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Orthogonal Genetic Systems John C Viewpoints ChemBioChem doi.org/10.1002/cbic.201900725 Orthogonal Genetic Systems John C. Chaput,*[a] Piet Herdewijn,*[b] and MarcelHollenstein*[c] Xenobiology is an emerging area of synthetic biologythat sidered when designing an orthogonal genetic system that aims to safeguard genetically engineered cells by storing syn- can replicate withoutinterference from the endogenous thetic biology information in xeno-nucleic acid polymers genome. In addition to practical value, these studies have the (XNAs). Critical to the success of this effort is the need to es- potentialtoprovide new fundamental insight into the struc- tablish cellular systems that can maintain an XNA chromosome ture andfunction properties of unnatural nucleic acidpoly- in actively dividing cells. Thisviewpoint discusses the structural mers. parameters of the nucleic acid backbone that should be con- Xenobiology is an area of synthetic biology that aims to create dogenousgenome. This means that the orthogonal genome model cellular organismsinwhich all synthetic biology infor- and enzymatic machinery required to replicatethe synthetic mation is encoded in artificial genetic polymers, commonly genome cannot interferewith the natural biosynthetic path- referred to as xeno-nucleic acids or XNAs.[1] Xenobiology cells ways of the cell and, vice versa, the natural systemcannot createdinthis waywould safeguardsynthetic biologyefforts interfere with the replication of the syntheticsystem. Herethe by establishing afirewall separating synthetic biology informa- term “orthogonality” refers to geneticorthogonality,which tion from natural biological information.[2] This challenging en- describes the separation of genetic information between two deavor requires chemical synthesis to produce nucleic acid different classes of nucleic acid molecule (e.g.,XNA and DNA). monomers (xNTPs)that are not commercially availableand This use of the term “orthogonality” differs from its chemical protein engineering to generate the requisite enzymatic ma- biologyusage, which describes chemical or enzymatic reac- chinery neededtosynthesize and propagate geneticinforma- tions that occur with high specificity, such as the site-specific tion encoded in strands of XNA polymers. To date, asignificant labelingofabiological macromolecule or the introduction of a number of advances have been made to support the develop- noncanonical residue into DNA or aprotein.[4] ment of XNA systems, including the generationoflaboratory- Thus far,most of the work in this area has focusedonthe evolvedpolymerases that have enabled the isolation of aptam- evolution of synthetic geneticpolymers by in vitro selection.[5] ers and catalysts with backbonestructures that are distinct The XNAs evaluated in these systems represent aspecial from those found in nature.[3] subset of synthetic geneticpolymers because they have the Extending this work to cellular systemsthat can maintain an unique ability to form stable antiparallel Watson–Crick duplex XNA chromosome (episome) in actively dividing cells is a structures with DNA and RNA, thus allowing information to daunting task that will require new technological advances. pass between different classes of nucleic acid molecules. This Amongthe various problems facing those wishingtopursue feature of biocompatibility simplifies the selectionprocess by this challenging endeavor is the need to establish an orthogo- allowing genetic information present in the starting DNA li- nal geneticsystem that can replicate separately from the en- brary to be transcribed into XNA by using an engineered DNA- dependentXNA polymerase,and allowing XNA members cap- tured during the selectionstep to be reverse transcribed back [a] Prof. J. C. Chaput into DNA by using an XNA-dependent DNA polymerase.[3c,6] Departments of Pharmaceutical Sciences, Chemistry,and Molecular Biology and Biochemistry For each cycle of selectionand amplification, the recovered University of California cDNA molecules are amplified by PCR, made single-stranded, 101 Theory,Irvine, CA 92617 (USA) and used as templates to construct anew population of XNA E-mail:[email protected] that has becomeenriched in molecules that exhibit adesired [b] Prof. P. Herdewijn functional property.[7] An important aspect of this strategy is Medicinal Chemistry,Rega Institute for Medical Research, KU Leuven Herestraat 49, Box 1041, 3000 Leuven(Belgium) that users can take advantage of infrastructure that is already E-mail:[email protected] availablefor the writing and reading DNA sequences by solid- [c] Prof. M. Hollenstein phase synthesis and next-generation sequencing, respective- Department of Structural Biology and Chemistry,Institut Pasteur ly.[8] 28 rue du Docteur Roux, 75724 Paris (France) The ability of certain XNAs to crosspairwith DNA and RNA E-mail:[email protected] depends on the sugar moiety.Bydefinition, nucleic acids con- The ORCID identification numbers for the authors of this article can be found under https://doi.org/10.1002/cbic.201900725. sist of three main parts:the nucleobase, the phosphodiester This article is part of aSpecial Collection on Xenobiology.Toview the linkage,and the sugar moiety.Although each group plays an complete collection, visit our homepage important role in the structural and functional properties of ChemBioChem 2020, 21,1408 –1411 1408 2019 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Viewpoints ChemBioChem doi.org/10.1002/cbic.201900725 nucleic acid polymers, it is the sugar moiety that determines the helical geometry of the duplex.[9] DNA, for example, favors aB-form helix with deoxyribose adoptinga2’-endo sugar pucker,whereas RNA prefers an A-form helix with ribose adoptinga3’-endo sugar pucker.Asexpected, changes to the helical conformationoccur when the naturalsugar is replaced with adifferent type of sugar.For example, arabino nucleic acid (ANA), an RNA analogue in which the stereochemistry of the 2’ carbon atom positions the 2’-hydroxy group in an Figure 2. Schematic representation of A) helical rise and B) helical twist in upward configuration, adopts aB-form helical geometryrather nucleic acid duplexes. than the standard A-form geometry commonly observedfor RNA.[10] Similarly,when a4’-thio group is introduced into the an averagehelicaltwist of 368 and an average helical rise of deoxyribose sugar,the corresponding DNA analogue adopts 3.37 ,whichleads to aduplex with 10 nucleotides per helical an A-form helix rather than the standard B-form geometry turn.[9] By comparison, standard A-form RNA has an average commonlyobservedfor DNA.[11] Figure 1provides acompari- helical twist of 318 and an averagehelical rise per residueof son of the helices formed by these natural and slightly modi- 2.9 ,which produces aduplex with 11.5 nucleotides per heli- fied nucleic acid systems. cal turn.[9] The structuralparameters that define the helical geometry Although areasonably large number of XNAs have been re- of DNA and RNA duplexes have been characterized in detail by portedinthe literature, most are incapable of crosspairing using information obtained by NMR spectroscopy and X-ray with DNA and RNA.[14] These systems have structural parame- crystallography.[12] The parameters that describe the helicity of ters that lie outside the conformational space defined by A- adouble-stranded nucleic acid structure in the most straight- and B-form helices.[15] However,two prominent cases in which forwardway are the average helical rise and the averageheli- XNA crosspairing with DNA and RNA occurs with high efficien- cal twist (Figure 2).[13] These parameters provide the average cy have been studied in detail. Thefirst is threose nucleic acid stacking distances between bases and the number of nucleo- (TNA),which is an artificial geneticpolymer in which the natu- tides per helical turn. Standard B-form DNA,for example, has ral five-carbonribose has been replaced with asynthetic four- Figure 1. Helical structures of natural and closely related synthetic genetic systems. DNA and ANA adopt B-formhelicalgeometries, whereas RNA and 4’-thio- DNA adopt A-form helical structures. ChemBioChem 2020, 21,1408 –1411 www.chembiochem.org 1409 2019 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Viewpoints ChemBioChem doi.org/10.1002/cbic.201900725 carbon threose.[16] TNA forms aB-like helical conformation that short segments of DNA on XNA templates,even when the two is similar to DNA with an average helical twist of 368 and an systemsdonot undergo Watson–Crick base pairing.[22] average helical rise of 3.2 ,which leads to aduplex with ten Orthogonal genetic polymers are incapable of crosspairing nucleotides per helical turn.[17] The second case is anhydrohexi- with natural genetic polymers because they have structural tol (HNA), which derives from asix-carbonpyranosyl sugar.[18] parameters that are incompatible with A- and B-form helices. HNA formsanA-like helical conformation that is similar to RNA Deoxyxylo nucleic acid (dXyloNA), for example, is an epimer of with an average helical twist of 318 and an average helical rise DNA in which the 3’-hydroxyl group is in the upward configu- of 2.6 ,which leads to aduplex with 11.5 nucleotidesper heli- ration and the sugar adopts a3’-endo conformation rather cal turn.[19] than the 2’-endo sugar pucker typicallyobservedfor DNA.[23] Interestingly,both TNA andHNA show signs of structural This relativelysubtle chemical change leads to an enormous heterogeneity depending on whether they are base paired structuralchange with self-pairing duplexes adopting left- with themselvesorcrosspaired
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