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International Edition: DOI: 10.1002/anie.201905999
Synthetic Biology German Edition: DOI: 10.1002/ange.201905999
What Is XNA?
John C. Chaput* and Piet Herdewijn*
synthetic biology · xeno-nucleic acid · XNA
Abstract: The term “xeno-nucleic acids”, abbreviated XNA, XNA modification into an essential gene (e.g., thymidylate
has grown in popularity to the point that it has become a catch- synthase, ThyA) or gene that codes for a fluorescent protein,
all phrase for almost any unnatural nucleic acid, raising the cells that either grow on an agar plate or become fluorescent
question: what is XNA and how does it differ from chemically indicate the successful readthrough of an XNA nucleotide by
modified DNA? a natural DNA polymerase present in the cellular milieu. This
assay has since been extended to other modifications,[3]
The abbreviation XNA first appeared in the literature in including unnatural base pairs that can be maintained in
2009 in a theoretical paper on nucleic acid polymers whose actively dividing cells.[4]
backbones are constructed from sugars that are distinct from Although the first XNA-modified organisms may be years
the natural deoxyribose and ribose sugars found in DNA and away, advances in the design of engineered polymerases that
RNA.[1] The authors envisioned a new strategy for creating recognize XNA substrates offer a new avenue for pursuing
genetically modified organisms (GMOs) in which all of the more immediate applications in biomedical research.[5] In
foreign DNA used to establish a desired non-biological 2012, the first examples of XNA-derived affinity reagents,
property would be stored in an artificial genetic system that more commonly known as aptamers, were developed using in
is orthogonal to natures genetic material. Such XNA-based vitro selection techniques that require engineered poly-
organisms would overcome the biocontainment problem of merases to synthesize XNA libraries and reverse transcribe
current DNA-based GMOs by providing a genetic firewall the selected variants back into DNA.[6] In the years that
separating the synthetic biology information required for followed, the quality of aptamers with XNA backbones has
non-biological activity from the biological information re- steadily improved leading to a short but growing list of
quired for life. Organisms based on xenobiological frame- reagents that have been selected to bind a range of small
works would be further safeguarded by an absolute depend- molecule and protein targets.[7] More recently, in vitro
ence on laboratory derived chemicals that are required to selection systems have been established to identify XNA
maintain a replicating XNA chromosome. Thus, in the event molecules that can catalyze a chemical reaction, giving rise to
that an XNA-based organism escaped into the wild, it would the first examples of XNA enzymes (XNAzymes), which are
be deprived of vital XNA nutrients and either die or lose its catalysts that can cut and ligate RNA substrates with
synthetic biology information. sequence-specific precision.[8] Although these same functions
Progress toward XNA-based GMOs has so far been have been demonstrated many times by natural and in vitro
limited to single template copying events performed in evolved DNA and RNA enzymes, XNAs offer the oppor-
bacteria cells.[2] In such cases, a DNA plasmid carrying one tunity to create biologically stable reagents that are no longer
or a small number of XNA nucleotides is transformed into susceptible to nuclease digestion.[9] Such reagents could find
a population of bacterial cells to determine whether a natu- practical utility in diagnostic and therapeutic applications that
rally occurring polymerase can read through the modification demand high biological stability, which has been a long-term
without stalling. In the first round of replication, the XNA goal of many academic and industrial laboratories.
modification in the (+) strand is replaced by a natural The rising popularity of XNA has spurred an unexpected
nucleotide in the ( ) complementary strand. By inserting the phenomenon where XNA has become a catch-all phrase to
describe almost any type of nucleic acid modification. In fact,
[*] Prof. J. C. Chaput molecules that were once clearly viewed as chemically
Departments of Pharmaceutical Sciences, Chemistry, and Molecular modified DNA or chemically modified RNA, such as 5-
Biology and Biochemistry alkynyl-uridine modified DNA, 2’-O-methyl RNA, or 2’-
University of California, Irvine fluoro-RNA, have been described in the literature and at
CA (USA) scientific conferences as XNA. While most chemists are
E-mail: jchaput@[Link]
acutely aware of the problem, the growing availability of
Prof. P. Herdewijn
chemically modified nucleic acids have made these reagents
Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven
Leuven (Belgium) available to a broader community that is less knowledgeable
E-mail: [Link]@[Link] about the intricacies of nucleic acid nomenclature. This use, or
The ORCID identification number(s) for the author(s) of this article more aptly misuse, of the term has caused widespread
can be found under: confusion over the definition of XNA, raising the question:
[Link]
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How does XNA differ from chemically modified DNA or HNA) or as prebiotic molecules in the evolution of life (e.g.,
RNA? TNA).[10] Nearly all of the XNAs developed to date were
In considering this question, it is important to remember named based on a specific sugar moiety found in the nucleic
the original intent of XNA, which was to establish a series of acid polymer. For example, threose is the sugar moiety in
artificial genetic polymers that retain the same nucleobase threose nucleic acid, which is abbreviated TNA. However,
(A,C,T,G) and phosphodiester linkage as natural DNA and others were named based on a larger family of nucleic acid
RNA, but differ in the chemical composition of the sugar molecules, like locked nucleic acids (LNA), which constitute
moiety.[1] This makes sense as the D in DNA and the R in a diverse group of bicyclic and tricyclic ring systems. In at least
RNA refer to the natural five-carbon atom sugars of 2’- one case, the XNA system lacks many of the features
deoxyribose and ribose, respectively, while the second (N) commonly found in a nucleic acid molecule. Peptide nucleic
and third (A) letters refer to the nucleobase and phospho- acid (PNA), for example, is an uncharged genetic polymer
diester linkage portions, respectively, of the molecule (Fig- that is missing both the sugar and phosphodiester linkage
ure 1). Thus, changing the first letter of the abbreviation DNA found in a natural genetic polymer yet retains the ability to
base pair with DNA and RNA.
With hundreds of chemical modifications described in the
literature, chemists are now struggling to understand how
nucleic acid molecules should be classified. One possible
approach is to use the generic abbreviations XNA, DZA/
RZA, and DNY/RNY, to signify nucleic acid polymers that
carry chemical modifications at the sugar, nucleobase, and
phosphodiester linkage. In this regard, XNA and DZA have
already been described in the literature as nucleic acid
systems with modified sugars[1] and modified bases,[11] re-
spectively, setting a precedence for moving forward. DNY
could designate modifications to the linker region, such as
sulfone DNA and guanidino DNA, which are known exam-
ples of neutral and positively charged DNA polymers.
Guanidino DNA, for example, has already been labeled as
DNG to signify replacement of the acid moiety with
a guanidine group.[12] In cases where nucleic acid molecules
carry a modification at more than one location, they could be
organized into other groups, like XZA or ZXY oligonucle-
otides. PNA, for example, could be classified as an XNY
system.
These, and many other examples, highlight the need for
Figure 1. Nucleic acid structure. All genetic polymers, like RNA shown a classification system that could be used to catalogue all of
here, are composed of building blocks called nucleotides that contain the various types of synthetic genetic polymers that have been
a sugar moiety (black), phosphodiester linkage (red), and nucleobase created to date. An equally important goal is to establish an
(green). The identity of the sugar determines whether the polymer is
agreed upon nomenclature for naming of new XNAs as they
DNA, RNA, or XNA.
are discovered. The XNA alphabet is limited to 26 letters, but
redundancies in the alphabet have already occurred. For
to an X literally means a synthetic genetic polymer based on example, A, the first letter of the XNA alphabet could refer to
a sugar moiety that is something other than 2’-deoxyribose arabino nucleic acid or altritol nucleic acid, both of which are
and the same is true for RNA. For historical reasons and abbreviated ANA. Since sugar modifications are essentially
because XNAs are meant to be structurally distinct from carbohydrate analogues, it may be advantageous to use a well-
DNA and RNA, close structural analogs of natural genetic established carbohydrate nomenclature when naming future
polymers that carry subtle modifications to the sugar and XNA molecules. For example, ANA could be revised so that
nucleobase moieties are best classified as chemically modified arabino nucleic acid stands for araNA and altritol nucleic acid
DNA or chemically modified RNA, as they are based on the stands for altNA. However, researchers may want to consider
structural framework of the natural ribose and 2’-deoxyribose other approaches as well. The goal of this Viewpoint is to
sugars. Often the oligonucleotide in question is a standard stimulate discussion among nucleic acid chemists about the
DNA or RNA molecule with one or a small number of structural differences between nucleic acid polymers and
modifications sprinkled throughout the sequence. In such possible strategies for naming and organizing these molecules
cases, the predominant backbone is natural DNA or natural into a future catalogue of nucleic acid systems.
RNA.
We realize many nucleic acid analogs now classified as
XNA have names that preceded the introduction of XNA into Conflict of interest
the literature. In most cases, these molecules were inves-
tigated as reagents for antisense therapeutics (e.g., LNA, The authors declare no conflict of interest.
Angew. Chem. Int. Ed. 2019, 58, 2 – 5 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim [Link] &&&&
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These are not the final page numbers!
Angewandte
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Viewpoint
Synthetic Biology X files: The term “xeno-nucleic acids”
(XNA) has grown in popularity to the
J. C. Chaput,* point that it has become a catch-all
P. Herdewijn* &&&&—&&&& phrase for almost any unnatural nucleic
acid, raising the question: what is XNA
What Is XNA? and how does it differ from chemically
modified DNA?
Angew. Chem. Int. Ed. 2019, 58, 2 – 5 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim [Link] &&&&
Ü
Ü
These are not the final page numbers!