Zunyi Yang

In vitro selection with artificial expanded genetic information systems

Significance Many chemicals are valuable because they bind to other molecules. Chemical theory cannot directly design “binders.” However, we might recreate in the laboratory the Darwinian processes that nature uses to create binders. This in vitro evolution uses nucleic acids as binders, libraries of DNA/RNA to survive a selection challenge before they can have “children” (systematic evolution of ligands by exponential enrichment, SELEX). Unfortunately, with only four nucleotides, natural DNA/RNA often yields only poor binders, perhaps because they are built from only four building blocks. Synthetic biology has increased the number of DNA/RNA building blocks, with tools to sequence, PCR amplify, and clone artificially expanded genetic information systems (AEGISs). We report here the first example of a SELEX using AEGIS, producing a molecule that binds to cancer cells. 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.

Enzymatic incorporation of a third nucleobase pair

DNA polymerases are identified that copy a non-standard nucleotide pair joined by a hydrogen bonding pattern different from the patterns joining the dA:T and dG:dC pairs. 6-Amino-5-nitro-3-(1′-β-d-2′-deoxyribofuranosyl)-2(1H)-pyridone (dZ) implements the non-standard ‘small’ donor–donor–acceptor (pyDDA) hydrogen bonding pattern. 2-Amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP) implements the ‘large’ acceptor–acceptor–donor (puAAD) pattern. These nucleobases were designed to present electron density to the minor groove, density hypothesized to help determine specificity for polymerases. Consistent with this hypothesis, both dZTP and dPTP are accepted by many polymerases from both Families A and B. Further, the dZ:dP pair participates in PCR reactions catalyzed by Taq, Vent (exo−) and Deep Vent (exo−) polymerases, with 94.4%, 97.5% and 97.5%, respectively, retention per round. The dZ:dP pair appears to be lost principally via transition to a dC:dG pair. This is consistent with a mechanistic hypothesis that deprotonated dZ (presenting a pyDAA pattern) complements dG (presenting a puADD pattern), while protonated dC (presenting a pyDDA pattern) complements dP (presenting a puAAD pattern). This hypothesis, grounded in the Watson–Crick model for nucleobase pairing, was confirmed by studies of the pH-dependence of mismatching. The dZ:dP pair and these polymerases, should be useful in dynamic architectures for sequencing, molecular-, systems- and synthetic-biology.

Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern

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.

Single-DNA Molecule Nanomotor Regulated by Photons

We report the design of a single-molecule nanomotor driven by photons. The nanomotor is a DNA hairpin-structured molecule incorporated with azobenzene moieties to facilitate reversible photocontrollable switching. Upon repeated UV-vis irradiation, this nanomotor displayed 40-50% open-close conversion efficiency. This type of nanomotor displays well-regulated responses and can be operated under mild conditions with no output of waste. In contrast to multiple-component DNA nanomachines, the intramolecular interaction in this single-molecule system offers unique concentration-independent motor functionality. Moreover, the hairpin structure of the motor backbone can significantly improve the efficiency of light-to-movement energy conversion. These results suggest that azobenzene-incorporated, hairpin-structured single-molecule DNA nanomotors have promising potential for applications which require highly efficient light-driven molecular motors.

Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages

The use of DNA polymerases to incorporate phosphorothioate linkages into DNA, and the use of exonuclease III to determine where those linkages have been incorporated, are re-examined in this work. The results presented here show that exonuclease III degrades single-stranded DNA as a substrate and digests through phosphorothioate linkages having one absolute stereochemistry, assigned (assuming inversion in the polymerase reaction) as S, but not the other absolute stereochemistry. This contrasts with a general view in the literature that exonuclease III favors double-stranded nucleic acid as a substrate and stops completely at phosphorothioate linkages. Furthermore, not all DNA polymerases appear to accept exclusively the (R) stereoisomer of nucleoside alpha-thiotriphosphates [and not the (S) diastereomer], a conclusion inferred two decades ago by examination of five Family-A polymerases and a reverse transcriptase. This suggests that caution is appropriate when extrapolating the detailed behavior of one polymerase from the behaviors of other polymerases. Furthermore, these results provide constraints on how exonuclease III–thiotriphosphate–polymerase combinations can be used to analyze the behavior of the components of a synthetic biology.

Aptamers against Cells Overexpressing Glypican 3 from Expanded Genetic Systems Combined with Cell Engineering and Laboratory Evolution

Laboratory in vitro evolution (LIVE) might deliver DNA aptamers that bind proteins expressed on the surface of cells. In this work, we used cell engineering to place glypican 3 (GPC3), a possible marker for liver cancer theranostics, on the surface of a liver cell line. Libraries were then built from a six-letter genetic alphabet containing the standard nucleobases and two added nucleobases (2-amino-8H-imidazo[1,2-a][1,3,5]triazin-4-one and 6-amino-5-nitropyridin-2-one), Watson-Crick complements from an artificially expanded genetic information system (AEGIS). With counterselection against non-engineered cells, eight AEGIS-containing aptamers were recovered. Five bound selectively to GPC3-overexpressing cells. This selection-counterselection scheme had acceptable statistics, notwithstanding the possibility that cells engineered to overexpress GPC3 might also express different off-target proteins. This is the first example of such a combination.

Conversion strategy using an expanded genetic alphabet to assay nucleic acids.

Methods to detect DNA and RNA (collectively xNA) are easily plagued by noise, false positives, and false negatives, especially with increasing levels of multiplexing in complex assay mixtures. Here, we describe assay architectures that mitigate these problems by converting standard xNA analyte sequences into sequences that incorporate nonstandard nucleotides (Z and P). Z and P are extra DNA building blocks that form tight nonstandard base pairs without cross-binding to natural oligonucleotides containing G, A, C, and T (GACT). The resulting improvements are assessed in an assay that inverts the standard Luminex xTAG architecture, placing a biotin on a primer (rather than on a triphosphate). This primer is extended on the target to create a standard GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of competition from any natural oligonucleotides, even in complex biological samples.

Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity

To explore the possibility of using restriction enzymes in a synthetic biology based on artificially expanded genetic information systems (AEGIS), 24 type-II restriction endonucleases (REases) were challenged to digest DNA duplexes containing recognition sites where individual Cs and Gs were replaced by the AEGIS nucleotides Z and P [respectively, 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 AEGIS nucleotides implement complementary hydrogen bond donor–donor–acceptor and acceptor–acceptor–donor patterns. Results allowed us to classify type-II REases into five groups based on their performance, and to infer some specifics of their interactions with functional groups in the major and minor grooves of the target DNA. For three enzymes among these 24 where crystal structures are available (BcnI, EcoO109I and NotI), these interactions were modeled. Further, we applied a type-II REase to quantitate the fidelity polymerases challenged to maintain in a DNA duplex C:G, T:A and Z:P pairs through repetitive PCR cycles. This work thus adds tools that are able to manipulate this expanded genetic alphabet in vitro, provides some structural insights into the working of restriction enzymes, and offers some preliminary data needed to take the next step in synthetic biology to use an artificial genetic system inside of living bacterial cells.

Helicase‐Dependent Isothermal Amplification of DNA and RNA by Using Self‐Avoiding Molecular Recognition Systems

Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low‐resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off‐target priming, and other artifacts. Here, we show how a “self avoiding molecular recognition system” (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase‐dependent isothermal amplification (SAMRS‐HDA). We also show that incorporating SAMRS into the 3′‐ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS‐HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS‐HDA is a more versatile approach than standard HDA, with a broader applicability for xNA‐targeted diagnostics and research.

A Crystal Structure of a Functional RNA Molecule Containing an Artificial Nucleobase Pair.

As one of its goals, synthetic biology seeks to increase the number of building blocks in nucleic acids. While efforts towards this goal are well advanced for DNA, they have hardly begun for RNA. Herein, we present a crystal structure for an RNA riboswitch where a stem C:G pair has been replaced by a pair between two components of an artificially expanded genetic-information system (AEGIS), Z and P, (6-amino-5-nitro-2(1H)-pyridone and 2-amino-imidazo[1,2-a]-1,3,5-triazin-4-(8H)-one). The structure shows that the Z:P pair does not greatly change the conformation of the RNA molecule nor the details of its interaction with a hypoxanthine ligand. This was confirmed in solution by in-line probing, which also measured a 3.7 nM affinity of the riboswitch for guanine. These data show that the Z:P pair mimics the natural Watson-Crick geometry in RNA in the first example of a crystal structure of an RNA molecule that contains an orthogonal added nucleobase pair.