Xylonucleic acid: synthesis, structure, and orthogonal pairing properties

Abstract

There is a common interest for studying xeno-nucleic acid systems in the fields of synthetic biology and the origin of life, in particular, those with an engineered backbone and possessing novel properties. Along this line, we have investigated xylonucleic acid (XyloNA) containing a potentially prebiotic xylose sugar (a 3′-epimer of ribose) in its backbone. Herein, we report for the first time the synthesis of four XyloNA nucleotide building blocks and the assembly of XyloNA oligonucleotides containing all the natural nucleobases. A detailed investigation of pairing and structural properties of XyloNAs in comparison to DNA/RNA has been performed by thermal UV-melting, CD, and solution state NMR spectroscopic studies. XyloNA has been shown to be an orthogonal self-pairing system which adopts a slightly right-handed extended helical geometry. Our study on one hand, provides understanding for superior structure-function (-pairing) properties of DNA/RNA over XyloNA for selection as an informational polymer in the prebiotic context, while on the other hand, finds potential of XyloNA as an orthogonal genetic system for application in synthetic biology.

The structural diversity of artificial genetic polymers

“…For example, ( R )-GNA and hDNA have strong self-pairing properties, but do not cross-pair with natural DNA and RNA ( 61 , 68 ). Similarly, the recently published xylonucleic acid (XyNA) and deoxy-xylonucleic acids (dXyNA) also constitute an autonomous pairing system, with the caveat that stable triplexes can be formed with both, DNA and RNA poly-A strands ( 69 – 71 ). In the crystal structure of a homochiral DNA (hDNA) octamer duplex, the two strands form Watson–Crick base pairs, with one adenine per strand being extruded from the duplex and forming a reverse-Hoogsteen base pair with a thymine base from an adjacent duplex ( 56 ).…”

Orthogonal Genetic Systems

“…NMR analysis reveals the existence of a ladder‐like structure (Figure ) with an average helical twist of only 2.7°. A similar case is observed for xylo nucleic acid (XyloNA), an equivalent RNA analogue that adopts a stable, self‐pairing system that is also orthogonal to DNA and RNA . Pyranosyl RNA (pRNA) is a third example of a self‐pairing system that adopts a ladder‐like structure that is orthogonal to DNA and RNA .…”

A Single Amino Acid Substitution in Therminator DNA Polymerase Increases Incorporation Efficiency of Deoxyxylonucleotides

“…Despite their chemical similarity to natural nucleotides, dxNTPs are challenging substrates for DNA polymerases. The sugar ring conformation in dxNA has an N ‐type/C3′‐ endo pucker, as in RNA and A‐DNA, in contrast with the S ‐type/C2′‐ endo pucker of B‐DNA . The 3′‐hydroxy group is inverted in relation to DNA (Scheme ), and even though this structural difference from a dNTP is limited, they are still incorporated less efficiently in the presence of most tested polymerases .…”

“…Materials used : All oligonucleotides and a codon‐optimized gene for Therminator DNA polymerase exo − were purchased from Integrated DNA Technologies (IDT). dxTTP and dxATP were synthesized as described . Anhydrotetracyclin (AHT) and pASK‐IBA2 vector were purchased from IBA Life Sciences.…”