Xeno nucleic acid

Glycol nucleic acid (left) is an example of a xeno nucleic acid because it has a different backbone than DNA (right).

Xeno nucleic acids (XNA) are synthetic nucleic acid analogues that have a different backbone from the ribose and deoxyribose found in the nucleic acids of naturally occurring RNA and DNA.

The same nucleobases can be used to store genetic information and interact with DNA, RNA, or other XNA bases, but the different backbone gives the structure different stability, and it cannot be processed by naturally occurring cellular processes. For example, natural DNA polymerases cannot read and duplicate this information, thus the genetic information stored in XNA is invisible to DNA-based organisms.

As of 2011, at least six types of synthetic sugars have been shown to form nucleic acid backbones that can store and retrieve genetic information. Research is now being done to create synthetic polymerases to transform XNA. The study of the production and application of XNA molecules has created the field of current xenobiology.

Background

The structure of DNA was discovered in 1953. Around the early 2000s, researchers created a number of exotic DNA-like structures, XNA. These are synthetic polymers that can carry the same information as DNA, but with different molecular constituents. The "X" in XNA stands for "xeno-", meaning strange or alien, indicating the difference in the molecular structure as compared to DNA or RNA.

Not much was done with XNA until the development of special polymerase enzyme, capable of copying XNA from a DNA template as well as copying XNA back into DNA. Pinheiro et al. (2012), for example, has demonstrated such an XNA-capable polymerase that works on sequences of around 100 base pairs in length. More recently, synthetic biologists Philipp Holliger and Alexander Taylor succeeded in creating XNAzymes, the XNA equivalent of a ribozyme, enzymes made of RNA. This demonstrates that XNAs not only store hereditary information, but can also serve as enzymes, raising the possibility that life elsewhere could have begun with something other than RNA or DNA.

Structure

Strands of DNA and RNA are formed by stringing together long chains of molecules called nucleotides. A nucleotide is made up of three chemical components: a phosphate, a five-carbon sugar group (this can be either a deoxyribose sugar—which gives us the "D" in DNA—or a ribose sugar—the "R" in RNA), and one of five standard bases (adenine, guanine, cytosine, thymine or uracil).

The molecules that piece together to form the xeno nucleic acids are almost identical to those of DNA and RNA, with one exception: in XNA nucleotides, the deoxyribose and ribose sugar groups of DNA and RNA have been replaced with other chemical structures. These substitutions make XNAs functionally and structurally analogous to DNA and RNA despite being unnatural and artificial.

XNA exhibits a variety of structural chemical changes relative to its natural counterparts. Types of synthetic XNA created so far include:

• 1,5-Anhydrohexitol nucleic acid (HNA)
• Cyclohexene nucleic acid (CeNA)
• Threose nucleic acid (TNA)
• Glycol nucleic acid (GNA)
• Locked nucleic acid
• Peptide nucleic acid (PNA)
• Fluoroarabino nucleic acid (FANA)

HNA could potentially be used as a drug that can recognize and bind to specified sequences. Scientists have been able to isolate HNAs for the possible binding of sequences that target HIV. Research has also shown that CeNAs with stereochemistry similar to the D form of DNA can create stable duplexes with itself and RNA. It was shown that CeNAs are not as stable when they form duplexes with DNA.

Synthesis

XNA with a similar chemical structure like DNA can be synthesized by engineered polymerases. HNA, CeNA, LNA/BNA, ANA/FANA, and TNA is suitable for this process, while the Spiegelmers(consists of L-nucleic acids) is suitable for engineered polymerases to synthesize.

All XNAs can be synthesized chemically. Generally the repeating units in the XNA macromolecules are synthesized first, then they are connected by chemical synthesis method, or the biological processes.

For the convenience of discussion, the following concepts are defined:

Xeno Nucleoside: analog of the nucleoside, contains the analog of 5-carbon sugar, and a nucleotide base.

Xeno Nucleotide: analog of nucleotide, contains the analog of 5-carbon sugar, and a nucleotide base, and one phosphate group consisting of one to three phosphates attaching to the hydroxyl group in the 5-carbon sugar analog.

To synthesize the xeno nucleoside, the 5 carbon sugar analogs is synthesized in chemical method at first; then, the bases are attached to the sugar analogs in chemical method as well. It is also possible to design other organic synthesis ways to synthesize the xeno nucleoside though it is not preferred. To chemically synthesize the XNA oligomer from polymerization of xeno nucleoside, the hydroxyl group corresponding to 5'-OH of 5 carbon sugar needs activation by adding an active group(like MMTr, monomethoxytrityl), then the activated xeno nucleosides can be attached in polymerization designated chemically. One typical example is CeNA, where the xeno nucleoside repeating units 2′-Cyclohexenylnucleosides are chemically synthesized by attaching the protected base to the protected cyclohexenyl precursor.

To chemically synthesize the xeno nucleoside oligomer, the classical organic synthesis techniques(adding xeno nucleoside one-by-one) are employed and therefore a low yield(towards specific product) is expected when the oligomer chains are long. Uncontrolled polymerization of activated xeno nucleosides will give simple thermodynamically stable product(like AAA in similar nucleoside reaction).

The xeno nucleotides are analog of nucleotide, which has an phosphate group attached to the corresponding hydroxyl group. Xeno nucleotides can be chemically treated to attach the phosphate group. Since the similarity between xeno nucleotides and natural nucleotides, the xeno nucleotides can be used as blocks of the engineered polymerases to synthesize the XNA.

Biosynthesis of the XNAs usually requires templates like the DNA replication, and this process require the xeno nucleotide to be structural similar to natural nucleotide. XNA can be bio-synthesized with DNA templates, where the information in DNA templates instruct the XNA synthesis. XNA can also be bio-synthesized with XNA templates in some condition, where the XNA bahaves like DNA. The synthesis of DNA molecule of XNA templates are also important. Special engineered polymerases and some reverse transcriptase are utilized in the DNA-to-XNA, XNA-to-XNA, and XNA-to-DNA synthesis.

1,5-Anhydrohexitol Nucleic Acid (HNA) bio-synthesis: HNA polymerases(like TgoT_6G12[1], which is archaeal polymerase from Thermococcus gorgonarius) have been engineered to synthesize HNA polymers.

Cyclohexene Nucleic Acid (CeNA) and 2-F-CeNA bio-synthesis: Vent (exo−) DNA polymerase from the B-family polymerases, Taq DNA polymerase from the A-family polymerases, and HIV reverse transcriptase from the reverse transcriptase family have been developed to facilitate the synthesis of CeNA, enabling its use in synthetic genetics.

Locked Nucleic Acid (LNA) / Bridged Nucleic Acid (BNA) bio-synthesis: These nucleic acids are synthesized through the engineering of polymerases that can accommodate their unique structural features, which include modifications that lock the nucleic acid structure. KOD DNA polymerases, a family B DNA polymerase derived from Thermococcus kodakarensis KOD1, are effective LNA decoders and encoders.

Threofuranosyl Nucleic Acid (TNA) bio-synthesis: TNA has been synthesized using mutants of archaeal DNA polymerases, such as Kod-RI, Tgo and Therminator DNA polymerases (9°N, A485L).

Arabino-Nucleic Acid (ANA)/2′-Fluoro-Arabinonucleic Acid (FANA) bio-synthesis: ANA/FANA is synthesized using engineered polymerases that can handle its specific backbone chemistry.

Spiegelmers bio-synthesis: Spiegelmers are created by selecting RNA or DNA aptamers against enantiomeric target molecules, followed by the chemical synthesis of their non-natural L-RNA or L-DNA isomers. This process involves preparing mirror-image targets through chemical synthesis, which can be challenging.

Implications

The study of XNA is not intended to give scientists a better understanding of biological evolution as it has occurred historically, but rather to explore ways in which humans might control and reprogram the genetic makeup of biological organisms in future. XNA has shown significant potential in solving the current issue of genetic pollution in genetically modified organisms. While DNA is incredibly efficient in its ability to store genetic information and lend complex biological diversity, its four-letter genetic alphabet is relatively limited. Using a genetic code of six XNAs rather than the four naturally occurring DNA nucleotide bases yields greater opportunities for genetic modification and expansion of chemical functionality.

The development of various hypotheses and theories about XNAs have altered a key factor in the current understanding of nucleic acids: heredity and evolution are not limited to DNA and RNA as once thought, but are processes that have developed from polymers capable of storing information. Investigations into XNAs will allow researchers to assess whether DNA and RNA are the most efficient and desirable building blocks of life, or if these two molecules emerged randomly after evolving from a larger class of chemical ancestors.

Applications

One theory of XNA utilization is its incorporation into medicine as a disease-fighting agent. Some enzymes and antibodies that are currently administered for various disease treatments are broken down too quickly in the stomach or bloodstream. Because XNA is foreign and because it is believed that humans have not yet evolved the enzymes to break them down, XNAs may be able to serve as a more durable counterpart to the DNA and RNA-based treatment methodologies that are currently in use.

Experiments with XNA have already allowed for the replacement and enlargement of this genetic alphabet, and XNAs have shown complementarity with DNA and RNA nucleotides, suggesting potential for its transcription and recombination. One experiment conducted at the University of Florida led to the production of an XNA aptamer by the AEGIS-SELEX (artificially expanded genetic information system - systematic evolution of ligands by exponential enrichment) method, followed by successful binding to a line of breast cancer cells. Furthermore, experiments in the model bacterium E. coli have demonstrated the ability for XNA to serve as a biological template for DNA in vivo.

In moving forward with genetic research on XNAs, various questions must come into consideration regarding biosafety, biosecurity, ethics, and governance/regulation. One of the key questions here is whether XNA in an in vivo setting would intermix with DNA and RNA in its natural environment, thereby rendering scientists unable to control or predict its implications in genetic mutation.

XNA also has potential applications to be used as catalysts, much like RNA has the ability to be used as an enzyme. Researchers have shown XNA is able to cleave and ligate DNA, RNA and other XNA sequences, with the most activity being XNA catalyzed reactions on XNA molecules. This research may be used in determining whether DNA and RNA's role in life emerged through natural selection processes or if it was simply a coincidental occurrence.

XNA may be employed as molecular clamps in quantitative real-time polymerase chain reactions (qPCR) by hybridizing with target DNA sequences. In a study published in PLOS ONE, an XNA-mediated molecular clamping assay detected mutant cell-free DNA (cfDNA) from precancerous colorectal cancer (CRC) lesions and colorectal cancer. XNA may also act as highly specific molecular probes for detection of nucleic acid target sequence.