Crystal structure of homo-DNA and nature's choice of pentose over hexose in the genetic system

An experimental rationalization of the structure type encountered in DNA and RNA by systematically investigating the chemical and physical properties of alternative nucleic acids has identified systems with a variety of sugar-phosphate backbones that are capable of Watson-Crick base pairing and in some cases cross-pairing with the natural nucleic acids. The earliest among the model systems tested to date, (4' --> 6')-linked oligo(2',3'-dideoxy-beta-d-glucopyranosyl)nucleotides or homo-DNA, shows stable self-pairing, but the pairing rules for the four natural bases are not the same as those in DNA. However, a complete interpretation and understanding of the properties of the hexapyranosyl (4' --> 6') family of nucleic acids has been impeded until now by the lack of detailed 3D-structural data. We have determined the crystal structure of a homo-DNA octamer. It reveals a weakly twisted right-handed duplex with a strong inclination between the hexose-phosphate backbones and base-pair axes, and highly irregular values for helical rise and twist at individual base steps. The structure allows a rationalization of the inability of allo-, altro-, and glucopyranosyl-based oligonucleotides to form stable pairing systems.     

Warum pentose-und nicht hexose-nucleinsäuren? Teil III. Oligo(2′,3′-dideoxy-β-D-glucopyranosyl) nucleotide (‘homo-DNS’): Paarungesigenschaften

Why Pentose-And Not Hexose-Nucleic Acids? Part III. Oligo(2′,3′-dideoxy-β-D -glucopyranosyl)nucleotides. (‘Homo-DNA’): Base-Pairing Properties

1 Summary in collaboration with Prof. Dr. C. E. Wintner, Haverford College, Haverford, PA 19041-1392.

The paper presents results of a comprehensive investigation on the pairing properties of homo-DNA oligonucleotides, the preparation of which has been described in Part II of this series [2]. The investigation was carried out by using established methods described in the literature for the characterization of oligonucleotides in the natural series, such as determination of melting temperatures of oligonucleotide duplexes by temperature-dependent of melting temperatures, determination of pairing stoichiometry by ratio-dependent UV spectroscopy of binary mixtures of pairing partners, temperature-dependent CD spectroscopy, gel electrophoresis under non-denaturing conditions, and – in selected cases – ¹H – and³¹P-NMR spectroscopy. The systematic comparison of the paring properties of homo-DNA oligonucleotides with corresponding DNA nucleotides (up to dodecamers) indicates that homo-DNA is a highly efficient, autonomous, artificial pairing system with a pairing behavior that is in part similar to, but also, in part, strikingly different from, the pairing behavior of DNA. The pairing properties established so far are listed below in a manner that reflects the sequence of subtitles in Chapt.2 of the text; they were determined under the conditions: H²O, 0.15M NaCl, 0.01M Tris-HCl buffer, pH 7, oligonucleotide concentrations in the μM range, 1:1 ratio of single strands in the case of non-selfcompementary sequences.     

Gas-phase Dissociation of homo-DNA Oligonucleotides

Synthetic modified oligonucleotides are of interest for diagnostic and therapeutic applications, as their biological stability, pairing selectivity, and binding strength can be considerably increased by the incorporation of unnatural structural elements. Homo-DNA is an oligonucleotide homologue based on dideoxy-hexopyranosyl sugar moieties, which follows the Watson-Crick A-T and G-C base pairing system, but does not hybridize with complementary natural DNA and RNA. Homo-DNA has found application as a bioorthogonal element in templated chemistry applications. The gas-phase dissociation of homo-DNA has been investigated by ESI-MS/MS and MALDI-MS/MS, and mechanistic aspects of its gas-phase dissociation are discussed. Experiments revealed a charge state dependent preference for the loss of nucleobases, which are released either as neutrals or as anions. In contrast to DNA, nucleobase loss from homo-DNA was found to be decoupled from backbone cleavage, thus resulting in stable products. This renders an additional stage of ion activation necessary in order to generate sequence-defining fragment ions. Upon MS³ of the primary base-loss ion, homo-DNA was found to exhibit unspecific backbone dissociation resulting in a balanced distribution of all fragment ion series.

Crystal structure of double helical hexitol nucleic acids

A huge variety of chemically modified oligonucleotide derivatives has been synthesized for possible antisense applications. One such derivative, hexitol nucleic acid (HNA), is a DNA analogue containing the standard nucleoside bases, but with a phosphorylated 1',5'-anhydrohexitol backbone. Hexitol nucleic acids are some of the strongest hybridizing antisense compounds presently known, but HNA duplexes are even more stable. We present here the first high-resolution structure of a double helical nucleic acid with all sugars being hexitols. Although designed to have a restricted conformational flexibility, the hexitol oligomer h(GTGTACAC) is able to crystallize in two different double helical conformations. Both structures display a high x-displacement, normal Watson-Crick base pairing, similar base stacking patterns, and a very deep major groove together with a minor groove with increased hydrophobicity. One of the conformations displays a major groove which is wide enough to accommodate a second HNA double helix resulting in the formation of a double helix of HNA double helices. Both structures show most similarities with the A-type helical structure, the anhydrohexitol chair conformation thereby acting as a good mimic for the furanose C3'-endo conformation observed in RNA. As compared to the quasi-linear structure of homo-DNA, the axial position of the base in HNA allows efficient base stacking and hence double helix formation.     

An unusual mode of DNA duplex association: Watson-Crick interaction of all-purine deoxyribonucleic acids

Nucleic acid duplexes associating through purine-purine base pairing have been constructed and characterized in a remarkable demonstration of nucleic acids with mixed sequence and a natural backbone in an alternative duplex structure. The antiparallel deoxyribose all-purine duplexes associate specifically through Watson-Crick pairing, violating the nucleobase size-complementarity pairing convention found in Nature. Sequence-specific recognition displayed by these structures makes the duplexes suitable, in principle, for information storage and replication fundamental to molecular evolution in all living organisms. All-purine duplexes can be formed through association of purines found in natural ribonucleosides. Key to the formation of these duplexes is the N(3)-H tautomer of isoguanine, preferred in the duplex, but not in aqueous solution. The duplexes have relevance to evolution of the modern genetic code and can be used for molecular recognition of natural nucleic acids.     

On the structure and dynamics of duplex GNA

Glycol nucleic acid (GNA), with a nucleotide backbone comprising of just three carbons and the stereocenter derived from propylene glycol (1,2-propanediol), is a structural analog of nucleic acids with intriguing biophysical properties, such as formation of highly stable antiparallel duplexes with high Watson-Crick base pairing fidelity. Previous crystallographic studies of double stranded GNA (dsGNA) indicated two forms of backbone conformations, an elongated M-type (containing metallo-base pairs) and the condensed N-type (containing brominated base pairs). A herein presented new crystal structure of a GNA duplex at 1.8 ? resolution from self-complementary 3'-CTC(Br)UAGAG-2' GNA oligonucleotides reveals an N-type conformation with alternating gauche-anti torsions along its (O3'-C3'-C2'-O2') backbone. To elucidate the conformational state of dsGNA in solution, molecular dynamic simulations over a period of 20 ns were performed with the now available repertoire of structural information. Interestingly, dsGNA adopts conformational states in solution intermediate between experimentally observed backbone conformations: simulated dsGNA shows the all-gauche conformation characteristic of M-type GNA with the higher helical twist common to N-type GNA structures. The so far counterintuitive, smaller loss of entropy upon duplex formation as compared to DNA can be traced back to the conformational flexibility inherent to dsGNA but missing in dsDNA. Besides extensive interstrand base stacking and conformational preorganization of single strands, this flexibility contributes to the extraordinary thermal stability of GNA.     

Synthesis and Properties of Isobicyclo‐DNA

We present the synthesis of the isobicyclo‐DNA building blocks with the nucleobases A, C, G and T, as well as biophysical and biological properties of oligonucleotides derived thereof. The synthesis of the sugar part was achieved in 5 steps starting from a known intermediate of the tricyclo‐DNA synthesis. Dodecamers containing single isobicyclo‐thymidine incorporations, fully modified A‐ and T‐containing sequences, and fully modified oligonucleotides containing all four bases were synthesized and characterized. Isobicyclo‐DNA forms stable duplexes with natural nucleic acids with a pronounced preference for DNA over RNA as complements. The most stable duplexes, however, arise by self‐pairing. Isobicyclo‐DNA forms preferentially B‐DNA‐like duplexes with DNA and A‐like duplexes with complementary RNA as determined by circular dichroism (CD) spectroscopy. Self‐paired duplexes show a yet unknown structure, as judged from CD spectroscopy. Biochemical tests revealed that isobicyclo‐DNA is stable in fetal bovine serum and does not elicit RNaseH activity.     

Synthesis and Properties of 2′-Deoxy-1′,2′-seco-D-ribosyl (5′ → 3′)oligonucleotides (= 1′,2′-seco-DNA) containing adenine and thymine

Some 2′-deoxy-1′,2′-seco-D-ribosyl (5′→3′)oligonucleotides (= 1′,2′-seco-DNA), differing from natural DNA only by a bond scission between the centers C(1′) and C(2′), were synthesized and studied in order to compare their structure properties and pairing behavior with those of corresponding natural DNA and homo-DNA oligonucleotides (2′,3′-dideoxy-β-D-glucopyranosyl oligonucleotides). Starting from (?)-D-tartaric acid, 2′-deoxy-1′,2′-secoadenosine derivative 9a and 1′,2′-secothymidine ( 9b ) were obtained in pure crystalline form. Using the phosphoramidite variant of the phosphite-triester method, a dinucleotide monophosphate 1′,2′-seco-d(T₂) was synthesized in solution, while oligonucleotides 1′,2′-seco-d[(AT)₆], 1′,2′-seco-d(A₁₀) and 1′,2′-seco-d(T₁₀) were prepared on solid phase with either automated or manual techniques. Results of UV- and CD-spectroscopic as well as gel-electrophoretic studies indicated that neither adenine-thymine base pairing (as observed in natural DNA and homo-DNA), nor the adenine-adenine base pairing (as observed in homo-DNA) was effective in 1′,2′-seco-DNA, Furthermore, hybrid pairing was observed neither between 1′.2′-seco-DNA and natural DNA nor between 1′,2′-seco-DNA and homo-DNA.     

NMR solution structure of an oligonucleotide hairpin with a 2'F-ANA/RNA stem: implications for RNase H specificity toward DNA/RNA hybrid duplexes.

The first structure of a 2'-deoxy-2'-fluoro-D-arabinose nucleic acid (2'F-ANA)/RNA duplex is presented. We report the structural characterization by NMR spectroscopy of a small hybrid hairpin, r(GGAC)d(TTCG)2'F-a(GTCC), containing a 2'F-ANA/RNA stem and a four-residue DNA loop. Complete (1)H, (13)C, (19)F, and (31)P resonance assignments, scalar coupling constants, and NOE constraints were obtained from homonuclear and heteronuclear 2D spectra. In the chimeric duplex, the RNA strand adopts a classic A-form structure having C3' endo sugar puckers. The 2'F-ANA strand is neither A-form nor B-form and contains O4' endo sugar puckers. This contrasts strongly with the dynamic sugar conformations previously observed in the DNA strands of DNA/RNA hybrid duplexes. Structural parameters for the duplex, such as minor groove width, x-displacement, and inclination, were intermediate between those of A-form and B-form duplexes and similar to those of DNA/RNA duplexes. These results rationalize the enhanced stability of 2'F-ANA/RNA duplexes and their ability to elicit RNase H activity. The results are relevant for the design of new antisense drugs based on sugar-modified nucleic acids.     

Warum Pentose- und nicht Hexose-Nucleinsäuren??. Teil II. Oligonucleotide aus 2′,3′-Dideoxy-β-D-glucopyranosyl-Bausteinen (‘Homo-DNS’): Herstellung.

Why Pentose and Not Hexose Nucleic Acids? Part II . Preparation of Oligonucleotides Containing 2′,3′-Dideoxy-β-D -glucopyranosyl Building Blocks⁽⁷⁾ This paper describes the preparation of the 2′,3′-dideoxy-β-D -glucopyranosyl-( = 2′,3′-dideoxy-β-D -erythro-hexopyranosyl)-derived nucleosides of the five bases adenine, cytosine, guanine, thymine, and uracil ( = ‘homo-de-oxyribonucleosides’) as well as the synthesis of oligonucleotides derived from them. The methods used for both nucleoside and oligonucleotide synthesis closely follow the known methods of synthesis in the corresponding series of natural 2′-deoxyribonucleosides and oligonucleotides. The efficient methods of automated DNA synthesis proved to be fully applicable to the synthesis of homo-DNA oligonucleotides, the only change necessary for achieving satisfactory coupling yields being a slight lengthening of the coupling time. Homo-DNA oligonucleotides with chain lengths of up to twelve nucleoside units were assembled on solid support either manually or on a commercial DNA synthesizer in scales of 0.4 μmol to as much as 200 μmol and were purified by either reversed-phase or ion-exchange HPLC to single-peak purity according to both chromatographic systems (estimated purity > 95%). The choice of the specific base sequences to be synthesized was determined primarily by the constitutional problems of base pairing that emerged from experimental observations made in the course of systematic studies of the pairing properties of homo-DNA oligonucleotides. About 100 homo-DNA sequences were prepared for this purpose. Their pairing properties will be described in Part III of this series; the present paper is restricted to the characterization of the purity and constitutional integrity of a few selected (single-stranded) oligonucleotides by ¹H-, ³¹P-, and ¹³C-NMR spectroscopy as well as by FAB and time-of-flight mass spectroscopy. The English Footnotes to Schemes 1–9, Fig. 1–12, and Table 1 provide an extension of this summary.     

Warum Pentose- und nicht Hexose-Nucleinsäuren?? Teil I. Einleitung und Problemstellung,Konformationsanalyse für Oligonucleotid-Ketten aus 2′,3′-Dideoxyglucopyranosyl-Bausteinen (‘Homo-DNS’) sowie Betrachtungen zur Konformation von A- und B-DNS

Why Pentose and Not Hexose Nucleic Acids? Part I . Introduction to the Problem, Conformational Analysis of Oligonucleotide Single Strands Containing 2′,3′-Dideoxyglucopyranosyl Building Blocks (‘Homo-DNA’), and Reflections on the Conformation of A- and B-DNA Summary in collaboration with Prof. Dr. C.E. Wintner, Haverford College, Haverford, PA 19041-1392; academic guest, ETH, March and June/July, 1991. Chemical rationalization of the structure of a biomolecule can be sought through consideration of two criteria: first, the relationship between the structure and its biological function; and second, the structure's potential for constitutional self assembly. The latter criterion convers the judgment, by chemical reasoning, of the chance of its preformation, that is, a synthetic event which must have been undergone by any molecular structure in order to have been selected (or to have selected itself) to become a biomolecule. One way to further the task of rationalizing a biomolecule's structure by experimental means is the design, synthesis, and study of structural alternatives which might have become biomolecules on the basis of either criterion, but which do not, in fact, appear in Nature today. In the formation of sugar phosphates from glycolaldehyde phosphate under basic conditions, straightforward and selective formation of rac-hexose 2,4,6-triphosphates is observed in the absence of formaldehyde, while rac-pentose 2,4-diphosphates are dominant, when (0.5 equiv.) formaldehyde is present [1]. This and other observations indicate that hexose sugars should be regarded to have had a chance of preformation comparable with that of pentose sugars. Why, then, did Nature choose pentoses and not hexoses as the sugar building blocks of nucleic acids? The reason must be functional; it must be the case that pentose nucleic acids are biologically superior to potential hexose alternatives. To the extent that biological function is a consequence of molecular structure and reactivity, the origin of this superiority should be decipherable through chemical experiment, that is, through synthesis of hexose nucleic acids, systematic study of their chemical properties, and comparison of these properties with those of their natural counterparts. This has been the object of the present investigation, initiated in 1986. The paper introduces a series of papers which will describe the results of a model study, namely, the synthesis, pairing properties and structure of homo-DNA oligonucleotides.     

Why Pentose- and Not Hexose-Nucleic Acids??. Part VII. Pyranosyl-RNA (‘p-RNA’). Preliminary communication

Qualitative conformational analysis of the entirety of conceivable hexo- and pentopyranosyl oligonucleotide systems derived from the diastereoisomeric aldohexoses (CH₂O)₆ and aldopentoses (CH₂O)₅ predicts the existence of a variety of pairing systems which have not been experimentally investigated so far. In particular, the analysis foresees the existence of a ribopyranosyl isomer of RNA (‘p-RNA’), containing the phosphodiester linkage between the positions C(4′) and C(2′) of neighboring ribopyranosyl units. Double strands of p-RNA oligonucleotides are expected to have a linear structure and to show purine-pyrimidine and purine-purine (Watson-Crick) pairing comparable in strength to that observed in homo-DNA. Experimentally, synthetic β-D -ribopyranosyl (4′→2′)-oligonucleotides derived from adenine and uracil confirm this prognosis: adenine-uracil pairing in p-RNA duplexes is stronger than in the corresponding RNA duplexes. Importantly, adenine in p-Ribo(A₈) does not show (reverse-Hoogsteen) self-pairing, in sharp contrast to its behavior in the homo-DNA series. The sheer existence of strong and selective pairing in a system that is constitutionally isomeric to RNA and can be predicted to have a linear structure has implications for the problem of RNA's origin. In this context, a comprehensive experimental study of the pairing properties of p-RNA, of its potential for constitutional assembly, self-replication, and intra-duplex isomerization to RNA seems mandatory.     

alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) recognition of DNA: an NMR spectroscopic study

We have used NMR and CD spectroscopy to study and characterise two alpha-L-LNA:DNA duplexes, a nonamer that incorporates three alpha-L-LNA nucleotides and a decamer that incorporates four alpha-L-LNA nucleotides, in which alpha-L-LNA is alpha-L-ribo-configured locked nucleic acid. Both duplexes adopt right-handed helical conformations and form normal Watson-Crick base pairing with all nucleobases in the anti conformation. Deoxyribose conformations were determined from measurements of scalar coupling constants in the sugar rings, and for the decamer duplex, distance information was derived from 1H-1H NOE measurements. In general, the deoxyriboses in both of the alpha-L-LNA:DNA duplexes adopt S-type (B-type structure) sugar puckers, that is the inclusion of the modified alpha-L-LNA nucleotides does not perturb the local native B-like double-stranded DNA (dsDNA) structure. The CD spectra of the duplexes confirm these findings, as these display B-type characteristic features that allow us to characterise the overall duplex type as B-like. The 1H-1H NOE distances which were determined for the decamer duplex were employed in a simulated annealing protocol to generate a model structure for this duplex, thus allowing a more detailed inspection of the impact of the alpha-L-ribo-configured nucleotides. In this structure, it is evident that the malleable DNA backbone rearranges in the vicinity of the modified nucleotides in order to accommodate them and present their nucleobases in a geometry suitable for Watson-Crick base pairing.     

Synthesis of aminoglycoside-modified oligonucleotides

[structure: see text] To study the structural requirements of aminoglycoside binding to nucleic acids, compound 1-an analogue of the naturally occurring nucleoside J-was synthesized. When incorporated into oligodeoxynucleotides, 1 leads to thermal stabilization of the resulting duplexes. The increase in pairing affinity is stronger with complementary RNA than with DNA.     

Solution structure and conformational dynamics of deoxyxylonucleic acids (dXNA): an orthogonal nucleic acid candidate

Orthogonal nucleic acids are chemically modified nucleic acid polymers that are unable to transfer information with natural nucleic acids and thus can be used in synthetic biology to store and transfer genetic information independently. Recently, it was proposed that xylose-DNA (dXNA) can be considered to be a potential candidate for an orthogonal system. Herein, we present the structure in solution and conformational analysis of two self-complementary, fully modified dXNA oligonucleotides, as determined by CD and NMR spectroscopy. These studies are the initial experimental proof of the structural orthogonality of dXNAs. In aqueous solution, dXNA duplexes predominantly form a linear ladderlike (type-1) structure. This is the first example of a furanose nucleic acid that adopts a ladderlike structure. In the presence of salt, an equilibrium exists between two types of duplex form. The corresponding nucleoside triphosphates (dXNTPs) were synthesized and evaluated for their ability to be incorporated into a growing DNA chain by using several natural and mutant DNA polymerases. Despite the structural orthogonality of dXNA, DNA polymerase β mutant is able to incorporate the dXNTPs, showing DNA-dependent dXNA polymerase activity.     

Reinforced HNA backbone hydration in the crystal structure of a decameric HNA/RNA hybrid

The crystal structure of a decameric HNA/RNA (HNA = 2',3'-dideoxy-1',5'-anhydro-d-arabinohexitol nucleic acid) hybrid with the RNA sequence 5'-GGCAUUACGG-3' is the first crystal structure of a hybrid duplex between a naturally occurring nucleic acid and a strand, which is fully modified to contain a six-membered ring instead of ribose. The presence of four duplex helices in the asymmetric unit allows for a detailed discussion of hydration, which revealed a tighter spinelike backbone hydration for the HNA- than for the RNA-strands. The reinforced backbone hydration is suggested to contribute significantly to the exceptional stability of HNA-containing duplexes and might be one of the causes for the evolutionary preference for ribose-derived nucleic acids.     

Quadracyclic Adenine: A Non‐Perturbing Fluorescent Adenine Analogue

Fluorescent‐base analogues (FBAs) comprise a group of increasingly important molecules for the investigation of nucleic acid structure and dynamics as well as of interactions between nucleic acids and other molecules. Here, we report on the synthesis, detailed spectroscopic characterisation and base‐pairing properties of a new environment‐sensitive fluorescent adenine analogue, quadracyclic adenine (qA). After developing an efficient route of synthesis for the phosphoramidite of qA it was incorporated into DNA in high yield by using standard solid‐phase synthesis procedures. In DNA qA serves as an adenine analogue that preserves the B‐form and, in contrast to most currently available FBAs, maintains or even increases the stability of the duplex. We demonstrate that, unlike fluorescent adenine analogues, such as the most commonly used one, 2‐aminopurine, and the recently developed triazole adenine, qA shows highly specific base‐pairing with thymine. Moreover, qA has an absorption band outside the absorption of the natural nucleobases (>300 nm) and can thus be selectively excited. Upon excitation the qA monomer displays a fluorescence quantum yield of 6.8 % with an emission maximum at 456 nm. More importantly, upon incorporation into DNA the fluorescence of qA is significantly less quenched than most FBAs. This results in quantum yields that in some sequences reach values that are up to fourfold higher than maximum values reported for 2‐aminopurine. To facilitate future utilisation of qA in biochemical and biophysical studies we investigated its fluorescence properties in greater detail and resolved its absorption band outside the DNA absorption region into distinct transition dipole moments. In conclusion, the unique combination of properties of qA make it a promising alternative to current fluorescent adenine analogues for future detailed studies of nucleic acid‐containing systems.     

An exocyclic methylene group acts as a bioisostere of the 2'-oxygen atom in LNA

We show for the first time that it is possible to obtain LNA-like (Locked Nucleic Acid 1) binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2'-oxygen atom in LNA with an exocyclic methylene group. Synthesis of the methylene-cLNA nucleoside was accomplished by an intramolecular cyclization reaction between a radical at the 2'-position and a propynyl group at the C-4' position. Only methylene-cLNA modified oligonucleotides showed similar thermal stability and mismatch discrimination properties for complementary nucleic acids as LNA. In contrast, the close structurally related methyl-cLNA analogs showed diminished hybridization properties. Analysis of crystal structures of cLNA modified self-complementary DNA decamer duplexes revealed that the methylene group participates in a tight interaction with a 2'-deoxyribose residue of the 5'-terminal G of a neighboring duplex, resulting in the formation of a CH...O type hydrogen bond. This indicates that the methylene group retains a negative polarization at the edge of the minor groove in the absence of a hydrophilic 2'-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA antisense oligonucleotides (ASOs) showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moieties in nucleic acid structure and emphasizes greater use of this bioisostere to improve the properties of nucleic acids for therapeutic and diagnostic applications.     

A left-handed supramolecular assembly around a right-handed screw axis in the crystal structure of homo-DNA

Duplexes of homo-DNA, a hexose analogue of DNA and autonomous pairing system, exhibit unusual conformational features, and in the crystal structure create a unique double-helical supramolecular motif whose main characteristic is a handedness that is opposite to that of the underlying crystallographic symmetry.     

Highly Stable Duplex Formation by Artificial Nucleic Acids Acyclic Threoninol Nucleic Acid (aTNA) and Serinol Nucleic Acid (SNA) with Acyclic Scaffolds

The stabilities of duplexes formed by strands of novel artificial nucleic acids composed of acyclic threoninol nucleic acid (aTNA) and serinol nucleic acid (SNA) building blocks were compared with duplexes formed by the acyclic glycol nucleic acid (GNA), peptide nucleic acid (PNA), and native DNA and RNA. All acyclic nucleic acid homoduplexes examined in this study had significantly higher thermal stability than DNA and RNA duplexes. Melting temperatures of homoduplexes were in the order of aTNA>PNA≈GNA≥SNA?RNA>DNA. Thermodynamic analyses revealed that high stabilities of duplexes formed by aTNA and SNA were due to large enthalpy changes upon formation of duplexes compared with DNA and RNA duplexes. The higher stability of the aTNA homoduplex than the SNA duplex was attributed to the less flexible backbone due to the methyl group of D ‐threoninol on aTNA, which induced clockwise winding. Unlike aTNA, the more flexible SNA was able to cross‐hybridize with RNA and DNA. Similarly, the SNA/PNA heteroduplex was more stable than the aTNA/PNA duplex. A 15‐mer SNA/RNA was more stable than an RNA/DNA duplex of the same sequence.