Conformational Morphing by a DNA Analogue Featuring 7-Deazapurines and 5-Halogenpyrimidines and the Origins of Adenine-Tract Geometry.

Several efforts are currently directed at the creation and cellular implementation of alternative genetic systems composed of pairing components that are orthogonal to the natural dA/dT and dG/dC base pairs. In an alternative approach, Watson-Crick-type pairing is conserved, but one or all of the four letters of the A, C, G, and T alphabet are substituted by modified components. Thus, all four nucleobases were altered to create halogenated deazanucleic acid (DZA): dA was replaced by 7-deaza-2'-deoxyadenosine (dzA), dG by 7-deaza-2'-deoxyguanosine (dzG), dC by 5-fluoro-2'-deoxycytidine (FdC), and dT by 5-chloro-2'-deoxyuridine (CldU). This base-pairing system was previously shown to retain function in Escherichia coli. Here, we analyze the stability, hydration, structure, and dynamics of a DZA Dickerson-Drew Dodecamer (DDD) of sequence 5'-FdC-dzG-FdC-dzG-dzA-dzA-CldU-CldU-FdC-dzG-FdC-dzG-3'. Contrary to similar stabilities of DDD and DZA-DDD, osmotic stressing revealed a dramatic loss of hydration for the DZA-DDD relative to that for the DDD. The parent DDD 5'-d(CGCGAATTCGCG)-3' features an A-tract, a run of adenosines uninterrupted by a TpA step, and exhibits a hallmark narrow minor groove. Crystal structures─in the presence of RNase H─and MD simulations show increased conformational plasticity ("morphing") of DZA-DDD relative to that of the DDD. The narrow dzA-tract minor groove in one structure widens to resemble that in canonical B-DNA in a second structure. These changes reflect an indirect consequence of altered DZA major groove electrostatics (less negatively polarized compared to that in DNA) and hydration (reduced compared to that in DNA). Therefore, chemical modifications outside the minor groove that lead to collapse of major groove electrostatics and hydration can modulate A-tract geometry.

Characterization of an HNA aptamer suggests a non-canonical G-quadruplex motif.

Nucleic acids not only form the basis of heredity, but are increasingly a source of novel nano-structures, -devices and drugs. This has spurred the development of chemically modified alternatives (xeno nucleic acids (XNAs)) comprising chemical configurations not found in nature to extend their chemical and functional scope. XNAs can be evolved into ligands (XNA aptamers) that bind their targets with high affinity and specificity. However, detailed investigations into structural and functional aspects of XNA aptamers have been limited. Here we describe a detailed structure-function analysis of LYS-S8-19, a 1',5'-anhydrohexitol nucleic acid (HNA) aptamer to hen egg-white lysozyme (HEL). Mapping of the aptamer interaction interface with its cognate HEL target antigen revealed interaction epitopes, affinities, kinetics and hot-spots of binding energy similar to protein ligands such as anti-HEL-nanobodies. Truncation analysis and molecular dynamics (MD) simulations suggest that the HNA aptamer core motif folds into a novel and not previously observed HNA tertiary structure, comprising non-canonical hT-hA-hT/hT-hT-hT triplet and hG4-quadruplex structures, consistent with its recognition by two different G4-specific antibodies.

Controlled E. coli Aggregation Mediated by DNA and XNA Hybridization.

Chemical cell surface modification is a fast-growing field of research, due to its enormous potential in tissue engineering, cell-based immunotherapy, and regenerative medicine. However, engineering of bacterial tissues by chemical cell surface modification has been vastly underexplored and the identification of suitable molecular handles is in dire need. We present here, an orthogonal nucleic acid-protein conjugation strategy to promote artificial bacterial aggregation. This system gathers the high selectivity and stability of linkage to a protein Tag expressed at the cell surface and the modularity and reversibility of aggregation due to oligonucleotide hybridization. For the first time, XNA (xeno nucleic acids in the form of 1,5-anhydrohexitol nucleic acids) were immobilized via covalent, SNAP-tag-mediated interactions on cell surfaces to induce bacterial aggregation.

Amino-acyl tXNA as inhibitors or amino acid donors in peptide synthesis.

Xenobiotic nucleic acids (XNAs) offer tremendous potential for synthetic biology, biotechnology, and molecular medicine but their ability to mimic nucleic acids still needs to be explored. Here, to study the ability of XNA oligonucleotides to mimic tRNA, we synthesized three L-Ala-tXNAs analogs. These molecules were used in a non-ribosomal peptide synthesis involving a bacterial Fem transferase. We compared the ability of this enzyme to use amino-acyl tXNAs containing 1',5'-anhydrohexitol (HNA), 2'-fluoro ribose (2'F-RNA) and 2'-fluoro arabinose. L-Ala-tXNA containing HNA or 2'F-RNA were substrates of the Fem enzyme. The synthesis of peptidyl-XNA and the resolution of their structures in complex with the enzyme show the impact of the XNA on protein binding. For the first time we describe functional tXNA in an in vitro assay. These results invite to test tXNA also as substitute for tRNA in translation.

Stable Hairpin Structures Formed by Xylose-Based Nucleic Acids.

Xenobiology explores synthetic nucleic acid polymers as alternative carriers of genetic information to expand the central dogma. The xylo- and deoxyxylo-nucleic acids (XyNA and dXyNA), containing 3' epimers of riboses and deoxyriboses, are considered to be potential candidates for an orthogonal system. In this study, thermal and spectroscopic analyses show that XyNA and dXyNA form stable hairpins. The dXyNA hairpin structure determined by NMR spectroscopy contains a flexible loop that locks the stem into a stable ladder-like duplex with marginal right-handed helicity. The reduced flexibility of the dXyNA duplex observed in the stem of the hairpin demonstrates that folding of dXyNA yields more stable structures described so far.

Chimeric siRNAs with chemically modified pentofuranose and hexopyranose nucleotides: altritol-nucleotide (ANA) containing GalNAc-siRNA conjugates: in vitro and in vivo RNAi activity and resistance to 5'-exonuclease.

In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2'-deoxy-2'-fluoro and 2'-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro. Although ANA residues were detrimental at the 5' end of the antisense strand, the siRNAs with ANA at position 6 or 7 in the seed region had activity comparable to the parent. The siRNA with ANA at position 7 in the seed region was active in a mouse model. An Oligonucleotide with ANA at the 5' end was more stable in the presence of 5'-exonuclease than an oligonucleotide of the same sequence and chemical composition without the ANA modification. Modeling studies provide insight into the origins of regiospecific changes in potency of siRNAs and the increased protection against 5'-exonuclease degradation afforded by the ANA modification.

N8-Glycosylated 8-Azapurine and Methylated Purine Nucleobases: Synthesis and Study of Base Pairing Properties.

In this report, we present the synthesis of N8-glycosylated 8-aza-2-methylhypoxanthine and 8-aza-6-thiohypoxanthine 2'-deoxynucleosides as well as methylated 2'-deoxynebularine derivatives. In vitro base pairing properties between each modified and canonical nucleobase were studied. As demonstrated by Tm, incorporation of the modified bases in DNA resulted, with few exceptions, in low stability of duplexes. Modified bases studied in this report are preferentially recognized by T (for N8-glycosylated 8-aza-2-methylhypoxanthine and methylated purines) and G (N8-glycosylated 8-aza-2-methylhypoxanthine). The base pair formed between N8-glycosylated 8-aza-6-thiohypoxanthine and N9-glycosylated 2-methyl-6-thiohypoxanthine (X2:X6) showed, to some extent, an orthogonal interaction. Based on Tm studies, the only potential self-pairing system is formed by the N8-glycosylated 8-aza-6-thiohypoxanthine nucleoside (X2) but only in the absence of canonical G and T. This study indicated that the canonical thymine base is the preferential base partner of methylated purine bases.

Rational design of an XNA ligase through docking of unbound nucleic acids to toroidal proteins.

Xenobiotic nucleic acids (XNA) are nucleic acid analogues not present in nature that can be used for the storage of genetic information. In vivo XNA applications could be developed into novel biocontainment strategies, but are currently limited by the challenge of developing XNA processing enzymes such as polymerases, ligases and nucleases. Here, we present a structure-guided modelling-based strategy for the rational design of those enzymes essential for the development of XNA molecular biology. Docking of protein domains to unbound double-stranded nucleic acids is used to generate a first approximation of the extensive interaction of nucleic acid processing enzymes with their substrate. Molecular dynamics is used to optimise that prediction allowing, for the first time, the accurate prediction of how proteins that form toroidal complexes with nucleic acids interact with their substrate. Using the Chlorella virus DNA ligase as a proof of principle, we recapitulate the ligase's substrate specificity and successfully predict how to convert it into an XNA-templated XNA ligase.

Invading Escherichia coli Genetics with a Xenobiotic Nucleic Acid Carrying an Acyclic Phosphonate Backbone (ZNA).

A synthetic orthogonal polymer embracing a chiral acyclic-phosphonate backbone [(S)-ZNA] is presented that uniquely adds to the emerging family of xenobiotic nucleic acids (XNAs). (S)-ZNA consists of reiterating six-atom structural units and can be accessed in few synthetic steps from readily available phophonomethylglycerol nucleoside (PMGN) precursors. Comparative thermal stability experiments conducted on homo- and heteroduplexes made of (S)-ZNA are described that evince its high self-hybridization efficiency in contrast to poor binding of natural complements. Although preliminary and not conclusive, circular dichroism data and dynamic modeling computations provide support to a left-handed geometry of double-stranded (S)-ZNA. Nonetheless, PMGN diphosphate monomers were recognized as substrates by Escherichia coli (E. coli) polymerase I as well as being imported into E. coli cells equipped with an algal nucleotide transporter. A further investigation into the in vivo propagation of (S)-ZNA culminated with the demonstration of the first synthetic nucleic acid with an acyclic backbone that can be transliterated to DNA by the E. coli cellular machinery.

Methylated Nucleobases: Synthesis and Evaluation for Base Pairing In†Vitro and In†Vivo.

The synthesis, base pairing properties and in vitro (polymerase) and in vivo (E. coli) recognition of 2'-deoxynucleotides with a 2-amino-6-methyl-8-oxo-7,8-dihydro-purine (X), a 2-methyl-6-thiopurine (Y) and a 6-methyl-4-pyrimidone (Z) base moiety are described. As demonstrated by Tm measurements, the X and Y bases fail to form a self-complementary base pair. Despite this failure, enzymatic incorporation experiments show that selected DNA polymerases recognize the X nucleotide and incorporate this modified nucleotide versus X in the template. In vivo, X is mainly recognized as a A/G or C base; Y is recognized as a G or C base and Z is mostly recognized as T or C. Replacing functional groups in nucleobases normally involved in W-C recognition (6-carbonyl and 2-amino group of purine; 6-carbonyl of pyrimidine) readily leads to orthogonality (absence of base pairing with natural bases).

XNA ligation using T4 DNA ligase in crowding conditions.

T4 DNA ligase is capable of ligating 2'OMe-RNA duplexes, HNA, LNA and FANA mixed sequences in the presence of 10% w/v PEG8000 and 3 M betaine. The enzymatic joining of oligonucleotides containing multiple consecutive XNA nucleotides at the ligation site has not been reported before.

Correction: Base pairing involving artificial bases in vitro and in vivo.

[This corrects the article DOI: 10.1039/C5SC03474D.].

Base pairing involving artificial bases in vitro and in vivo.

Herein we report the synthesis of N8-glycosylated 8-aza-deoxyguanosine (N8-8-aza-dG) and 8-aza-9-deaza-deoxyguanosine (N8-8-aza-9-deaza-dG) nucleotides and their base pairing properties with 5-methyl-isocytosine (d-isoCMe), 8-amino-deoxyinosine (8-NH2-dI), 1-N-methyl-8-amino-deoxyinosine (1-Me-8-NH2-dI), 7,8-dihydro-8-oxo-deoxyinosine (8-Oxo-dI), 7,8-dihydro-8-oxo-deoxyadenosine (8-Oxo-dA), and 7,8-dihydro-8-oxo-deoxyguanosine (8-Oxo-dG), in comparison with the d-isoCMe:d-isoG artificial genetic system. As demonstrated by Tm measurements, the N8-8-aza-dG:d-isoCMe base pair formed less stable duplexes as the C:G and d-isoCMe:d-isoG pairs. Incorporation of 8-NH2-dI versus the N8-8-aza-dG nucleoside resulted in a greater reduction in Tm stability, compared to d-isoCMe:d-isoG. Insertion of the methyl group at the N1 position of 8-NH2-dI did not affect duplex stability with N8-8-aza-dG, thus suggesting that the base paring takes place through Hoogsteen base pairing. The cellular interpretation of the nucleosides was studied, whereby a lack of recognition or mispairing of the incorporated nucleotides with the canonical DNA bases indicated the extent of orthogonality in vivo. The most biologically orthogonal nucleosides identified included the 8-amino-deoxyinosines (1-Me-8-NH2-dI and 8-NH2-dI) and N8-8-aza-9-deaza-dG. The 8-oxo modifications mimic oxidative damage ahead of cancer development, and the impact of the MutM mediated recognition of these 8-oxo-deoxynucleosides was studied, finding no significant impact in their in vivo assay.

Oligonucleotides containing a ribo-configured cyclohexanyl nucleoside: probing the role of sugar conformation in base pairing selectivity.

The synthesis and a preliminary evaluation of the pairing properties of ribo-cyclohexanyl nucleic acids (r-CNA) is herein reported. Incorporation of a single r-CNA nucleotide into natural duplexes did not enhance their stability, while a very high pairing selectivity for RNA was found. As deduced by comparative analysis of Tm and NMR data, a relationship between pairing selectivity and conformational preferences of the "sugar" moiety of r-CNA (and more generally of six-membered nucleic acids) was suggested.

1',5'-Anhydro-L-ribo-hexitol Adenine Nucleic Acids (α-L-HNA-A): Synthesis and Chiral Selection Properties in the Mirror Image World.

The synthesis and a preliminary investigation of the base pairing properties of (6' → 4')-linked 1',5'-anhydro-L-ribo-hexitol nucleic acids (α-L-HNA) have herein been reported through the study of a model oligoadenylate system in the mirror image world. Despite its considerable preorganization due to the rigidity of the "all equatorial" pyranyl sugar backbone, α-L-HNA represents a versatile informational biopolymer, in view of its capability to cross-communicate with natural and unnatural complements in both enantiomeric forms. This seems the result of an inherent flexibility of the oligonucleotide system, as witnessed by the singular formation of iso- and heterochiral associations composed of regular, enantiomorphic helical structures. The peculiar properties of α-L-HNA (and most generally of the α-HNA system) provide new elements in our understanding of the structural prerequisites ruling the stereoselectivity of the hybridization processes of nucleic acids.

Hybridisation potential of 1',3'-Di-O-methylaltropyranoside nucleic acids.

In further study of our series of six-membered ring-containing nucleic acids, different 1',3'-di-O-methyl altropyranoside nucleoside analogs (DMANA) were synthesized comprising all four base moieties, adenine, cytosine, uracil and guanine. Following assembly into oligonucleotides (ONs), their affinity for natural oligonucleotides was evaluated by thermal denaturation of the respective duplexes. Data were compared with results obtained previously for both anhydrohexitol (HNAs) and 3'-O-methylated altrohexitol modified ONs (MANAs). We hereby demonstrate that ONs modified with DMANA monomers, unlike some of our previously described analogues with constrained 6-membered hexitol rings, did not improve thermodynamic stability of dsRNA complexes, most probably in view of an energetic penalty when forced in the required 1C4 pairing conformation. Overall, a single incorporation was more or less tolerated or even positive for the adenine congener, but incorporation of a second modification afforded a slight destabilization (except for A), while a fully modified sequence displayed a thermal stability of -0.3 °C per modification. The selectivity of pairing remained very high, and the new modification upon incorporation into a DNA strand, strongly destabilized the corresponding DNA duplexes. Unfortunately, this new modification does not bring any advantage to be further evaluated for antisense or siRNA applications.

Isoguanine and 5-methyl-isocytosine bases, in vitro and in vivo.

The synthesis, base-pairing properties and in vitro and in vivo characteristics of 5-methyl-isocytosine (isoC(Me) ) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic acid)-DNA(d) mosaic backbone, are described. The required h-isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by Tm measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d-isoG base mispairing follows the order T>G>C while the h-isoG base mispairing follows the order G>C>T. The h- and d-isoC(Me) bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoC(Me) misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base-pair interpretation for the deoxyribose and hexitol isoC(Me) /isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality.

Probing ambiguous base-pairs by genetic transformation with XNA templates.

The templating potential of anhydrohexitol oligonucleotides bearing ambiguous bases was studied in vivo, by using a selection screen for mosaic heteroduplex plasmids in Escherichia coli. 1,5-Anhydro-2,3-dideoxy-2-(5-nitroindazol-1-yl)-D-arabino-hexitol showed the greatest ambiguity among the three nucleosides tested. At most two successive ambiguous bases could be tolerated on hexitol templates read in bacterial cells. Hexitol nucleosides bearing simplified heterocycles thus stand as promising monomers for generating random DNA sequences in vivo from defined synthetic oligonucleotides.