Inspired by nature, chemists have spent the last 50 years systematically designing and synthesizing a vast array of sugar-modified nucleic acids, so-called xenonucleic acids (XNAs), collectively forming what we now describe as the XNA alphabet. Within the alphabet, systems can be categorized into two major groups: those capable of interacting with natural nucleic acids and those that do not cross-pair with DNA or RNA. The sugar component of XNAs plays a crucial role in defining their conformational space, which, in turn, influences their hybridization properties and potential applications across biotechnology and synthetic biology. This review provides an overview of sugar-modified XNA systems developed to date as well as the geometric parameters and physicochemical principles that have enhanced our understanding of XNA conformational behavior, particularly in relation to their orthogonality to (i.e. inability to cross-pair with) natural nucleic acids. These insights are essential for developing a more rational approach to key processes such as XNA replication and evolution, ultimately paving the way for applications in areas including synthetic genetics, nucleic acid therapeutics, diagnostics, and nanotechnology.

Synthetic genetics is a subdiscipline of synthetic biology that aims to develop artificial genetic polymers (also referred to as xeno-nucleic acids or XNAs) that can replicate in vitro and eventually in model cellular organisms. This field of science combines organic chemistry with polymerase engineering to create alternative forms of DNA that can store genetic information and evolve in response to external stimuli. Practitioners of synthetic genetics postulate that XNA could be used to safeguard synthetic biology organisms by storing genetic information in orthogonal chromosomes. XNA polymers are also under active investigation as a source of nuclease resistant affinity reagents (aptamers) and catalysts (xenozymes) with practical applications in disease diagnosis and treatment. In this review, we provide a structural perspective on known antiparallel duplex structures in which at least one strand of the Watson-Crick duplex is composed entirely of XNA. Currently, only a handful of XNA structures have been archived in the Protein Data Bank as compared to the more than 100 000 structures that are now available. Given the growing interest in xenobiology projects, we chose to compare the structural features of XNA polymers and discuss their potential to access new regions of nucleic acid fold space.

This chapter looks at the physical and structural techniques that can be applied to nucleic acids. The first few sections focus on spectroscopic techniques, nuclear magnetic resonance, mass spectrometry and diffraction techniques. The following sections explore cryogenic electron microscopy, optical microscopy, atomic force microscopy and electrophoresis. Chromatographic methods, centrifugation and light scattering techniques are also discussed. Finally, the chapter concludes with sections on thermodynamic analysis, molecular mechanics and dynamics, and QM/MM methods for modelling nucleic acids reactions.

Revealing the positions of all the atoms in large macromolecules is powerful but only possible with neutron macromolecular crystallography (NMC). Neutrons provide a sensitive and gentle probe for the direct detection of protonation states at near-physiological temperatures and clean of artifacts caused by x rays or electrons. Currently, NMC use is restricted by the requirement for large crystal volumes even at state-of-the-art instruments such as the macromolecular neutron diffractometer at the Spallation Neutron Source. EWALD's design will break the crystal volume barrier and, thus, open the door for new types of experiments, the study of grand challenge systems, and the more routine use of NMC in biology. EWALD is a single crystal diffractometer capable of collecting data from macromolecular crystals on orders of magnitude smaller than what is currently feasible. The construction of EWALD at the Second Target Station will cause a revolution in NMC by enabling key discoveries in the biological, biomedical, and bioenergy sciences.

α-l-(3-2)-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitro primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase η (hPol η) opposite a TNA-modified template strand without and in combination with O4-alkyl thymine lesions. Across TNA-T (tT), hPol η inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol η inserted dAMP opposite O4-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol η complexes with template tT and O4-methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3 → 2 versus 5 → 3, respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3-OH of the primers terminal thymine was positioned at 3.4 Å on average from the α-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol η. Conversely, the crystal structure of a ternary hPol η·DNA·tTTP complex revealed that the primers terminal 3-OH was too distant from the tTTP α-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-à-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.

Threose nucleic acid (TNA) is an artificial genetic polymer capable of heredity and evolution, and is studied in the context of RNA chemical etiology. It has a four-carbon threose backbone in place of the five-carbon ribose of natural nucleic acids, yet forms stable antiparallel complementary Watson-Crick homoduplexes and heteroduplexes with DNA and RNA. TNA base-pairs more favorably with RNA than with DNA but the reason is unknown. Here, we employed NMR, ITC, UV, and CD to probe the structural and dynamic properties of heteroduplexes of RNA/TNA and DNA/TNA. The results indicate that TNA templates the structure of heteroduplexes, thereby forcing an A-like helical geometry. NMR measurement of kinetic and thermodynamic parameters for individual base pair opening events reveal unexpected asymmetric "breathing" fluctuations of the DNA/TNA helix. The results suggest that DNA is unable to fully adapt to the conformational constraints of the rigid TNA backbone and that nucleic acid breathing dynamics are determined from both backbone and base contributions.

Pre-mRNA encoding human NEIL1 undergoes editing by adenosine deaminase ADAR1 that converts a single adenosine to inosine, and this conversion results in an amino acid change of lysine 242 to arginine. Previous investigations of the catalytic efficiencies of the two forms of the enzyme revealed differential release of thymine glycol (ThyGly) from synthetic oligodeoxynucleotides, with the unedited form, NEIL1 K242 being ≈30-fold more efficient than the edited NEIL1 K242R. In contrast, when these enzymes were reacted with oligodeoxynucleotides containing guanidinohydantoin or spiroiminohydantoin, the edited K242R form was ≈3-fold more efficient than the unedited NEIL1. However, no prior studies have investigated the efficiencies of these two forms of NEIL1 on either high-molecular weight DNA containing multiple oxidatively-induced base damages, or oligodeoxynucleotides containing a bulky alkylated formamidopyrimidine. To understand the extent of changes in substrate recognition, γ-irradiated calf thymus DNA was treated with either edited or unedited NEIL1 and the released DNA base lesions analyzed by gas chromatography-tandem mass spectrometry. Of all the measured DNA lesions, imidazole ring-opened 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were preferentially released by both NEIL1 enzymes with K242R being ≈1.3 and 1.2-fold more efficient than K242 on excision of FapyAde and FapyGua, respectively. Consistent with the prior literature, large differences (≈7.5 to 12-fold) were measured in the excision of ThyGly from genomic DNA by the unedited versus edited NEIL1. In contrast, the edited NEIL1 was more efficient (≈3 to 5-fold) on release of 5-hydroxycytosine. Excision kinetics on DNA containing a site-specific aflatoxin B₁-FapyGua adduct revealed an ≈1.4-fold higher rate by the unedited NEIL1. Molecular modeling provides insight into these differential substrate specificities. The results of this study and in particular, the comparison of substrate specificities of unedited and edited NEIL1 using biologically and clinically important base lesions, are critical for defining its role in preservation of genomic integrity.

Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.