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Functional nucleic acids can be evolved in vitro using cycles of selection and amplification, starting from diverse-sequence libraries, which are typically restricted to natural or partially-modified polymer chemistries. Here, we describe the efficient DNA-templated synthesis and reverse transcription of libraries entirely composed of serum nuclease resistant alternative nucleic acid chemistries validated in nucleic acid therapeutics; locked nucleic acid (LNA), 2'-O-methyl-RNA (2'OMe-RNA), or mixtures of the two. We evaluate yield and diversity of synthesised libraries and measure the aggregate error rate of a selection cycle. We find that in addition to pure 2'-O-methyl-RNA and LNA, several 2'OMe-RNA/LNA blends seem suitable and promising for discovery of biostable functional nucleic acids for biomedical applications.

The ability of reverse transcriptases (RTs) to synthesize a complementary DNA from natural RNA and a range of unnatural xeno nucleic acid (XNA) template chemistries, underpins key methods in molecular and synthetic genetics. However, RTs have proven challenging to discover and engineer, in particular for the more divergent XNA chemistries. Here we describe a general strategy for the directed evolution of RT function for any template chemistry called compartmentalized bead labelling and demonstrate it by the directed evolution of efficient RTs for 2'-O-methyl RNA and hexitol nucleic acids and the discovery of RTs for the orphan XNA chemistries D-altritol nucleic acid and 2'-methoxyethyl RNA, for which previously no RTs existed. Finally, we describe the engineering of XNA RTs with active exonucleolytic proofreading as well as the directed evolution of RNA RTs with very high complementary DNA synthesis fidelities, even in the absence of proofreading.

Nucleic-acid catalysts (ribozymes, DNA- and XNAzymes) cleave target (m)RNAs with high specificity but have shown limited efficacy in clinical applications. Here we report on the in vitro evolution and engineering of a highly specific modular RNA endonuclease XNAzyme, FR6_1, composed of 2'-deoxy-2'-fluoro-β-D-arabino nucleic acid (FANA). FR6_1 overcomes the activity limitations of previous DNA- and XNAzymes and can be retargeted to cleave highly structured full-length (5 kb) BRAF and KRAS mRNAs at physiological Mg

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. ispartof: Chembiochem vol:17 issue:12 pages:1107-1110 ispartof: location:Germany status: published

Recombination, the exchange of information between different genetic polymer strands, is of fundamental importance in biology for genome maintenance and genetic diversification and is mediated by dedicated recombinase enzymes. Here, we describe an innate capacity for non-enzymatic recombination (and ligation) in random-sequence genetic oligomer pools. Specifically, we examine random and semi-random eicosamer (N20) pools of RNA, DNA and the unnatural genetic polymers ANA (arabino-), HNA (hexitol-) and AtNA (altritol-nucleic acids). While DNA, ANA and HNA pools proved inert, RNA (and to a lesser extent AtNA) pools displayed diverse modes of spontaneous intermolecular recombination, connecting recombination mechanistically to the vicinal ring cis-diol configuration shared by RNA and AtNA. Thus, the chemical constitution that renders both susceptible to hydrolysis emerges as the fundamental determinant of an innate capacity for recombination, which is shown to promote a concomitant increase in compositional, informational and structural pool complexity and hence evolutionary potential. ispartof: ELIFE vol:7 ispartof: location:England status: published

This unit describes the application of "synthetic genetics," i.e., the replication of xeno nucleic acids (XNAs), artificial analogs of DNA and RNA bearing alternative backbone or sugar congeners, to the directed evolution of synthetic oligonucleotide ligands (XNA aptamers) specific for target proteins or nucleic acid motifs, using a cross-chemistry selective exponential enrichment (X-SELEX) approach. Protocols are described for synthesis of diverse-sequence XNA repertoires (typically 1014 molecules) using DNA templates, isolation and panning for functional XNA sequences using targets immobilized on solid phase or gel shift induced by target binding in solution, and XNA reverse transcription to allow cDNA amplification or sequencing. The method may be generally applied to select fully-modified XNA aptamers specific for a wide range of target molecules. © 2018 by John Wiley & Sons, Inc.

The spontaneous emergence of function from prebiotic pools of informational polymers is a central conjecture of current origin of life scenarios. However, the innate functional capacity of random genetic polymer pools is unknown. Here, we have examined the ab initio activity of random and semi-random eicosamer pools of RNA, DNA and the unnatural genetic polymers ANA (arabino-), HNA (hexitol-) and AtNA (altritol-nucleic acids) with respect to a simple functional test: the capacity for intermolecular ligation and recombination. While DNA, ANA and HNA pools proved inert, naïve RNA and AtNA pools displayed diverse modes of intermolecular recombination in eutectic ice phases. Recombination appears linked to the vicinal ring cis-diol shared by RNA and AtNA. Thus, the chemical configuration that renders both susceptible to hydrolysis also enables substantial spontaneous intrapool recombination in the absence of activation chemistry with a concomitant increase in the compositional and structural complexity of recombined pools.

Informationentragende Nukleinsauren sind universell uber 3′-5′-Bindungen verknupft. Regioisomere 2′-5′-Verknupfungen bilden sich dagegen unregelmasig wahrend der nichtenzymatischen RNA-Synthese und waren womoglich hilfreich fur die prabiotische RNA-Replikation. Hier berichten wir uber die enzymatische Synthese von DNA und RNA mit ortsspezifischen 2′-5′-Verknupfungen durch ein gezielt verandertes Polymerase-Enzym, das 3′-Desoxy- oder 3′-O-Methyl-NTPs als Substrat verwendet. Zusatzlich vermelden wir die reverse Transkription der so hergestellten, modifizierten Nukleinsauren zuruck in 3′-5′-verknupfte DNA mit guter Fidelitat. Somit ergibt sich eine schnelle und einfache Methode zur “strukturellen Mutagenese” durch positionsselektives Einfugen von 2′-5′-Verknupfungen, durch die, ohne Anderung der Basensequenz, die Struktur und Funktion von Nukleinsauren anhand lokaler Deformationen durch regioisomere Bindungen untersucht werden kann. Beispielhaft wenden wir diese Methode auf das 10-23-RNA-Endonuklease-DNAzym an.

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.

The remarkable physicochemical properties of the natural nucleic acids, DNA and RNA, define modern biology at the molecular level and are widely believed to have been central to life's origins. However, their ability to form repositories of information as well as functional structures such as ligands (aptamers) and catalysts (ribozymes/DNAzymes) is not unique. A range of nonnatural alternatives, collectively termed xeno nucleic acids (XNAs), are also capable of supporting genetic information storage and propagation as well as evolution. This gives rise to a new field of "synthetic genetics," which seeks to expand the nucleic acid chemical toolbox for applications in both biotechnology and molecular medicine. In this review, we outline XNA polymerase and reverse transcriptase engineering as a key enabling technology and summarize the application of "synthetic genetics" to the development of aptamers, enzymes, and nanostructures.

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.

Unnatural Bases The genetic basis of all life on the planet is comprised of deoxyribonucleic acid (DNA) with four nitrogenous nucleotide bases, abbreviated to A, G, C, and T. But there are variations on this theme, and Pinheiro et al. (p. 341 ; see the Perspective by Joyce ) describe the directed evolution of unnatural nucleic acid–like genetic polymers. Variant enzymes were developed that efficiently transcribed DNA to anhydrohexitol (HNA), cyclohexenyl (CeNA), locked (LNA), and threofuranosyl (TNA) nuceic acid analogs. Further variant enzymes were developed to reverse-transcribe these analogs back to DNA. Thus, man-made nucleic acid analogs can be designed and selected that have the potential to operate in a way analogous to the natural process of heredity and evolution.

An ancestor of avian IgY was the evolutionary precursor of mammalian IgG and IgE, and present day chicken IgY performs the function of human IgG despite having the domain structure of human IgE. The kinetics of IgY binding to its receptor on a chicken monocyte cell line, MQ-NCSU, were measured, the first time that the binding of a non-mammalian antibody to a non-mammalian cell has been investigated (k(+1) = 1.14 +/- 0.46 x 10(5) mol(-1)sec(-1), k(-1) = 2.30 +/- 0.14 x 10(-3) s(-1), and K(a) = 4.95 x 10(7) m(-1)). This is a lower affinity than that recorded for mammalian IgE-high affinity receptor interactions (Ka approximately 10(10) m(-1)) but is within the range of mammalian IgG-high affinity receptor interactions (human: Ka approximately 10(8)-10(9) m(-1) mouse: Ka approximately 10(7)-10(8) m(-1). IgE has an extra pair of immunoglobulin domains when compared with IgG. Their presence reduces the dissociation rate of IgE from its receptor 20-fold, thus contributing to the high affinity of IgE. To assess the effect of the equivalent domains on the kinetics of IgY binding, IgY-Fc fragments with and without this domain were cloned and expressed in mammalian cells. In contrast to IgE, their presence in IgY has little effect on the association rate and no effect on dissociation. Whatever the function of this extra domain pair in avian IgY, it has persisted for at least 310 million years and has been co-opted in mammalian IgE to generate a uniquely slow dissociation rate and high affinity.

This protocol describes the directed evolution of artificial endonuclease and ligase enzymes composed of synthetic genetic polymers (XNAzymes), using 'cross-chemistry selective enrichment by exponential amplification' (X-SELEX). The protocol is analogous to (deoxy)ribozyme selections, but it enables the development of fully substituted catalysts. X-SELEX is initiated by the synthesis of diverse repertoires (here 10(14) different sequences), using xeno nucleic acid (XNA) polymerases, on DNA templates primed with DNA, RNA or XNA oligonucleotides that double as substrates, allowing selection for XNA-catalyzed cleavage or ligation. XNAzymes are reverse-transcribed into cDNA using XNA-dependent DNA polymerases, and then PCR-amplified to generate templates for subsequent rounds or deep sequencing. We describe methods developed for four XNA chemistries, arabino nucleic acids (ANAs), 2'-fluoroarabino nucleic acids (FANAs), hexitol nucleic acids (HNAs) and cyclohexene nucleic acids (CeNAs), which require ∼1 week per round, and typically 10-20 rounds; in principle, these methods are scalable and applicable to a wide range of novel XNAzyme chemistries, substrates and reactions.

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. ispartof: Nature vol:518 issue:7539 pages:427-430 ispartof: location:England status: published

Aptamers are a class of single-stranded nucleic acid ligands that can bind their targets with high specificity and affinities rivalling those of antibodies. First described over 20 years ago by Tuerk & Gold [1] and Ellington & Szostak [2] (who coined the name), their promise as both diagnostic and therapeutic agents remains to be realised. Key problems include the generally low biostability of the standard DNA/RNA or mixed RNA/2'F-DNA backbones under physiological conditions, limited chemical diversity of functional groups on the natural nucleobases, and the difficulty in reliably discovering aptamer ligands to some therapeutic targets. This review will describe recent progress in developing aptamer selection technology as well as expanding aptamer chemistry and informational complexity to improve aptamer discovery and properties.

IgY is the principal serum antibody in birds and reptiles, and an IgY-like molecule was the evolutionary precursor of both mammalian IgG and IgE. A receptor for IgY on chicken monocytes, chicken leukocyte receptor AB1 (CHIR-AB1), lies in the avian leukocyte receptor cluster rather than the classical Fc receptor cluster where the genes for mammalian IgE and IgG receptors are found. IgG and IgE receptors bind to the lower hinge region of their respective antibodies with 1:1 stoichiometry, whereas the myeloid receptor for IgA, FcalphaRI, and the IgG homeostasis receptor, FcRn, which are found in the mammalian leukocyte receptor cluster, bind with 2:1 stoichiometry between the heavy chain constant domains 2 and 3 of each heavy chain. In this paper, the extracellular domain of CHIR-AB1 was expressed in a soluble form and shown to be a monomer that binds to IgY-Fc with 2:1 stoichiometry. The two binding sites have similar affinities: K(a)(1) = 7.22 +/- 0.22 x 10(5) m(-1) and K(a)(2) = 3.63 +/- 1.03 x 10(6) m(-1) (comparable with the values reported for IgA binding to its receptor). The affinity constants for IgY and IgY-Fc binding to immobilized CHIR-AB1 are 9.07 +/- 0.07 x 10(7) and 6.11 +/- 0.02 x 10(8) m(-1), respectively, in agreement with values obtained for IgY binding to chicken monocyte cells and comparable with reported values for human IgA binding to neutrophils. Although the binding site for CHIR-AB1 on IgY is not known, the data reported here with a monomeric receptor binding to IgY at two sites with low affinity suggest an IgA-like interaction.

Avian IgY is closely related to an ancestor of both mammalian IgG and IgE and thus provides insights into the evolution of antibody structure and function. A recombinant fragment of IgY-Fc consisting of a dimer of the Cupsilon3 and Cupsilon4 domains, Fcupsilon3-4, was expressed and crystallized and its X-ray structure determined to 1.75 A resolution. Fcupsilon3-4 is the only nonmammalian Fc fragment structure determined to date and provides the first structural evidence for an ancient origin of antibody architecture. The Fcupsilon3-4 structure reveals features common to both IgE-Fc and IgG-Fc, and the implications for IgY binding to its receptor are discussed.

The avian IgY antibody isotype shares a common ancestor with both mammalian IgG and IgE and so provides a means to study the evolution of their structural and functional specialisations. Although both IgG and IgE bind to their leukocyte Fc receptors with 1:1 stoichiometry, IgY binds to CHIR-AB1, a receptor expressed in avian monocytes, with 2:1 stoichiometry. The mutagenesis data reported here explain the structural basis for this difference, mapping the CHIR-AB1 binding site to the Cupsilon3/Cupsilon4 interface and not the N-terminal region of Cupsilon3 where, at equivalent locations, the IgG and IgE leukocyte Fc receptor binding sites lie. This finding, together with the phylogenetic relationship of the antibodies and their receptors, indicates that a substantial shift in the nature of Fc receptor binding occurred during the evolution of mammalian IgG and IgE.

The physicochemical properties of nucleic acids are dominated by their highly charged phosphodiester backbone chemistry. The polyelectrolyte structure decouples information content (base sequence) from bulk properties such as solubility and has been proposed as a defining trait of all informational polymers. However, this conjecture has not been tested experimentally. Here, we describe the encoded synthesis of a genetic polymer with an uncharged backbone chemistry: alkyl-phosphonate nucleic acids (phNA), in which the canonical, negatively charged phosphodiester is replaced by an uncharged P-alkylphosphonodiester backbone. Using synthetic chemistry and polymerase engineering, we describe the enzymatic, DNA-templated synthesis of P-methyl- and P-ethyl-phNAs, and the directed evolution of specific streptavidin-binding phNA aptamer ligands directly from random-sequence, mixed P-methyl- / P-ethyl-phNA repertoires. Our results establish a first example of the DNA-templated enzymatic synthesis and evolution of an uncharged genetic polymer and provide a foundational methodology for their exploration as a source of novel, functional molecules.

WILL you permit me, as a student for twenty years of the phenomena and laws of weather, to express my surprise that in meteorological tables or records, and weather notices in general, so little attention is bestowed upon the direction of the wind? It is true that in the daily forecasts issued from the Meteorological Office, this has been made for some time past a prominent, and, to my mind, the most valuable feature. Still the point has by no means been adequately dwelt upon by writers upon meteorology, the result being the loose and utterly unscientific talk we are accustomed to hear upon the very first principles of the problem of climate.