Cryo-EM structure and functional landscape of an RNA polymerase ribozyme.

The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) - including a variant that uses trinucleotide triphosphates (triplets) as substrates - have been created by in vitro evolution and are the closest functional analogues of the replicase, but the structural basis for their function is poorly understood. Here we use single-particle cryogenic electron microscopy (cryo-EM) and high-throughput mutation analysis to obtain the structure of a triplet polymerase ribozyme (TPR) apoenzyme and map its functional landscape. The cryo-EM structure at 5-Å resolution reveals the TPR as an RNA heterodimer comprising a catalytic subunit and a noncatalytic, auxiliary subunit, resembling the shape of a left hand with thumb and fingers at a 70° angle. The two subunits are connected by two distinct kissing-loop (KL) interactions that are essential for polymerase function. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme, whereby heterodimer formation and KL interactions preorganize the TPR for optimal primer-template duplex binding, triplet substrate discrimination, and templated RNA synthesis. These results provide a better understanding of TPR structure and function and should aid the engineering of more efficient PRs.

Characterization of an HNA aptamer suggests a non-canonical G-quadruplex motif.

Nucleic acids not only form the basis of heredity, but are increasingly a source of novel nano-structures, -devices and drugs. This has spurred the development of chemically modified alternatives (xeno nucleic acids (XNAs)) comprising chemical configurations not found in nature to extend their chemical and functional scope. XNAs can be evolved into ligands (XNA aptamers) that bind their targets with high affinity and specificity. However, detailed investigations into structural and functional aspects of XNA aptamers have been limited. Here we describe a detailed structure-function analysis of LYS-S8-19, a 1',5'-anhydrohexitol nucleic acid (HNA) aptamer to hen egg-white lysozyme (HEL). Mapping of the aptamer interaction interface with its cognate HEL target antigen revealed interaction epitopes, affinities, kinetics and hot-spots of binding energy similar to protein ligands such as anti-HEL-nanobodies. Truncation analysis and molecular dynamics (MD) simulations suggest that the HNA aptamer core motif folds into a novel and not previously observed HNA tertiary structure, comprising non-canonical hT-hA-hT/hT-hT-hT triplet and hG4-quadruplex structures, consistent with its recognition by two different G4-specific antibodies.

Direct Mapping of Higher-Order RNA Interactions by SHAPE-JuMP.

Higher-order structure governs function for many RNAs. However, discerning this structure for large RNA molecules in solution is an unresolved challenge. Here, we present SHAPE-JuMP (selective 2'-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) to interrogate through-space RNA tertiary interactions. A bifunctional small molecule is used to chemically link proximal nucleotides in an RNA structure. The RNA cross-link site is then encoded into complementary DNA (cDNA) in a single, direct step using an engineered reverse transcriptase that "jumps" across cross-linked nucleotides. The resulting cDNAs contain a deletion relative to the native RNA sequence, which can be detected by sequencing, that indicates the sites of cross-linked nucleotides. SHAPE-JuMP measures RNA tertiary structure proximity concisely across large RNA molecules at nanometer resolution. SHAPE-JuMP is especially effective at measuring interactions in multihelix junctions and loop-to-helix packing, enables modeling of the global fold for RNAs up to several hundred nucleotides in length, facilitates ranking of structural models by consistency with through-space restraints, and is poised to enable solution-phase structural interrogation and modeling of complex RNAs.

Catalysts from synthetic genetic polymers.

The emergence of catalysis in early genetic polymers such as RNA is considered a key transition in the origin of life, pre-dating the appearance of protein enzymes. DNA also demonstrates the capacity to fold into three-dimensional structures and form catalysts in vitro. However, to what degree these natural biopolymers comprise functionally privileged chemical scaffolds for folding or the evolution of catalysis is not known. The ability of synthetic genetic polymers (XNAs) with alternative backbone chemistries not found in nature to fold into defined structures and bind ligands raises the possibility that these too might be capable of forming catalysts (XNAzymes). Here we report the discovery of such XNAzymes, elaborated in four different chemistries (arabino nucleic acids, ANA; 2'-fluoroarabino nucleic acids, FANA; hexitol nucleic acids, HNA; and cyclohexene nucleic acids, CeNA) directly from random XNA oligomer pools, exhibiting in trans RNA endonuclease and ligase activities. We also describe an XNA-XNA ligase metalloenzyme in the FANA framework, establishing catalysis in an entirely synthetic system and enabling the synthesis of FANA oligomers and an active RNA endonuclease FANAzyme from its constituent parts. These results extend catalysis beyond biopolymers and establish technologies for the discovery of catalysts in a wide range of polymer scaffolds not found in nature. Evolution of catalysis independent of any natural polymer has implications for the definition of chemical boundary conditions for the emergence of life on Earth and elsewhere in the Universe.

Modified nucleic acids: replication, evolution, and next-generation therapeutics.

Modified nucleic acids, also called xeno nucleic acids (XNAs), offer a variety of advantages for biotechnological applications and address some of the limitations of first-generation nucleic acid therapeutics. Indeed, several therapeutics based on modified nucleic acids have recently been approved and many more are under clinical evaluation. XNAs can provide increased biostability and furthermore are now increasingly amenable to in vitro evolution, accelerating lead discovery. Here, we review the most recent discoveries in this dynamic field with a focus on progress in the enzymatic replication and functional exploration of XNAs.

Nucleic acids: function and potential for abiogenesis.

The emergence of functional cooperation between the three main classes of biomolecules - nucleic acids, peptides and lipids - defines life at the molecular level. However, how such mutually interdependent molecular systems emerged from prebiotic chemistry remains a mystery. A key hypothesis, formulated by Crick, Orgel and Woese over 40 year ago, posits that early life must have been simpler. Specifically, it proposed that an early primordial biology lacked proteins and DNA but instead relied on RNA as the key biopolymer responsible not just for genetic information storage and propagation, but also for catalysis, i.e. metabolism. Indeed, there is compelling evidence for such an 'RNA world', notably in the structure of the ribosome as a likely molecular fossil from that time. Nevertheless, one might justifiably ask whether RNA alone would be up to the task. From a purely chemical perspective, RNA is a molecule of rather uniform composition with all four bases comprising organic heterocycles of similar size and comparable polarity and pK a values. Thus, RNA molecules cover a much narrower range of steric, electronic and physicochemical properties than, e.g. the 20 amino acid side-chains of proteins. Herein we will examine the functional potential of RNA (and other nucleic acids) with respect to self-replication, catalysis and assembly into simple protocellular entities.

Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering.

Nucleic acids are a distinct form of sequence-defined biopolymer. What sets them apart from other biopolymers such as polypeptides or polysaccharides is their unique capacity to encode, store, and propagate genetic information (molecular heredity). In nature, just two closely related nucleic acids, DNA and RNA, function as repositories and carriers of genetic information. They therefore are the molecular embodiment of biological information. This naturally leads to questions regarding the degree of variation from this seemingly ideal "Goldilocks" chemistry that would still be compatible with the fundamental property of molecular heredity. To address this question, chemists have created a panoply of synthetic nucleic acids comprising unnatural sugar ring congeners, backbone linkages, and nucleobases in order to establish the molecular parameters for encoding genetic information and its emergence at the origin of life. A deeper analysis of the potential of these synthetic genetic polymers for molecular heredity requires a means of replication and a determination of the fidelity of information transfer. While non-enzymatic synthesis is an increasingly powerful method, it currently remains restricted to short polymers. Here we discuss efforts toward establishing enzymatic synthesis, replication, and evolution of synthetic genetic polymers through the engineering of polymerase enzymes found in nature. To endow natural polymerases with the ability to efficiently utilize non-cognate nucleotide substrates, novel strategies for the screening and directed evolution of polymerase function have been realized. High throughput plate-based screens, phage display, and water-in-oil emulsion technology based methods have yielded a number of engineered polymerases, some of which can synthesize and reverse transcribe synthetic genetic polymers with good efficiency and fidelity. The inception of such polymerases demonstrates that, at a basic level at least, molecular heredity is not restricted to the natural nucleic acids DNA and RNA, but may be found in a large (if finite) number of synthetic genetic polymers. And it has opened up these novel sequence spaces for investigation. Although largely unexplored, first tentative forays have yielded ligands (aptamers) against a range of targets and several catalysts elaborated in a range of different chemistries. Finally, taking the lead from established DNA designs, simple polyhedron nanostructures have been described. We anticipate that further progress in this area will expand the range of synthetic genetic polymers that can be synthesized, replicated, and evolved providing access to a rich sequence, structure, and phenotypic space. "Synthetic genetics", that is, the exploration of these spaces, will illuminate the chemical parameter range for en- and decoding information, 3D folding, and catalysis and yield novel ligands, catalysts, and nanostructures and devices for applications in biotechnology and medicine.

A synthetic approach to abiogenesis.

Synthetic biology seeks to probe fundamental aspects of biological form and function by construction (resynthesis) rather than deconstruction (analysis). Here we discuss how such an approach could be applied to assemble synthetic quasibiological systems able to replicate and evolve, illuminating universal properties of life and the search for its origins.

A polymerase engineered for bisulfite sequencing.

Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains.

Selection of 2'-deoxy-2'-fluoroarabinonucleotide (FANA) aptamers that bind HIV-1 reverse transcriptase with picomolar affinity.

Using a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol capable of selecting xeno-nucleic acid (XNA) aptamers, a 2'-deoxy-2'-fluoroarabinonucleotide (FANA) aptamer (referred to as FA1) to HIV-1 reverse transcriptase (HIV-1 RT) was selected. FA1 bound HIV-1 RT with KD,app values in the low pM range under different ionic conditions. Comparisons to published HIV-1 RT RNA and DNA aptamers indicated that FA1 bound at least as well as these aptamers. FA1 contained a 20 nucleotide 5' DNA sequence followed by a 57 nucleotide region of FANA nucleotides. Removal of the fourteen 5' DNA nucleotides did not affect binding. FA1's predicted structure was composed of four stems and four loops. All stem nucleotides could be modified to G-C base pairs (14 total changes) with a small effect on binding. Eliminating or altering most loop sequences reduced or abolished tight binding. Overall, results suggested that the structure and the sequence of FA1 were important for binding. FA1 showed strong inhibition of HIV-1 RT in extension assays while no specific binding to avian myeloblastosis or Moloney murine leukemia RTs was detected. A complete DNA version of FA1 showed low binding to HIV-1 RT, emphasizing the unique properties of FANA in HIV-1 RT binding.

Nanostructures from Synthetic Genetic Polymers.

Nanoscale objects of increasing complexity can be constructed from DNA or RNA. However, the scope of potential applications could be enhanced by expanding beyond the moderate chemical diversity of natural nucleic acids. Here, we explore the construction of nano-objects made entirely from alternative building blocks: synthetic genetic polymers not found in nature, also called xeno nucleic acids (XNAs). Specifically, we describe assembly of 70 kDa tetrahedra elaborated in four different XNA chemistries (2'-fluro-2'-deoxy-ribofuranose nucleic acid (2'F-RNA), 2'-fluoroarabino nucleic acids (FANA), hexitol nucleic acids (HNA), and cyclohexene nucleic acids (CeNA)), as well as mixed designs, and a ∼600 kDa all-FANA octahedron, visualised by electron microscopy. Our results extend the chemical scope for programmable nanostructure assembly, with implications for the design of nano-objects and materials with an expanded range of structural and physicochemical properties, including enhanced biostability.

Isoguanine and 5-methyl-isocytosine bases, in vitro and in vivo.

The synthesis, base-pairing properties and in vitro and in vivo characteristics of 5-methyl-isocytosine (isoC(Me) ) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic acid)-DNA(d) mosaic backbone, are described. The required h-isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by Tm measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d-isoG base mispairing follows the order T>G>C while the h-isoG base mispairing follows the order G>C>T. The h- and d-isoC(Me) bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoC(Me) misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base-pair interpretation for the deoxyribose and hexitol isoC(Me) /isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality.

Synthetic polymers and their potential as genetic materials.

DNA and RNA are the only known natural genetic materials. Systematic modification of each of their chemical building blocks (nucleobase, sugar, and phosphate) has enabled the study of the key properties that make those nucleic acids genetic materials. All three moieties contribute to replication and, significantly, all three moieties can be replaced by synthetic analogs without loss of function. Synthetic nucleic acid polymers capable of storing and propagating information not only expand the central dogma, but also highlight that DNA and RNA are not unique chemical solutions for genetic information storage. By considering replication as a question of information transfer, we propose that any polymer that can be replicated could serve as a genetic material.

A short adaptive path from DNA to RNA polymerases.

DNA polymerase substrate specificity is fundamental to genome integrity and to polymerase applications in biotechnology. In the current paradigm, active site geometry is the main site of specificity control. Here, we describe the discovery of a distinct specificity checkpoint located over 25 Å from the active site in the polymerase thumb subdomain. In Tgo, the replicative DNA polymerase from Thermococcus gorgonarius, we identify a single mutation (E664K) within this region that enables translesion synthesis across a template abasic site or a cyclobutane thymidine dimer. In conjunction with a classic "steric-gate" mutation (Y409G) in the active site, E664K transforms Tgo DNA polymerase into an RNA polymerase capable of synthesizing RNAs up to 1.7 kb long as well as fully pseudouridine-, 5-methyl-C-, 2'-fluoro-, or 2'-azido-modified RNAs primed from a wide range of primer chemistries comprising DNA, RNA, locked nucleic acid (LNA), or 2'O-methyl-DNA. We find that E664K enables RNA synthesis by selectively increasing polymerase affinity for the noncognate RNA/DNA duplex as well as lowering the K(m) for ribonucleotide triphosphate incorporation. This gatekeeper mutation therefore identifies a key missing step in the adaptive path from DNA to RNA polymerases and defines a previously unknown postsynthetic determinant of polymerase substrate specificity with implications for the synthesis and replication of noncognate nucleic acid polymers.

Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2'-5' Linkages.

Information-bearing nucleic acids display universal 3'-5' linkages, but regioisomeric 2'-5' linkages occur sporadically in non-enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site-specific 2'-5' linkages by an engineered polymerase using 3'-deoxy- or 3'-O-methyl-NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3'-5' linked DNA with good fidelity. This enables a fast and simple method for "structural mutagenesis" by the position-selective incorporation of 2'-5' linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild-type base sequence as we demonstrate for the 10-23 RNA endonuclease DNAzyme.

Non-canonical 3'-5' extension of RNA with prebiotically plausible ribonucleoside 2',3'-cyclic phosphates.

Ribonucleoside 2',3'-cyclic phosphates (N>p's) are generated by multiple prebiotically plausible processes and are credible building blocks for the assembly of early RNA oligomers. While N>p's can be polymerized into short RNAs by non-enzymatic processes with variable efficiency and regioselectivity, no enzymatic route for RNA synthesis had been described. Here we report such a non-canonical 3'-5' nucleotidyl transferase activity. We engineered a variant of the hairpin ribozyme to catalyze addition of all four N>p's (2',3'-cyclic A-, G-, U-, and CMP) to the 5'-hydroxyl termini of RNA strands with 5' nucleotide addition enhanced in all cases by eutectic ice phase formation at -7 °C. We also observed 5' addition of 2',3'-cyclic phosphate-activated ß-nicotinamide adenine dinucleotide (NAD>p) and ACA>p RNA trinucleotide, and multiple additions of GUCCA>p RNA pentamers. Our results establish a new mode of RNA 3'-5' extension with implications for RNA oligomer synthesis from prebiotic nucleotide pools.

Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening.

Developing therapeutic antibodies is laborious and costly. Here we report a method for antibody discovery that leverages the Illumina HiSeq platform to, within 3 days, screen in the order of 108 antibody-antigen interactions. The method, which we named 'deep screening', involves the clustering and sequencing of antibody libraries, the conversion of the DNA clusters into complementary RNA clusters covalently linked to the instrument's flow-cell surface on the same location, the in situ translation of the clusters into antibodies tethered via ribosome display, and their screening via fluorescently labelled antigens. By using deep screening, we discovered low-nanomolar nanobodies to a model antigen using 4 × 106 unique variants from yeast-display-enriched libraries, and high-picomolar single-chain antibody fragment leads for human interleukin-7 directly from unselected synthetic repertoires. We also leveraged deep screening of a library of 2.4 × 105 sequences of the third complementarity-determining region of the heavy chain of an anti-human epidermal growth factor receptor 2 (HER2) antibody as input for a large language model that generated new single-chain antibody fragment sequences with higher affinity for HER2 than those in the original library.

Molecular breeding of polymerases for resistance to environmental inhibitors.

Potent inhibitors limit the use of PCR assays in a wide spectrum of specimens. Here, we describe the engineering of polymerases with a broad resistance to complex environmental inhibitors using molecular breeding of eight different polymerase orthologues from the genus Thermus and directed evolution by CSR in the presence of inhibitors. Selecting for resistance to the inhibitory effects of Neomylodon bone powder, we isolated 2D9, a chimeric polymerase comprising sequence elements derived from DNA polymerases from Thermus aquaticus, Thermus oshimai, Thermus thermophilus and Thermus brockianus. 2D9 displayed a striking resistance to a broad spectrum of complex inhibitors of highly divergent composition including humic acid, bone dust, coprolite, peat extract, clay-rich soil, cave sediment and tar. The selected polymerase promises to have utility in PCR-based applications in a wide range of fields including palaeobiology, archaeology, conservation biology, forensic and historic medicine.

Emergence of ATP- and GTP-Binding Aptamers from Single RNA Sequences by Error-Prone Replication and Selection.

The spontaneous emergence of function from diverse RNA sequence pools is widely considered an important transition in the origin of life. Here we show that diverse sequence pools are not a prerequisite for the emergence of function. Starting five independent selection experiments each from a single RNA seed sequence - comprising a central homopolymeric poly-A (or poly-U) segment flanked by different conserved primer binding sites - we observe transformation (continuous drift) of the seeds into low diversity sequence pools by mutation, truncation and recombination without ever reaching that of a random pool even after 24 rounds. Upon continuous error prone replication and selection for ATP binding we isolate specific ATP- or GTP-binding aptamers with low micromolar affinities. Our results have implications for early RNA evolution in the light of the high mutation rates associated with both non-enzymatic and enzymatic prebiotic RNA replication.