Artificially expanded genetic information systems (AEGISs) as potent inhibitors of the RNA-dependent RNA polymerase of the SARS-CoV-2

Standard and AEGIS nicking molecular beacons detect amplicons from the Middle East respiratory syndrome coronavirus

Deoxynucleoside kinase from D.melanogaster (DmdNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidine more efficiently than natural purine nucleosides, it accepts all four 2'-deoxynucleosides and many analogues, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in DmdNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of an artificially expanded genetic information system (AEGIS). By shuffling hydrogen bonding groups on the nucleobases, AEGIS adds potentially as many as four additional nucleobase pairs to the genetic "alphabet". Specifically, we show that DmdNK Q81E creates the monophosphates from the AEGIS nucleosides dP, dZ, dX, and dK (respectively 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, dP; 6-amino-3-(1'-β-d-2'-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one, dZ; 8-(1'β-d-2'-deoxy-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione, dX; and 2,4-diamino-5-(1'-β-d-2'-deoxyribofuranosyl)-pyrimidine, dK). Using a coupled enzyme assay, in vitro kinetic parameters were obtained for three of these nucleosides (dP, dX, and dK; the UV absorbance of dZ made it impossible to get its precise kinetic parameters). Thus, DmdNK Q81E appears to be a suitable enzyme to catalyze the first step in the biosynthesis of AEGIS 2'-deoxynucleoside triphosphates in vitro and, perhaps, in vivo, in a cell able to manage plasmids containing AEGIS DNA.

One frontier in synthetic biology seeks to move artificially expanded genetic information systems (AEGIS) into natural living cells and to arrange the metabolism of those cells to allow them to replicate plasmids built from these unnatural genetic systems. In addition to requiring polymerases that replicate AEGIS oligonucleotides, such cells require metabolic pathways that biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those polymerases. Such pathways generally require nucleoside and nucleotide kinases to phosphorylate AEGIS nucleosides and nucleotides on the path to these triphosphates. Thus, constructing such pathways focuses on engineering natural nucleoside and nucleotide kinases, which often do not accept the unnatural AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the enzyme engineer to follow the kinase reaction, assays that are easily confused by ATPase and other spurious activities that might arise through "site-directed damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides.

Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide “letters”, the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson–Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so.Results: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson–Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2’-deoxyisocytidine and 2’-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted.Conclusion: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology.

Assays that target DNA and RNA (xNA) are regarded as the “gold standards” in pathogen detection, surveillance, and diagnostics. However, they are often considered inappropriate for use at points-of-sampling and in low resource environments. This paper discusses innovations created by scientists at the FfAME and Firebird that promise to change this. The first is an artificially expanded genetic information system (AEGIS), a species of DNA having eight nucleotide letters added to the four found naturally in DNA. AEGIS nucleobases pair with geometries similar to standard Watson- Crick pairs, but with hydrogen bonding patterns different from (and orthogonal to) patterns that join the A:T and G:C pairs. Thus, AEGIS DNA allows xNA capture and amplification to have very high specificity and very low noise. A second innovation is a self-avoiding molecular recognition system (SAMRS). SAMRS is a species of DNA that behaves the opposite of AEGIS; SAMRS oligonucleotides bind with Watson-Crick complementarity to natural DNA, but not to other SAMRS oligonucleotides. A third innovation is a molecule beacon design that signals the presence of a target xNA even in complex biological mixtures. A fourth innovation is isothermal amplification of xNA targets, without PCR instruments or the skills needed to interpret their output. Here, levels of detection are as few as 30 molecules. Finally, we offer a sample preparation architecture that, as its very first step, sterilizes a sample, rendering it non-hazardous to inexperienced users. It then allows complete xNA capture and CLIA-waivable amplification.

Cleaning up polymerase chain reactions: Artificially expanded genetic information systems (AEGIS) add extra nucleotide letters to DNA alphabets; oligonucleotides containing AEGIS nucleotides do not bind to natural DNA. This orthogonality is exploited here by placing two AEGIS nucleotides (P and Z) in external tags for primers targeting three cancer genes in a nested PCR architecture. AEGIS tags support multiplexed PCR with fewer primer dimers and off-target amplicons than multiplexed PCR without AEGIS components.

To support efforts to develop a ‘synthetic biology’ based on an artificially expanded genetic information system (AEGIS), we have developed a route to two components of a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson–Crick complement, the purine analog 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP). These implement the pyDDA:puAAD hydrogen bonding pattern (where ‘py’ indicates a pyrimidine analog and ‘pu’ indicates a purine analog, while A and D indicate the hydrogen bonding patterns of acceptor and donor groups presented to the complementary nucleobases, from the major to the minor groove). Also described is the synthesis of the triphosphates and protected phosphoramidites of these two nucleosides. We also describe the use of the protected phosphoramidites to synthesize DNA oligonucleotides containing these AEGIS components, verify the absence of epimerization of dZ in those oligonucleotides, and report some hybridization properties of the dZ:dP nucleobase pair, which is rather strong, and the ability of each to effectively discriminate against mismatches in short duplex DNA.

Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson-Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS-SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS-SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS-SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS-SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins.

Although the fundamental properties of DNA as first proposed by Watson and Crick in 1953 provided a basic understanding of how duplex DNA was organized and might be replicated, it was not until the first crystal structures of DNA (Z-DNA in 1979, B-DNA in 1980, and A-DNA in 1982) that the true complexity of the molecule began to be appreciated. Many crystal structures of oligonucleotides have since shed light on the helical forms that "Watson-Crick" DNA can adopt, their associated groove widths, and the properties of the nucleobase pairs and their interactions in all three helical forms. Additional understanding of the properties of Watson-Crick DNA has been provided by computational studies employing a variety of theoretical methods. Together with these studies devoted to understanding Watson-Crick DNA, recent efforts to expand the genetic alphabet have founded a new field in synthetic biology. One of these efforts, the artificially expanded genetic information system (AEGIS) developed by Steven Benner and co-workers, takes advantage of orthogonal hydrogen bonding to produce DNA comprised of six nucleobase pairs, of which the most extensively studied is referred to as P:Z with P being 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one) and Z being 6-amino-5-nitro-2(1H)-pyridone. P:Z forms three edge-on hydrogen bonds that differ from standard Watson-Crick pairs in the arrangement of acceptors and donor groups; P presents acceptor, acceptor, donor, and Z presents donor, donor, acceptor. Z is unique among the AEGIS nucleobases in having a nitro group present in the major groove. PZ-containing DNA has been exploited in a number of clinical applications and is being used to develop receptors and catalysts. Ultimately, the grand challenge will be to create a semisynthetic organism with an expanded genome. Furthermore, just as our understanding of the properties of natural DNA have benefited from structural and computational characterization, so too will our understanding of artificial DNA. This Account focuses on the structural and biophysical properties of AEGIS DNA containing P:Z pairs. We begin with the fundamental properties of P:Z nucleobase pairs, including their electrostatic potential and hydrogen-bonding energies, as elucidated by quantum mechanical calculations. We then examine the impact of including multiple consecutive P:Z pairs into duplex DNA providing an opportunity to investigate stacking interactions between P:Z pairs. The self-complementary 5'-CTTATPPTAZZATAAG was crystallized in B-form using the host-guest system along with analogous natural sequences including Gs or As. Use of the host-guest system to characterize B-DNA obviates a number of limitations on the structural characterization of sequences of interest; these include the ability to crystallize the desired sequences and to distinguish structural effects imparted by the lattice constraints from those inherent in the sequence itself. On the other hand, 3/6ZP, 5'-CTTATPPPZZZATAAG, was crystallized in A-form in a DNA-only lattice allowing a comparative analysis of P:Z pairs in two of the biologically relevant helical forms: A- and B-DNA. Computational studies on the 3/6ZP sequence starting in A-form provide additional evidence for a more energetically favorable stacking interaction, which we term the "slide" conformer, observed in the A-form crystal structure; this unusual stacking interaction plays a major role in altering the conformational dynamics observed for the PZ-containing duplex as compared to a GC-containing "control" duplex in long time scale molecular dynamics simulations. This combined use of structural and computational strategies paves the way for obtaining a detailed description of artificial DNA, both in how it differs from Watson-Crick DNA and in the rational discovery of proteins, such as endonucleases, transcription factors, and polymerases, which can specifically manipulate DNA containing AEGIS nucleobase pairs.

The Watson-Crick-Franklin (WCF) rules describing nucleobase pairing in antiparallel strands of DNA and RNA can be exploited to create artificially expanded genetic information systems (AEGIS) with as many as 12 independently replicable nucleotides joined by six hydrogen bond pairing schemes. One of these additional pairs joins two nucleotides trivially designated as Z (6-amino-5-nitro-(1H)-pyridin-2-one) and P (2-amino-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one). The Z:P pair has supported 6-nucleotide PCR to give diagnostics products, in environmental surveillance kits, and for laboratory in vitro evolution (LIVE) that has generated, inter alia, molecules that inactivate toxins, antibody analogs that bind cancer cells, therapeutic candidates that deliver drugs to those cells, reagents to identify targets on those cells' surfaces, reagents to move cargoes across the blood-brain barrier, and catalysts with ribonuclease activity. However, the Z nucleoside is acidic, with a pKa of ∼7.8. In its deprotonated form, Z- forms a WCF pair with G. This leads to the slow replacement of Z:P pairs by C:G pairs during PCR or, in the reverse process, their introduction. Here, we examine analogs of Z that retain the same donor:donor:acceptor hydrogen bonding pattern as earlier generations of the Z heterocycle, still form a WCF pair with P, but have a higher pKa. Experiments with Taq polymerase show that the rate of loss of Z:P pairs decreases markedly as the pKa of the Z heterocycle increases. This provides direct support for the hypothesis that Z:P pairs are in fact lost via deprotonated Z-:G mismatches. Further, it provides a Z:P system that can be replicated with very high fidelity, with >97% retention of the Z:P pairs over 10,000-fold amplification.

In addition to completing the Watson-Crick nucleobase matching "concept" (big pairs with small, hydrogen bond donors pair with hydrogen bond acceptors), artificially expanded genetic information systems (AEGIS) also challenge DNA polymerases with a complete set of mismatches, including wobble mismatches. Here, we explore wobble mismatches with AEGIS with DNA polymerase 1 from Escherichia coli. Remarkably, we find that the polymerase tolerates an AEGIS:standard wobble that has the same geometry as the G:T wobble that polymerases have evolved to exclude but excludes a wobble geometry that polymerases have never encountered in natural history. These results suggest certain limits to "structural analogy" and "evolutionary guidance" as tools to help synthetic biologists expand DNA alphabets.

Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show that E. coli RNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition.

C-Glycoside DNA bases from an artificially expanded genetic information system reduce processivity in a SF2 helicase as revealed by nanopore tweezers

The addition of the unnatural P:Z base pair to the four naturally occurring DNA bases expands the genetic alphabet and yields an artificially expanded genetic information system (AEGIS). Herein, the structural feature of oligonucleotides containing a novel unnatural P:Z base pair is characterized using both molecular dynamics and quantum chemistry. The results show that the incorporation of the novel artificial base pair (P:Z) preserves the global conformational feature of duplex DNA except for some local structures. The Z-nitro group imparts new properties to the groove width, which widens the major groove. The unnatural oligonucleotides containing mismatched base pairs exhibit low stability. This ensures efficient and high-fidelity replication. In general, the incorporation of the P:Z pair strengthens the stability of the corresponding DNA duplex. The calculated results also show that the thermostability originates from both hydrogen interaction and stacking interaction. The Z-nitro group plays an important role in enhancing the stability of the H-bonds and stacking strength of the P:Z pair. Overall, the present results provide theoretical insights in the exploration of artificially expanded genetic information systems.

Here we are reporting, for the first time, a ligand-guided selection (LIGS) experiment using an artificially expanded genetic information system (AEGIS) to successfully identify an AEGIS-DNA aptamer against T cell receptor-CD3ε expressed on Jurkat.E6 cells. Thus, we have effectively combined the enhanced diversity of an AEGIS DNA library with LIGS to develop a superior screening platform to discover superior aptamers. Libraries of DNA molecules from highly diversified building blocks will provide better ligands due to more functional diversity and better-controlled folding. Thus, a DNA library with AEGIS components (dZ and dP) was used in LIGS experiments against TCR-CD3ε in its native state using two clinically relevant monoclonal antibodies to identify an aptamer termed JZPO-10, with nanomolar affinity. Multiple specificity assays using knockout cells, and competition experiments using monoclonal antibodies utilized in LIGS, show unprecedented specificity of JZPO-10, suggesting that the combination of LIGS with AEGIS-DNA libraries will provide a superior screening platform to discover artificial ligands against critical cellular targets.