Synthesis and RNA-selective hybridization of α-l-ribo- and β-d-lyxo-configured oligonucleotides

Three α-l-ribofuranosyl analogues of RNA nucleotides (α-l-RNA analogues) have been synthesized and incorporated into oligonucleotides using the phosphoramide approach on an automated DNA synthesizer. The 4′-C-hydroxymethyl-α-l-ribofuranosyl thymine monomer was furthermore synthesized. Relative to the unmodified duplexes, incorporation of a single α-l-RNA monomer into a DNA strand leads to reduced thermal stability of duplexes with DNA complements but unchanged thermal stability of duplexes with RNA complements, whereas incorporation of more than one α-l-RNA monomer lead to moderately decreased thermal stability also of duplexes with RNA complements. Efficient hybridization with an RNA complement and no melting transition with a DNA complement were observed with stereoregular chimeric oligonucleotides composed of a mixture of α-l-RNA and affinity enhancing α-l-LNA monomers (α-l-ribo-configured locked nucleic acid). Furthermore, duplexes formed between oligodeoxynucleotides containing an α-l-RNA monomer and complementary RNA were good substrates for Escherichia coli RNase H. RNA-selective hybridization was also achieved by the incorporation of 1-(4-C-hydroxymethyl-β-d-lyxofuranosyl)thymine monomers into a DNA strand, whereas stable duplexes were formed with both complementary DNA and RNA when these monomers were incorporated into an RNA strand.     

Synthesis and thermal denaturation studies of novel 2'-O,3'-C-linked bicyclic oligonucleotides with a methoxy or a piperazino group facing the major groove of nucleic acid duplexes

With the aim of evaluating duplex stabilities of oligonucleotides (ONs) with major groove facing functionalities, two novel 2'-O,3'-C-linked bicyclic nucleoside phosphoramidite building blocks were synthesized by routes involving regioselective O-methylation or piperazine attachment using carbonyldiimidazole coupling chemistry. The novel monomers were incorporated into 9-mer mixed base ONs and the thermal stability toward complementary single stranded DNA and RNA was evaluated by thermal denaturation experiments. O-methylated ONs confirmed the applicability of the functionalized bicylic sugar unit for attachment of groups facing the major groove and satisfactory binding properties towards the RNA complement were observed. For the piperazino modified ONs, experiments were performed in aqueous buffers with low (40 mM) and medium (110 mM) salt concentrations, at pH 5 and pH 7. A change from a medium to a low salt concentration induced a significant relative increase in the thermal stability of modified duplexes toward both DNA and RNA complements, which suggests protonation of the piperazino group under the experimental conditions applied.     

Oligonucleotides with 1,4-dioxane-based nucleotide monomers

An epimeric mixture of H-phosphonates 5R and 5S has been synthesized in three steps from known secouridine 1. Separation of the epimers has been accomplished by RP-HPLC, allowing full characterization and incorporation of monomers X and Y into 9-mer oligonucleotides using H-phosphonates building blocks 5R and 5S, respectively. A single incorporation of either monomer X or monomer Y in the central position of a DNA 9-mer results in decreased thermal affinity toward both DNA and RNA complements (ΔT(m) = -3.5 °C/-3.5 °C for monomer X and ΔT(m) = -11.0 °C/-6.5 °C for monomer Y). CD measurements do not reveal major rearrangements of the duplexes formed, but molecular modeling suggests that local rearrangement of the sugar phosphate backbone and decreased base interactions with neighboring bases might be the origin of the decreased stability of duplexes.     

Synthesis and pairing properties of alpha-tricyclo-DNA

We describe the synthesis of the phosphoramidite building blocks of alpha-tricyclo-DNA (alpha-tc-DNA) covering all four natural bases, starting from the already known corresponding alpha-tc-nucleosides. These building blocks were used for the preparation of three alpha-tc-oligonucleotide 10-mers representing a homopurine, a homopyrimidine, and a mixed purine/pyrimidine base sequence. The base-pairing properties with complementary parallel and antiparallel oriented DNA and RNA were studied by UV-melting analysis and CD spectroscopy. We found that alpha-tc-DNA binds preferentially to parallel nucleic acid complements through Watson-Crick duplex formation, with a preference for RNA over DNA. In comparison with natural DNA, alpha-tc-DNA shows equal to enhanced affinity to RNA and also pairs to antiparallel DNA or RNA complements, although with much lower affinity. In the mixed-base sequence these antiparallel duplexes are of the reversed Watson-Crick type, while in the homopurine/homopyrimidine sequences Hoogsteen and/or reversed Hoogsteen pairing is observed. Antiparallel duplex formation of two alpha-tc-oligonucleotides was also observed, although the thermal stability of this duplex was surprisingly low. The base-pairing properties of alpha-tc-DNA are discussed in the context of alpha-DNA, alpha-RNA, and alpha-LNA.     

C‐5 Propynyl Modifications Enhance the Mechanical Stability of DNA

Increased thermal or mechanical stability of DNA duplexes is desired for many applications in nanotechnology or ‐medicine where DNA is used as a programmable building block. Modifications of pyrimidine bases are known to enhance thermal stability and have the advantage of standard base‐pairing and easy integration during chemical DNA synthesis. Through single‐molecule force spectroscopy experiments with atomic force microscopy and the molecular force assay we investigated the effect of pyrimidines harboring C‐5 propynyl modifications on the mechanical stability of double‐stranded DNA. Utilizing these complementary techniques, we show that propynyl bases significantly increase the mechanical stability if the DNA is annealed at high temperature. In contrast, modified DNA complexes formed at room temperature and short incubation times display the same stability as non‐modified DNA duplexes.     

Bicyclo[3.2.1]amide-DNA: a chiral,nonchiroselective base-pairing system

The design, synthesis, and base-pairing properties of bicyclo[3.2.1]amide-DNA (bca-DNA), a novel phosphodiester-based DNA analogue, are reported. This analogue consists of a conformationally constrained backbone entity, which emulates a B-DNA geometry, to which the nucleo-bases were attached through an extended, acyclic amide linker. Homobasic adenine-containing bca decamers form duplexes with complementary oligonucleotides containing bca, DNA, RNA, and, surprisingly, also L-RNA backbones. UV and CD spectroscopic investigations revealed the duplexes with D- or L-complements to be of similar stability and enantiomorphic in structure. Bca oligonucleotides that contain all four bases form strictly antiparallel, left-handed complementary duplexes with themselves and with complementary DNA, but not with RNA. Base-mismatch discrimination is comparable to that of DNA, while the overall thermal stabilities of bca-oligonucleotide duplexes are inferior to those of DNA or RNA. A detailed molecular modeling study of left- and right-handed bca-DNA-containing duplexes showed only minor changes in the backbone structure and revealed a structural switch around the base-linker unit to be responsible for the generation of enantiomorphic duplex structures. The obtained data are discussed with respect to the structural and energetic role of the ribofuranose entities in DNA and RNA association.     

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.     

Synthesis and properties of nucleic acid analogues consisting of a benzene-phosphate backbone

[Chemical reaction: See text] The synthesis and properties of a nucleic acid analogue consisting of a benzene-phosphate backbone are described. The building blocks of the nucleic acid analogue are composed of bis(hydroxymethyl)benzene residues connected to nucleobases via the biaryl-like axis. Stabilities of the duplexes were studied by thermal denaturation. It was found that the thermal stabilities of the duplexes composed of the benzene-phosphate backbone are highly dependent on their sequences. The duplexes with the benzene-phosphate backbone comprised of the mixed sequences were thermally less stable than the natural DNA duplexes, whereas that composed of the homopyrimidine and homopurine sequences was thermally and thermodynamically more stable than the corresponding natural DNA duplex. It was suggested that the analogues more efficiently stabilize the duplexes in a B-form duplex rather than in an A-form duplex. Thus, the duplexes consisting of the benzene-phosphate backbone, especially composed of the homopyrimidine and homopurine sequences, may offer a novel structural motif useful for developing novel materials applicable in the fields of bio- and nanotechnologies.     

Multilabeled pyrene-functionalized 2'-amino-LNA probes for nucleic acid detection in homogeneous fluorescence assays

Homogeneous fluorescence assays for detection of nucleic acids are widely used in biological sciences. Typically, probes such as molecular beacons that rely on distance-dependent fluorescence quenching are used for such assays. Less attention has been devoted to tethering a single kind of fluorophores to oligonucleotides and exploiting hybridization-induced modulation of fluorescence intensity for nucleic acid detection. Herein, thermal denaturation experiments and fluorescence properties of oligodeoxyribonucleotides containing one or more 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA monomer(s) X are described. These pyrene-functionalized 2'-amino-LNAs display large increases in thermal stability against DNA/RNA complements with excellent Watson-Crick mismatch discrimination. Upon duplex formation of appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA probes and complementary DNA/RNA, intensive fluorescence emission with quantum yields between 0.28 and 0.99 are observed. Quantum yields of such magnitudes are unprecedented among pyrene-labeled oligonucleotides. Molecular modeling studies suggest that the dioxabicyclo[2.2.1]heptane skeleton and amide linkage of monomer X fix the orientation of the pyrene moiety in the minor groove of a nucleic acid duplex. Interactions between pyrene and nucleobases, which typically lead to quenching of fluorescence, are thereby reduced. Duplexes between multiple modified probes and DNA/RNA complements exhibit additive increases in fluorescence intensity, while the fluorescence of single stranded probes becomes increasingly quenched. Up to 69-fold increase in fluorescence intensity (measured at lambda(em) = 383 nm) is observed upon hybridization to DNA/RNA. The emission from duplexes of multiple modified probes and DNA/RNA at concentrations down to less than 500 nM can easily be seen by the naked eye using standard illumination intensities.     

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 modelling of DNA junction and minor groove zipper motifs incorporating the double-headed nucleoside 5'(S)-C-(thymine-1-ylmethyl)thymidine

A nucleoside with two nucleobases, a so-called double-headed nucleoside, 5'(S)-C-(thymine-1-ylmethyl)thymidine 3, is synthesised and incorporated into oligonucleotides. The additional nucleobase is hereby positioned in the minor groove of the duplexes, which are formed with complementary DNA and RNA-sequences. Slight thermal destabilisation of these duplexes as compared to unmodified duplexes is observed. With other target sequences forming bulged duplexes or three-way junctions, no additional influence of the additional base on the thermal stability is observed. On the other hand, a base-base stacking interaction and subsequent stabilisation is observed when two double-headed nucleotide moieties are positioned in two complementary DNA-sequences forming a DNA-zipper motif.     

Metal binding to ligand-containing peptide nucleic acids

The substitution of nucleobases in nucleic acid duplexes with ligands that have high affinity for transition metal ions creates metal-binding sites at specific locations within the duplexes. Several studies on the incorporation of metal ions into DNA and peptide nucleic acid (PNA) duplexes have suggested that the stability constant of the metal complex formed within the duplexes is a primary determinant of the thermal stability of the duplexes. To understand this relationship, we have synthesized two PNA monomers that carry the same ligand, namely 8-hydroxyquinoline, but have this ligand attached differently to the PNA backbone. The PNA monomers have been incorporated into PNA duplexes. UV and CD spectroscopy and calorimetric studies of the 8-hydroxyquinoline-PNA duplexes showed that the effect of the stability of the metal complex on the PNA duplexes was significantly modulated by the steric relationship between the complex and the duplex. This information is useful for the construction of hybrid inorganic-nucleic acid nanostructures.     

Stability of Hoogsteen‐Type Triplexes – Electrostatic Attraction between Duplex Backbone and Triplex‐Forming Oligonucleotide (TFO) Using an Intercalating Conjugate

Syntheses are described for two novel twisted intercalating nucleic acid (TINA) monomers where the intercalator comprises a benzene ring linked to a naphthalimide moiety via an ethynediyl bridge. The intercalators Y and Z have a 2‐(dimethylamino)ethyl and a methyl residue on the naphthalimide moiety, respectively. When used as triplex‐forming oligonucleotides (TFOs), the novel naphthalimide TINAs show extraordinary high thermal stability in Hoogsteen‐type triplexes and duplexes with high discrimination of mismatch strands. DNA Strands containing the intercalator Y show higher thermal triplex stability than DNA strands containing the intercalator Z . This observation can be explained by the ionic interaction of the protonated dimethylamino group under physiological conditions, targeting the negatively charged phosphate backbone of the duplex. This interaction leads to an extra binding mode between the TFO and the duplex, in agreement with molecular‐modeling studies. We believe that this is the first example of an intercalator linking the TFO to the phosphate backbone of the duplex by an ionic interaction, which is a promising tool to achieve a higher triplex stability.     

N-methylpiperazinocarbonyl-2',3'-BcNA and 4'-C-(N-methylpiperazino)methyl-DNA: introduction of basic functionalities facing the major groove and the minor groove of a DNA:DNA duplex

Piperazino-functionalized 2',3'-BcNA and 4'-C-hydroxymethyl-DNA are appropriate molecular architectures for the introduction of basic functionalities facing the major groove and the minor groove of nucleic acid duplexes, respectively. 4'-C-(N-Methylpiperazino)methyl-DNA monomers induce significantly increased thermal stability of a DNA:DNA duplex.     

The role of organic linkers in directing DNA self-assembly and significantly stabilizing DNA duplexes

We show a simple method to control both the stability and the self-assembly behavior of DNA structures. By connecting two adjacent duplexes with small synthetic linkers, factors such as linker rigidity and DNA strand orientation can increase the thermal denaturation temperature of 17 base-pair duplexes by up to 10 °C, and significantly increase the cooperativity of melting of the two duplexes. The same DNA sequence can thus be tuned to melt at vastly different temperatures by selecting the linker structure and DNA-to-linker connectivity. In addition, a small rigid m-triphenylene linker directly affects the self-assembly product distribution. With this linker, changes in the orientation of the linked strands (e.g., 5'3' vs 3'3') can lead to dramatic changes in the self-assembly behavior, from the formation of cyclic dimer and tetramer to higher-order oligomers. These variations can be readily predicted using a simple strand-end alignment model.     

2'-Ribose-ferrocene oligonucleotides for electronic detection of nucleic acids

We have synthesized two novel phosphoramidites with a ferrocenyl moiety at the 2'-ribose position linked through a butoxy linker. Using automated DNA/RNA synthesis techniques, oligonucleotides containing ferrocene at various positions were prepared and characterized by HPLC, MALDI-TOF mass spectrometry, and electrochemistry. Thermal stability studies of the ferrocene-modified DNA duplexes revealed that introduction of one or two ferrocenyl complexes does not result in an observed change of the T(m) values of the corresponding DNA duplexes when compared to the nonmodified hybrids. These data indicate that the introduction of a ferrocenyl group at the 2'-position of the ribose ring containing either a purine or pyrimidine base has no effect on the stability of the modified DNA. The electrochemical behavior of the ferrocene-containing DNA was examined by cyclic voltammetry. The modified 2'-ferrocene-oligonucleotides are electrochemically active and can be used as signaling probes for the electronic detection of nucleic acids on bioelectronic sensors.     

Synthesis,Characterization, and Repair of a Flexible O6‐2′‐Deoxyguanosine‐alkylene‐O6‐2′‐deoxyguanosine Intrastrand Cross‐Link

Oligonucleotides tethered by an alkylene linkage between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines, which lack a phosphodiester linkage between these residues, have been synthesized as a model system of intrastrand cross‐linked (IaCL) DNA. UV thermal denaturation studies of duplexes formed between these butylene‐ and heptylene‐linked oligonucleotides with their complementary DNA sequences revealed about 20 °C reduction in stability relative to the unmodified duplex. Circular dichroism spectra of the model IaCL duplexes displayed a signature characteristic of B‐form DNA, suggesting minimal global perturbations are induced by the lesion. The model IaCL containing duplexes were investigated as substrates of O⁶‐alkylguanine DNA alkyltransferase (AGT) proteins from human and E. coli (Ada‐C and OGT). Human AGT was found to repair both model IaCL duplexes with greater efficiency towards the heptylene versus butylene analog adding to our knowledge of substrates this protein can repair.     

Synthesis and conformational properties of oligonucleotides incorporating 2'-O-phosphorylated ribonucleotides as structural motifs of pre-tRNA splicing intermediates

To synthesize oligonucleotides containing 2'-O-phosphate groups, four kinds of ribonucleoside 3'-phosphoramidite building blocks 6a-d having the bis(2-cyano-1,1-dimethylethoxy)thiophosphoryl (BCMETP) group were prepared according to our previous phosphorylation procedure. These phosphoramidite units 6a-d were not contaminated with 3'-regioisomers and were successfully applied to solid-phase synthesis to give oligodeoxyuridylates 15, 16 and oligouridylates 21, 22. Self-complementary Drew-Dickerson DNA 12mers 24-28 replaced by a 2'-O-phosphorylated ribonucleotide at various positions were similarly synthesized. In these syntheses, it turned out that KI(3) was the most effective reagent for oxidative desulfurization of the initially generated thiophosphate group to the phosphate group on polymer supports. Without using this conversion step, a tridecadeoxyuridylate 17 incorporating a 2'-O-thiophosphorylated uridine derivative was also synthesized. To investigate the effect of the 2'-phosphate group on the thermal stability and 3D-structure of DNA(RNA) duplexes, T(m) measurement of the self-complementary oligonucleotides obtained and MD simulation of heptamer duplexes 33-36 were carried out. According to these analyses, it was suggested that the nucleoside ribose moiety phosphorylated at the 2'-hydroxyl function predominantly preferred C2'-endo to C3'-endo conformation in DNA duplexes so that it did not significantly affect the stability of the DNA duplex. On the other hand, the 2'-modified ribose moiety was expelled to give a C3'-endo conformation in RNA duplexes so that the RNA duplexes were extremely destabilized.     

7,8-Dihydropyrido[2,3-d]pyrimidin-2-one; a bicyclic cytosine analogue capable of enhanced stabilisation of DNA duplexes

Incorporation of a bicyclic cytosine analogue, 3-beta-D-(2'-deoxyribofuranosyl)-7,8-dihydropyrido[2,3-d]pyrimidine, into synthetic DNA duplexes results in a greatly enhanced thermal stability (3-4 degrees C per modification) compared to the corresponding unmodified duplex.