Efficient synthesis and replication of diverse sequence libraries composed of biostable nucleic acid analogues.

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.

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.

Discovery and evolution of RNA and XNA reverse transcriptase function and fidelity.

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.

Beyond DNA and RNA: The Expanding Toolbox of Synthetic Genetics.

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.

Random-sequence genetic oligomer pools display an innate potential for ligation and recombination.

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.

Engineering and application of polymerases for synthetic genetics.

Organic chemistry has systematically probed the chemical determinants of function in nucleic acids by variation to the nucleobase, sugar ring and backbone moieties to build synthetic genetic polymers. Concomitantly, protein engineering has advanced to allow the discovery of polymerases capable of utilizing modified nucleotide analogs. A conjunction of these two lines of investigation in nucleotide chemistry and molecular biology has given rise to a new field of synthetic genetics dedicated to the exploration of the capacity of these novel, synthetic nucleic acids for the storage and propagation of genetic information, for evolution and for crosstalk, that is, information exchange with the natural genetic system. Here we summarize recent progress in synthetic genetics, specifically in the design of novel unnatural basepairs to expand the genetic alphabet as well as progress in engineering polymerases capable of templated synthesis, reverse transcription and evolution of synthetic genetic polymers.

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.

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.

Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.

In vitro display technologies have proved to be powerful tools for obtaining high-affinity protein binders. We recently described SNAP display, an entirely in vitro DNA display system that uses the SNAP-tag to link protein with its encoding DNA in water-in-oil emulsions. Here, we apply SNAP display for the affinity maturation of a designed ankyrin repeat proteins (DARPin) that binds to the extracellular domain of HER2 previously isolated by ribosome display. After four SNAP display selection cycles, proteins that bound specifically to HER2 in vitro, with dissociation constants in the low- to sub-nanomolar range, were isolated. In vitro affinities of the panel of evolved DARPins directly correlated with the fluorescence intensities of evolved DARPins bound to HER2 on a breast cancer cell line. A stability trade-off is observed as the most improved DARPins have decreased thermostability, when compared with the parent DARPin used as a starting point for affinity maturation. Dissection of the framework mutations of the highest affinity variant, DARPin F1, shows that functionally destabilising and compensatory mutations accumulated throughout the four rounds of evolution.

An experimental framework for improved selection of binding proteins using SNAP display.

Display technologies (e.g. phage and ribosome display) are powerful tools for selecting and evolving protein binders against various target molecules. SNAP display is a DNA display technology that is conducted entirely in vitro: DNA encoding a library of variants is encapsulated in water-in-oil droplets wherein in vitro protein expression and covalent coupling to the encoding DNA occurs. Here, we explore critical factors for the successful performance of SNAP display based on a set of experiments designed to measure and quantify to what extent they affect selection efficiency. We find that, in SNAP display, the reconstituted cell free expression system PURExpress led to 1.5-fold more active protein and achieved 3.5-fold greater DNA recovery in model selections compared to the RTS 100 Escherichia coli lysate based expression system. We report on the influence parameters including droplet occupancy, valency and selection stringency have on recovery and enrichment. An improved procedure involving bivalent display and stringent selection against a model target, Her2, led to a 10(7)-fold enrichment of a DARPin (H10-2-G3, known to bind Her2 with picomolar affinity) over a non-binding DARPin after three rounds of selection. Furthermore, when spiked into a mixture of DARPins with different affinities, DARPin H10-2-G3 outcompeted all other variants demonstrating SNAP display's ability to efficiently resolve clones with affinities in the nano- to picomolar range. These data establish SNAP display as an in vitro protein engineering tool for isolating protein binders and provide a framework for troubleshooting affinity selections.

SNAP display - an in vitro method for the selection of protein binders.

Display technologies are powerful tools that are widely used for selecting protein binders against different targets by mimicking natural selection. Such in vitro display methods have been pivotal in the isolation and evolution of antibodies with affinities greater than those produced by the natural immune response. Here we describe SNAP display, which covalently links genotype with phenotype by the action of the SNAP-tag in an emulsion microdroplet. Successful model selections suggest that SNAP display, has potential as a robust display platform suitable for selecting protein binders. A multivalent format of SNAP display using dendrimer-like DNA has also been established to probe avidity effects in multivalent protein display selections. This review describes the principles of SNAP display and extrapolates its scope for in vitro selections.