A plausible process for non-enzymatic RNA replication would greatly simplify models of the transition from prebiotic chemistry to simple biology. However, all known conditions for the chemical copying of an RNA template result in the synthesis of a complementary strand containing a mixture of 2′-5′ and 3′-5′ linkages, rather than the selective synthesis of only 3′-5′ linkages as found in contemporary RNA. Here we show that such backbone heterogeneity is compatible with RNA folding into defined three-dimensional structures that retain molecular recognition and catalytic properties and, therefore, would not prevent the evolution of functional RNAs such as ribozymes. Moreover, the same backbone heterogeneity lowers the melting temperature of RNA duplexes that would otherwise be too stable for thermal strand separation. By allowing copied strands to dissociate, this heterogeneity may have been one of the essential features that allowed RNA to emerge as the first biopolymer.The ability of RNA molecules to fold into defined three-dimensional structures with exquisitely specific molecular recognition and catalytic properties is the conceptual basis of the RNA World hypothesis, an early stage in the evolution of life in which RNA served not only as the polymer of inheritance, but as the central functional polymer of biochemistry [1][2][3] . This model is most strikingly supported by the observation that all modern proteins are synthesized by the peptidyl transferase ribozyme at the heart of the ribosome 4,5 . With the RNA World hypothesis so strongly supported by this and other evidence 1 , the central question in the origin of life field concerns the pathway from the prebiotic chemistry of the early Earth to the emergence of simple forms of cellular life containing RNA genomes coding for RNA enzymes. While there has been considerable recent progress towards the elucidation of potentially prebiotic pathways for ribonucleotide synthesis 6-8 and the assembly of activated nucleotides into oligonucleotides 9,10 , the non-enzymatic replication of RNA oligonucleotides remains problematic. A series of seemingly intractable difficulties * Correspondence to: szostak@molbio.mgh.harvard.edu. † Current Address: Department of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street, London, WC1H 0AJ, UK Author ContributionsAll authors contributed to the design of the experiments and to writing the paper. Experiments were conducted by A.E.E. and M.W.P. Competing Financial Interests StatementThe authors declare no competing financial interests.Published as: Nat Chem. 2013 May ; 5(5): 390-394. HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptcontinues to make a robust system for the chemical replication of RNA elusive [11][12][13][14] . These problems include the slow rate, poor fidelity and low regioselectivity of non-enzymatic RNA template copying; in addition, activated substrates typically hydrolyze on the same timescale as polymerization. The importance of the latter point ...

Biopolymers exist within living cells as far-fromequilibrium metastable polymers. Living systems must constantly invest energy for biopolymer synthesis. In the earliest stages of life on Earth, the complex molecular machinery that contemporary life employs for the synthesis and maintenance of polymers did not exist. Thus, a major question regarding the origin of life is how the first far-from-equilibrium polymers emerged from a prebiotic "pool" of monomers. Here, we describe a proof-of-principle system, in which L-malic acid monomers form far-from-equilibrium, metastable oligoesters via repeated, cyclic changes in hydration and temperature. Such cycles would have been associated with day−night and/or seasonal cycles on the early Earth. In our model system, sample heating, which promotes water evaporation and ester bond formation, drives polymerization. Even though periodic sample rehydration and heating in the hydrated state promotes ester bond hydrolysis, successive iterations of wet−dry cycles result in polymer yields and molecular weight distributions in excess of that observed after a single drying cycle. We term this phenomenon a "polymerization ratchet". We have quantitatively characterized the "ratchet" of our particular system. Ester bond formation rates and oligoester hydrolysis rates were determined for temperatures ranging from 60 to 95°C. Based on these rates, a mathematical model was developed using polycondensation kinetics, from which conditions were predicted for oligoester growth. This model was verified experimentally by the demonstration that L-malic acid monomers subjected to multiple wet−dry cycles form oligoesters, which reach a steady-state concentration and mean length after several cycles. The concentration of oligoesters that persist between subsequent steady-state cycles depends on the temperature and durations of the dry and wet phases of the cycle. These results provide insights regarding the potential for very simple systems to exhibit features that would have been necessary for initiation of polymer evolution, before the emergence of genomes or enzymes.

Hold the water! Deep‐eutectic solvents (DESs) are nonvolatile media suitable for a wide range of chemical reactions. The discovery that nucleic acids can form duplex, triplex, and G‐quadruplex structures (which in some cases differ from those adopted in aqueous media) in a water‐free DES (see picture) opens the enticing possibility that catalytic nucleic acids and enzyme–nucleic acid complexes could also be used in these solvents.

The emergence of homeostatic mechanisms that enabled maintenance of an intracellular steady-state during growth was critical to the advent of cellular life. Here, we show that concentration-dependent reversible binding of short oligonucleotides, of both specific and random sequence, can modulate ribozyme activity. In both cases, catalysis is inhibited at high concentrations, and dilution activates the ribozyme via inhibitor dissociation, thus maintaining near-constant ribozyme specific activity throughout protocell growth. To mimic the result of RNA synthesis within non-growing protocells, we co-encapsulated high concentrations of ribozyme and oligonucleotides within fatty acid vesicles; ribozyme activity was inhibited. Following vesicle growth, the resulting internal dilution produced ribozyme activation. This simple physical system enables a primitive homeostatic behavior: the maintenance of constant ribozyme activity per unit volume during protocell volume changes. We suggest such systems, wherein short oligonucleotides reversibly inhibit functional RNAs, could have preceded sophisticated modern RNA regulatory mechanisms, such as those involving miRNAs.

The mixture of 2′-5′ and 3′-5′ linkages generated during the nonenzymatic replication of RNA has long been regarded as a central problem for the origin of the RNA world. However, we recently observed that both a ribozyme and an RNA aptamer retain considerable functionality in the presence of prebiotically plausible levels of linkage heterogeneity. To better understand the RNA structure and function in the presence of backbone linkage heterogeneity, we obtained high-resolution X-ray crystal structures of a native 10-mer RNA duplex (1.32 Å) and two variants: one containing one 2′-5′ linkage per strand (1.55 Å) and one containing three such linkages per strand (1.20 Å). We found that RNA duplexes adjust their local structures to accommodate the perturbation caused by 2′-5′ linkages, with the flanking nucleotides buffering the disruptive effects of the isomeric linkage and resulting in a minimally altered global structure. Although most 2′-linked sugars were in the expected 2′-endo conformation, some were partially or fully in the 3′-endo conformation, suggesting that the energy difference between these conformations was relatively small. Our structural and molecular dynamic studies also provide insight into the diminished thermal and chemical stability of the duplex state associated with the presence of 2′-5′ linkages. Our results contribute to the view that a low level of 2′-5′ substitution would not have been fatal in an early RNA world and may in contrast have been helpful for both the emergence of nonenzymatic RNA replication and the early evolution of functional RNAs.origin of life | backbone heterogeneity | X-ray crystallography T he capacity of RNA to act as both a carrier of genetic information and as a catalyst has led many to investigate its potential role as the first biopolymer (1-4). An early stage involving nonenzymatic replication simplifies RNA-first scenarios, but known nonenzymatic copying reactions generate a mixture of 3′-5′ and 2′-5′ backbone linkages because of the similar nucleophilicity and orientation of the 2′ and 3′ hydroxyl groups on ribose (Fig. 1). Although regioselectivity for the 3′-5′ linkage can be improved by using different metal ions or activated monomers, it reaches, at most, ∼90% (5-11). This lack of regiospecificity has been regarded as a central problem for the emergence of the RNA world, because the resulting backbone heterogeneity was expected to disrupt the folding, molecular recognition, and catalytic properties of functional RNAs. However, we recently observed that functional nucleic acid molecules can still evolve in the presence of nonheritable mixed DNA/RNA backbone heterogeneity (12), and known functional RNAs retain catalytic and ligand binding behavior in the presence of 2′-5′/3′-5′ backbone linkage heterogeneity (13).The well-known duplex-destabilizing property of 2′-5′ linkages can enable thermal strand separation of long RNA duplexes in the presence of the high Mg 2+ concentrations required for known prebiotic copying reactions (13-16). However, the mechanism r...

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, poses grave threats to both the global economy and health. The predominant diagnostic screens in use for SARS-CoV-2 detection are molecular techniques such as nucleic acid amplification tests. In this Review, we compare current and emerging isothermal diagnostic methods for COVID-19. We outline the molecular and serological techniques currently being used to detect SARS-CoV-2 infection, past or present, in patients. We also discuss ongoing research on isothermal techniques, CRISPR-mediated detection assays, and point-of-care diagnostics that have potential for use in SARS-CoV-2 detection. Large-scale viral testing during a global pandemic presents unique challenges, chief among them the simultaneous need for testing supplies, durable equipment, and personnel in many regions worldwide, with each of these regions possessing testing needs that vary as the pandemic progresses. The low-cost isothermal technologies described in this Review provide a promising means by which to address these needs and meet the global need for testing of symptomatic individuals as well as provide a possible means for routine testing of asymptomatic individuals, providing a potential means of safely enabling reopenings and early monitoring of outbreaks.

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We found that several major chromosomal fragile sites in human lymphomas, including the bcl-2 major breakpoint region, bcl-1 major translocation cluster, and c-Myc exon 1-intron 1 boundary, contain distinctive sequences of consecutive cytosines exhibiting a high degree of reactivity with the structure-specific chemical probe bisulfite. To assess the inherent structural variability of duplex DNA in these regions and to determine the range of structures reactive to bisulfite, we have performed bisulfite probing on genomic DNA in vitro and in situ; on duplex DNA in supercoiled and linearized plasmids; and on oligonucleotide DNA/DNA and DNA/2-O-methyl RNA duplexes. Bisulfite is significantly more reactive at the frayed ends of DNA duplexes, which is expected given that bisulfite is an established probe of single-stranded DNA. We observed that bisulfite also distinguishes between more subtle sequence/structural differences in duplex DNA. Supercoiled plasmids are more reactive than linear DNA; and sequences containing consecutive cytosines, namely GGGCCC, are more reactive than those with alternating guanine and cytosine, namely GCGCGC. Circular dichroism and x-ray crystallography show that the GGGCCC sequence forms an intermediate B/A structure. Molecular dynamics simulations also predict an intermediate B/A structure for this sequence, and probe calculations suggest greater bisulfite accessibility of cytosine bases in the intermediate B/A structure over canonical B-or A-form DNA. Electrostatic calculations reveal that consecutive cytosine bases create electropositive patches in the major groove, predicting enhanced localization of the bisulfite anion at homo-C tracts over alternating G/C sequences. These characteristics of homo-C tracts in duplex DNA may be associated with DNA-protein interactions in vivo that predispose certain genomic regions to chromosomal fragility.The sequence-specific structural variations of the dsDNA 5 helix are well documented (1-4). For example, DNA sequences with consecutive A (or T) bases can adopt a variation of the B-form helix with a narrower minor groove, whereas sequences with consecutive G (or C) bases are more prone to adopt the A-form helix under dehydrating conditions (5). The A-form helix observed in RNA and DNA-RNA hybrid duplexes is present during transcription and replication and sometimes when DNA is bound to proteins (6 -10). Most reported DNA crystal structures exhibit either the B-form or A-form helical structure, with a small fraction exhibiting an intermediate structure.A strong correlation exists between the propensity of a given DNA sequence to adopt a non-B-form structure and the interaction of solvent (water and ions) with the major and minor grooves (5), but neither the natural range of DNA conformations under physiologic solution-phase conditions nor how subtle variations in DNA structure may influence the activity of DNA binding proteins is well understood.The structure of a particular sequence of dsDNA is often inferred from its pattern of reactivity with struc...