Radical SAM enzymes have only recently been recognized as an ancient family sharing an unusual radical-based reaction mechanism. This late appreciation is due to the extreme oxygen sensitivity of most radical SAM enzymes, making their characterization particularly arduous. Nevertheless, realization that the novel apposition of the established cofactors S-adenosylmethionine and [4Fe-4S] cluster creates an explosive source of catalytic radicals, the appreciation of the sheer size of this previously neglected family, and the rapid succession of three successfully solved crystal structures within a year have ensured that this family has belatedly been noted. In this review, we report the characterization of two enzymes: the established radical SAM enzyme, HemN or oxygen-independent coproporphyrinogen III oxidase from Escherichia coli, and littorine mutase, a presumed radical SAM enzyme, responsible for the conversion of littorine to hyoscyamine in plants. The enzymes are compared to other radical SAM enzymes and in particular the three reported crystal structures from this family, HemN, biotin synthase and MoaA, are discussed.