Stability Of Oligonucleotides Research Articles

Assignment and NOE Analysis of 2‘-Hydroxyl Protons in RNA: Implications for Stabilization of RNA A-Form Duplexes

The ribose 2'-OH hydroxyl group distinguishes RNA from DNA. The 2'-OH hydroxyl protons are responsible for differences in conformation, hydration, and thermodynamic stability of RNA and DNA oligonucleotides. Additionally, the 2'-OH group plays a central role in RNA catalysis. This important group lies in the shallow groove of RNA, where it is involved in a network of hydrogen bonds with water molecules stabilizing RNA A-form duplexes. Structural and dynamical information on 2'-OH hydroxyl protons is essential to understand their respective roles. Here we report the 2'-OH hydroxyl proton assignments for a 30mer RNA, the HIV-2 transactivation region, in water using solution NMR techniques. We provide structural information on 2'-OH hydroxyl groups in the form of orientational preferences contradicting the paradigm that the 2'-OH hydroxyl typically points away from the ribose H1' proton.

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.

Clamping down on weak terminal base pairs: oligonucleotides with molecular caps as fidelity-enhancing elements at the 5'- and 3'-terminal residues.

The base-pairing fidelity of oligonucleotides depends on the identity of the nucleobases involved and the position of matched or mismatched base pairs in the duplex. Nucleobases forming weak base pairs, as well as a terminal position favor mispairing. We have searched for 5'-appended acylamido caps that enhance the stability and base-pairing fidelity of oligonucleotides with a 5'-terminal 2'-deoxyadenosine residue using combinatorial synthesis and MALDI-monitored nuclease selections. This provided the residue of 4-(pyren-1-yl)butyric acid as a lead. Lead optimization gave (S)-N-(pyren-1-ylmethyl)pyrrolidine-3-phosphate as a cap that increases duplex stability and base-pairing fidelity. For the duplex of 5'-AGGTTGAC-3' with its fully complementary target, this cap gives an increase in the UV melting point T(m) of +10.9 degrees C. The T(m) is 6.3-8.3 degrees C lower when a mismatched nucleobase faces the 5'-terminal dA residue. The optimized cap can be introduced via automated DNA synthesis. It was combined with an anthraquinone carboxylic acid residue as a cap for the 3'-terminal residue. A doubly capped dodecamer thus prepared gives a melting point decrease for double-terminal mismatches that is 5.7-5.9 degrees C greater than that for the unmodified control duplex.

Synthesis and Stability of Oligonucleotides Containing Acyclic Achiral Nucleoside Analogues with Two Base Moieties.

Nucleotide building blocks with two base moieties were synthesized and incorporated into oligonucleotides. One of the two bases is involved in base pairing within the double helix, while the other base is sticking out of the minor groove. This system may be used for presenting sequence information at the outside of the helix.

Selective Circular Oligonucleotide Probes Improve Detection of Point Mutations in DNA

The synthesis of disulfide-cross-linked circular oligonucleotides, employing two different approaches, was accomplished. Several circular oligomers, which bear a C(5)-aminoalkyl-tethered thymidine unit, were labeled with photoluminescent europium(III) chelates. All circular structures were thoroughly characterized with denaturing PAGE and electrospray-ionization mass spectrometry. It was demonstrated that the disulfide cross-linking, resulting in circularization, considerably increases the enzymatic stability of phosphodiester oligonucleotides. In addition, UV melting experiments, followed, where possible, by extraction of thermodynamic parameters, revealed that several circular oligomers appear to be more selective towards their complementary targets than their corresponding linear precursors. Finally, the mixed-phase hybridization experiments have demonstrated that use of circular probes indeed improves the selectivity in the detection of DNA point mutations.

Locked nucleic acid: a potent nucleic acid analog in therapeutics and biotechnology.

Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing very high affinity and excellent specificity toward complementary DNA and RNA, and LNA oligonucleotides have been applied as antisense molecules both in vitro and in vivo. In this review, we briefly describe the basic physiochemical properties of LNA and some of the difficulties that may be encountered when applying LNA technology. The central part of the review focuses on the use of LNA molecules in regulation of gene expression, including delivery to cells, stability, unspecific effects, toxicity, pharmacokinetics, and design of LNA oligonucleotides. The last part evaluates LNA as a diagnostic tool in genotyping.

Synthesis and stability of oligonucleotides containing acyclic achiral nucleoside analogues with two base moieties.

[structure: see text] Nucleotide building blocks with two base moieties were synthesized and incorporated into oligonucleotides. One of the two bases is involved in base pairing within the double helix, while the other base is sticking out of the minor groove. This system may be used for presenting sequence information at the outside of the helix.

The influence of size on the thermal stability of oligonucleotides: the case of AT sequences

The influence of size on the thermal stability of oligonucleotides: the case of AT sequences

2′‐O‐[2‐(Amino)‐2‐oxoethyl] Oligonucleotides.

Oligonucleotides with novel modifications, 2‘-O-[2-(amino)-2-oxoethyl] (2‘-O-NAc), 2‘-O-[2-(methylamino)-2-oxoethyl] (2‘-O-NMAc), 2‘-O-[2-(dimethylamino)-2-oxoethyl] (2‘-O-DMAc), and 2‘-O-[2-[[2-(dimethylamino)ethyl]amino]-2-oxoethyl] (2‘-O-DMAEAc), have been synthesized. These modified oligonucleotides exhibit high binding affinity to complementary RNA (and not to DNA) and considerably enhance the nuclease stability of oligonucleotides with t1/2 > 24 h.

2'-O-[2-(amino)-2-oxoethyl] oligonucleotides.

[structure: see text] Oligonucleotides with novel modifications, 2'-O-[2-(amino)-2-oxoethyl] (2'-O-NAc), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMAc), 2'-O-[2-(dimethylamino)-2-oxoethyl] (2'-O-DMAc), and 2'-O-[2-[[2-(dimethylamino)ethyl]amino]-2-oxoethyl] (2'-O-DMAEAc), have been synthesized. These modified oligonucleotides exhibit high binding affinity to complementary RNA (and not to DNA) and considerably enhance the nuclease stability of oligonucleotides with t(1/2) > 24 h.

A frequency Domain Phase/Modulation Technique for Intracellular Multicomponent Fluorescence Analysis: Technical Approach and Pharmacological Applications

A UV confocal laser microspectrofluorimeter prototype has been adapted for fluorescence lifetime measurements by using a frequency-domain phase/modulation method (modulation frequency 1 to 200 MHz, lifetime resolution: tenth of a ns). This technique enables excited state lifetimes of several fluorescent components to be resolved and determined. Through a global analysis, specific spectral contribution of each species can be monitored with no need to use model spectra. This approach is efficient to distinguish strongly overlapping components (e.g., intracellular multicomponent fluorescence signal) which otherwise cannot easily be discriminated from each other. Experimental set‒up is first described. Application dealing with an antisense oligonucleotide (a synthetic dT15oligomer analogue containing isopolar, non-isosteric, phosphonate [3'-O-P-CH2-O-5'] internucleotide linkages) bound to a fluorescent label (tetramethylrhodamine dye), in various solutions and interacting with living cells, is then presented. This is of a major interest for antisense and/or antigene strategies which have recently been developed as efficient ways to cure viral and/or malignant diseases. The frequency-domain phase/modulation technique enabled oligonucleotide stability inside the cells to be checked.

Down-regulation of VEGF mRNA expression by 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) antisense oligonucleotides and investigation of non-target gene expression.

We studied the properties of 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) oligonucleotides as antisense molecules. Compared to a phosphorothioate (PS) DNA and RNA heteroduplex, a duplex of an ENA/PS/ENA gapmer with RNA was a more effective substrate for RNase H-mediated cleavage. We designed ENA antisense oligonucleotides (AON) targeting human vascular endothelial growth factor (VEGF) mRNA. ENA AON with a cationic polymer considerably down-regulated VEGF mRNA expression, while no down-regulation by PS AON was observed. We also investigated the influence on the gene expression of genes other than VEGF by a gene database homology search and RT-PCR analysis. This method turned out to be an efficient approach to search for non-target genes sequence-specifically down-regulated by AON. In rat plasma, an ENA/PS/ENA gapmer was very stable after 24 hr, while a bridged nucleic acid (BNA) or locked nucleic acid (LNA) oligonucleotide and a PS oligonucleotide were half degraded in 4 hr. The high stability of ENA oligonucleotides in plasma would make ENA oligonucleotides ideal for in vivo studies.

Role of 2'-OH on thermodynamic stability and structure of chimeric oligonucleotides.

Thermodynamic parameters of RNA/DNA chimeric duplexes were obtained systematically. Stability of the duplexes including chimeric junctions was context dependent, although CD spectra showed less structural perturbation by the junctions. Nearest-neighbor delta G degree 37 values for these chimeric RNA-DNA junctions suggested that 2'-OH on the 5'-side nucleotide at the junction may be the determinant for the nearest-neighbor stability of the chimeric junction.

HIV-1 nucleocapsid protein activates transient melting of least stable parts of the secondary structure of TAR and its complementary sequence

The nucleocapsid protein NCp7 of HIV-1 possesses a nucleic acid chaperone activity that is critical in minus and plus strand transfer during reverse transcription. The minus strand transfer notably relies on the ability of NCp7 to destabilize the stable stem with five contiguous, double-stranded segments of both the TAR sequence at the 3′ end of the viral genome and the complementary sequence, cTAR, in minus strong-stop DNA. In order to examine the nature and the extent of NCp7 destabilizing activity, we investigated, by absorbance and fluorescence spectroscopy, the interaction of TAR and cTAR with a (12–55)NCp7 peptide containing the zinc-finger motifs but lacking the ability to aggregate the oligonucleotides. The absorbance changes in the UV band of cTAR show that seven to eight base-pairs, on average, are melted per oligonucleotide at a ratio of one peptide to 7.5 nucleotides. In contrast, the melting of TAR does not exceed an average of one base-pair per oligonucleotide. This may be linked to the greater stability of TAR, since a strong correlation between NCp7 destabilizing effect and oligonucleotide stability was observed. The effect of (12–55)NCp7 on the stem terminus was investigated by using a cTAR molecule doubly labeled at the 3′ and 5′ ends by a donor/acceptor couple. In the absence of the peptide, about 80 % of the oligonucleotides are in a dark non-fluorescent state, having a close proximity of the two dyes. The remaining 20 % are distributed between three fluorescent species, having either the terminal segment, the two terminal segments or all segments of the stem melted. This is in line with a fraying mechanism wherein the stem terminus fluctuates rapidly between open and closed states. Addition of (12–55)NCp7 shifts the equilibrium toward the open species, suggesting that NC enhances fraying of the stem terminus. Taken together, our data suggest that NCp7 activates the transient opening of base-pairs in the least stable parts of the stem. Also, this activity of NCp7 was found to be dependent on the zinc-finger motifs, since no melting was observed with a fingerless NCp7 peptide.

Characterization of oligonucleotide/lipid interactions in submicron cationic emulsions: influence of the cationic lipid structure and the presence of PEG-lipids

We have recently described how oligonucleotide (ON) stability and release from O/W cationic emulsions are governed by the lipid composition. The aim of the present paper was to investigate the properties of the ON/lipid complexes through fluorescence resonance energy transfer (FRET), size, surface tension measurements and cryomicroscopy. Starting from a typical emulsion containing stearylamine as a cationic lipid, the influence of the lipid structure (monocationic molecules bearing mono or diacyl chains, or polycations) as well as of the presence of PEGylated lipids, were studied. The presence of a positive charge on the droplet surface clearly contributed to enhance the ON interaction with lipid monolayers and to bring the ON molecules closer to the interface. Hydrophobic interactions through the acyl chains were shown to further enhance the anchorage of the ON/lipid complexes. In contrast, the incorporation of PEGylated lipids acted as a barrier against the establishment of electrostatic bindings, the polyethyleneglycol chains acting themselves as interaction sites for the ON leading to hydrophilic complexes. Similar features were observed for the polycationic lipid, and cryomicroscopy revealed the existence of bridges of various intensities between the droplets of the emulsion containing either PEG or the polycation, probably because of the configuration of the ON at the interface.

ChemInform Abstract: Synthesis and Duplex Stability of Oligonucleotides Containing 7‐Vinyl‐7‐deazaguanine as a Strong Electron‐Donating Nucleobase.

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Phosphodiester and phosphorothioate oligonucleotide condensation and preparation of antisense nanoparticles

Protamine is a cationic peptide with a molecular mass of approx. 4000 Da that is able to condense DNA. In the present study it was used to complex antisense oligonucleotides (ODNs) and to form solid particles with initial diameters of 90–150 nm. The reaction was very rapid and occurred by simple mixing of diluted solutions of the polycation with the oligonucleotide. The aggregation was dependent on the oligonucleotide chain length and the protamine/ODN mass ratio. Particle formation required a minimal chain length of nine nucleotides and a mass ratio of 0.5:1. The particle surface charge and the number of particles depended on the mass ratio. With increasing amounts of the peptide, the number of particles and the ζ potential increased. Both negatively and positively charged particles improved the stability of oligonucleotides against DNase I digestion. Above a mass ratio of 2.5:1 no degradation was found. The uptake of unbound rhodamine-labelled ODNs and its complexes with protamine was determined with Vero cells under in vitro cell culture conditions at 37°C and 4°C. At 37°C the cellular uptake increased with increasing mass ratio. The internalized oligonucleotides were localized in the cytoplasm and in the nucleus of the cells. When Vero cells were treated with these samples at 4°C for 4 h, no fluorescence could be detected inside the cells. Therefore, our data indicate an energy dependent endocytotic uptake mechanism. In contrast, spermine and spermidine, which are also known condensation agents, did not aggregate with oligonucleotides into nanoparticles under the same conditions.

ChemInform Abstract: Molecular-Dynamics Studies of Single-Stranded Hexitol, Altritol, Mannitol, and Ribose Nucleic Acids (HNA, MNA, ANA, and RNA, Resp.) and of the Stability of HNA×RNA, ANA×RNA, and MNA×RNA Duplexes.

The influence of the orientation of a 3′-OH group on the conformation and stability of hexitol oligonucleotides in complexes with RNA and as single strands in aqueous solution was investigated by molecular-dynamics (MD) simulations with AMBER 4.1. The particle mesh Ewald (PME) method was used for the treatment of long-range electrostatic interactions. An equatorial orientation of the 3′-OH group in the single-stranded D-mannitol nucleic acid (MNA) m(GCGTAGCG) and in the complex with the RNA r(CGCAUCGC) has an unfavorable influence on the helical stability. Frequent H-bonds between the 3′-OH group and the O−C(6′) of the phosphate backbone of the following nucleotide explain the distorted conformation of the MNA⋅RNA complex as well as that of the single MNA strand. This is consistent with experimental results that show lowered hybridization potentials for MNA⋅RNA complexes. An axial orientation of the 3′-OH group in the D-altritol nucleic acid (ANA) a(GCGTAGCG) leads to a stable complex with the complementary RNA r(CGCAUCGC), as well as to a more highly preorganized single-stranded ANA chain. The averaged conformation of the ANA⋅RNA complex is similar to that of A-RNA, with only minor changes in groove width, helical curvature, and H-bonding pattern. The relative stabilities of ANA⋅RNA vs. HNA⋅RNA (HNA=D-hexitol nucleic acid without 3′-OH group) can be explained by differences in restricted movements, H-bonds, and solvation effects.

Synthesis and duplex stability of oligonucleotides containing 7-vinyl-7-deazaguanine as a strong electron-donating nucleobase

We synthesized 7-vinyl-7-deazaguanine ( vzG), an N7-modified guanine base, which has a smaller oxidation potential than G and 7-deazaG. The incorporation of vzG into oligonucleotides was achieved by phosphoramidite chemistry on a DNA synthesizer. The T m of the duplex containing vzG was higher than that of the corresponding duplex containing G or 7-deazaG. The stabilization of the duplex containing vzG is due to the favorable enthalpy change by increased π-stacking between the vinyl group and the 5′-flanking base.

2'-O-[2-[N,N-(dialkyl)aminooxy]ethyl]-modified antisense oligonucleotides.

[structure] Oligonucleotides with two novel modifications, 2'-O-¿2-[N, N-(dimethyl)aminooxy]ethyl¿ (2'-O-DMAOE) and 2'-O-¿2-[N, N-(diethyl)aminooxy]ethyl¿ (2'-O-DEAOE), have been synthesized. These modifications exhibit high binding affinity to target RNA (and not to DNA) and enhance the nuclease stability of oligonucleotides considerably with t(1/2) > 24 h as a phosphodiester.