Oligodeoxynucleotide Analogues Research Articles

Automated Synthesis and Purification of Guanidine-Backbone Oligonucleotides.

This protocol describes a method based on iodine and a base as mild coupling reagents to synthetize deoxyribonucleic guanidines (DNGs)-oligodeoxynucleotide analogues with a guanidine backbone. DNGs display unique properties, such as high cellular uptake with low toxicity and increased stability against nuclease degradation, but have been impeded in their development by the requirement for toxic and iterative manual synthesis protocols. The novel synthesis method reported here eliminates the need for the toxic mercuric chloride and pungent thiophenol that were critical to previous DNG synthesis methods and translates their synthesis to a MerMadeTM 12 automated oligonucleotide synthesizer. This method can be used to synthesize DNG strands up to 20 bases in length, along with 5'-DNG-DNA-3' chimeras, at 1- to 5-μmol scales in a fully automated manner. We also present detailed and accessible instructions to adapt the MerMadeTM 12 oligonucleotide synthesizer to enable the parallel synthesis of DNG and DNA/RNA oligonucleotides. Because DNG linkages alter the overall charge of the oligonucleotides, we also describe purification strategies to generate oligonucleotides with varying lengths and numbers of DNGs, based on extraction or preparative-scale gel electrophoresis, along with methods to characterize the final products. Overall, this article provides an overview of the synthesis, purification, and handling of DNGs and mixed-charge DNG-DNA oligonucleotides. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of a MerMadeTM synthesizer for guanidine couplings Basic Protocol 2: Synthesis of DNG strands on a MerMadeTM synthesizer Basic Protocol 3: Purification of DNG strands using preparative acetic acid urea (AU) PAGE Basic Protocol 4: Characterization of DNG strands using MALDI-TOF MS Basic Protocol 5: Characterization of DNG strands using AU PAGE Support Protocol 1: Synthesis of initiator-functionalized CPG Support Protocol 2: Synthesis of thiourea monomer.

Oligodeoxynucleotide Analogues of Circulating DNA Inhibit dsRNA-Induced Immune Response at the Early Stages of Signal Transduction Cascade in a Cell Type-Dependent Manner.

Oligodeoxynucleotide (ODN) analogues of cell-surface-bound circulating DNA inhibit the dsRNA-induced production of pro-inflammatory interleukin 6, interferon beta and antibacterial peptide beta-defensin 2 not only in human gingival fibroblasts, but also in human primary endothelial and transformed cells (Hela and A431). ODN analogues do not effect dendritic cells activation by poly(I:C). The data obtained indicate that the early stages of the signal transduction cascade are violated by ODN analogues and the effects depend on the cell type.

One-Electron Photooxidation and Site-Selective Strand Cleavage at 5-Methylcytosine in DNA by Sensitization with 2-Methyl-1,4-naphthoquinone-Tethered Oligonucleotides

Photosensitized one-electron oxidation was applied to discriminate a specific base site of 5-methylcytosine (mC) generated in DNA possessing a partial sequence of naturally occurring p53 gene, using a sensitizing 2-methyl-1,4-naphthoquinone (NQ) chromophore tethered to an interior of oligodeoxynucleotide (ODN) strands. Photoirradiation and subsequent hot piperidine treatment of the duplex consisting of mC-containing DNA and NQ-tethered complementary ODN led to oxidative strand cleavage selectively at the mC site, when the NQ chromophore was arranged so as to be in close contact with the target mC. The target mC is most likely to be one-electron oxidized into the radical cation intermediate by the sensitization of NQ. The resulting mC radical cation may undergo rapid deprotonation and subsequent addition of molecular oxygen, thereby leading to its degradation followed by strand cleavage at the target mC site. In contrast to mC-containing ODN, ODN analogs with replacement of normal cytosine, thymine, adenine, or guanine at the mC site underwent less amount of such an oxidative strand cleavage at the target base site, presumably due to occurrence of charge transfer and charge recombination processes between the base radical cation and the NQ radical anion. Furthermore, well designed incorporation of the NQ chromophore into an interior of ODN could suppress a competitive strand cleavage at consecutive guanines, which occurred as a result of positive charge transfer. Thus, photosensitization by an NQ-tethered ODN led to one-electron oxidative strand cleavage exclusively at the target mC site, providing a convenient method of discriminating mC in naturally occurring DNA such as human p53 gene as a positive band on a sequencing gel.

Synthesis and Structural Properties of New Oligodeoxynucleotide Analogues Containing a 2′,5′‐Internucleotidic Squaryldiamide Linkage Capable of Formation of a Watson−Crick Base Pair with Adenine and a Wobble Base Pair with Guanine at the 3′‐Downstream Junction Site

AbstractA TpT dimer analogue (U2′sq5′T), in which the 3′‐5′ phosphodiester linkage was replaced by a 2′‐5′ squaryldiamide linkage and the 5′‐upstream T was replaced by a 3′‐deoxyuridine, was synthesized in almost quantitative yield from diethyl squarate. This new dimer structural motif was designed to eliminate the squaryldiamide skeleton‐induced overall strain in T3′sq5′T, previously incorporated into DNA fragments as a new TpT mimic, through the change in the connection mode from the 3′‐5′ linkage to a 2′‐5′ linkage. Spectral analyses of U2′sq5′T suggest that the overall structure of this dimer mimic is basically similar to that of TpT. A DNA 10mer 5′‐d(CGCAU2′sq5′TAGCC)‐3′ incorporating this dimer was synthesized. From the CD analysis, it turned out that the overall structure of a DNA duplex of 5′‐d(CGCAU2′sq5′TAGCC)‐3′/3′‐d(GCGTAATCGG)‐5′ is closer to that of the unmodified duplex than the DNA duplex of 5′‐d(CGCAT3′sq5′TAGCC)‐3′/3′‐d(GCGTAATCGG)‐5′. Interestingly, extensive Tm experiments suggest that d(CGCAU2′sq5′TAGCC)‐3′ exhibits intriguing inherent hybridization affinity not only for the completely complementary oligodeoxynucleotide 3′‐d(GCGAATCGG)‐5′, but also for 3′‐d(GCGTAGTCGG)‐5′, with a mismatched dG. The unique property of the 3′‐downstream dT moiety of U2′sq5′T − the ability to recognize both dA and dG − was also supported by more detailed computational analysis of U2′sq5′T and TpT. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Synthesis and Properties of Oligodeoxynucleotide Analogues with Bis(methylene) Sulfone Bridges.

Synthesis and Properties of Oligodeoxynucleotide Analogues with Bis(methylene) Sulfone Bridges.

Synthesis and Properties of Oligodeoxynucleotide Analogs with Bis(methylene) Sulfone Bridges

AbstractA convergent, solution‐phase synthesis was developed for the bis(methylene) sulfone‐bridged oligodeoxynucleotide analogs (SNA) 5′‐d(HOCH2‐Tso2Tso2Tso2Cso2Tso2Tso2Tso2T‐CH2SO$\rm{_{3}^{-}}$)‐3′ (35b) and 5′‐d(HOCH2‐Tso2Tso2Tso2Tso2Tso2Tso2Tso2T‐CH2SO$\rm{_{3}^{-}}$)‐3′ (34c) (SO2 corresponds to CH2SO2CH2 instead of OP(O)(O−)(O). In these, the phosphodiester linkages are replaced by non‐ionic bis(methylene) sulfone linkers. The general strategy involved convergent coupling of 3′,5′‐bishomo‐β‐D‐deoxyribonucleotide analogs functionalized at the 6′‐end (CH2C(5′)) as bromides or mesylates and at the CH2C(3′) position as thiols, with the resulting thioether being oxidized to the corresponding sulfone. A single charge was introduced at the terminal CH2C(3′) position of the octamers to increase their solubility in water. During the synthesis, it became apparent that the key intermediates generated secondary structures through either folding or aggregation in a variety of solvents. This generated unusual reactivity and was unique for very similar structures. For example, although the dimeric thiol d(BzOCH2‐Tso2C‐CH2SH) (14b) was a well‐behaved synthetic intermediate, the tetrameric thiol d(TrOCH2‐Tso2Tso2Tso2toC‐CH2SH) derived from the corresponding thioacetate was rapidly converted to a disulfide by very small amounts of oxidant (28→29, Scheme 6), while the analogous tetrameric thiol d(BzOCH2‐Tso2TsTso2T‐CH2SH) (26), differing only by a single heterocycle, was oxidized much more slowly (Bz=PhCO, Tr=Ph3C, to=2‐MeC6H4CO (at N4 of dc)). The sequence‐dependent reactivity, well known in many classes of natural products (including polypeptides), is not prominent in natural oligonucleotides. These results are discussed in light of the proposal that the repeating negative charge in nucleic acids is key to their ability to serve as genetic molecules, in particular, their capability to support Darwinian evolution. The ability of 5′‐d(HOCH2‐Tso2Tso2Tso2Cso2Tso2Tso2Tso2T‐CH2SO$\rm{_{3}^{-}}$)‐3′ (35b) to bind as a third strand to duplex DNA was also examined. No triple‐helix‐forming propensity was detected in this molecule.

Squaryl group as a new mimic of phosphate group in modified oligodeoxynucleotides: synthesis and properties of new oligodeoxynucleotide analogues containing an internucleotidic squaryldiamide linkage.

This paper describes the synthesis and properties of a new type of modified oligodeoxynucleotide containing a neutral but highly polarized squaryl group as a novel mimic of the phosphate group. A modified thymidine dimer derivative (TsqT) having a squaryldiamide linkage was synthesized in almost quantitative yield by a two-step substitution of diethyl squarate with 3'-amino-5'-O-(4,4'-dimethoxytrityl)-3'-deoxythymidine and 5'-amino-5'-deoxythymidine. The CD and UV studies of TsqT suggest that this dimer has basically a structure similar to that of TpT. The NMR studies of TsqT show a unique property, namely, that the squaryl group of TsqT is influenced by Mg2+ concentration. The ab initio calculations of TsqT showed a highly polarized structure resembling that of a phosphate group. This dimer structural motif was finally incorporated into oligodeoxynucleotides by use of the phosphoramidite approach. The hybridization affinity of these modified oligodeoxynucleotides for the complementary and mismatched oligodeoxynucleotides was studied in detail by using Tm experiments. Consequently, it turned out that in a matched duplex of 5'-d(CGCATsqTAGCC)-3'/5'-d(GGCTAATGCG)-3' the A-T base pairs at the modified site can be preserved, but instead thermal destabilization of the overall structure was observed. To estimate the structure of the duplex, two kinds of fluorescein chromophores (fluorescein (FL) and cyanine 3 (Cy3)) were introduced into the 5'-terminal site of 5'-d(GACGCATsqTAGCCGAT)-3' and 5'-d(ATCGGCTAATGCGTC)-3', respectively. The fluorescence resonance energy transfer experiments using these functionalized oligodeoxynucleotides suggest that the matched duplexes have a bent structure at the modified site. This conclusion was also strongly supported by computational MM and MD simulations.

Watson−Crick Base-Pairing Properties of Tricyclo-DNA

Tricyclo-DNA belongs to the family of conformationally restricted oligodeoxynucleotide analogues. It differs structurally from DNA by an additional ethylene bridge between the centers C(3') and C(5') of the nucleosides, to which a cyclopropane unit is fused for further enhancement of structural rigidity. The synthesis of the hitherto unknown tricyclodeoxynucleosides containing the bases cytosine and guanine and of the corresponding phosphoramidite building blocks is described, as well as a structural description of a representative of an alpha- and a beta-tricyclodeoxynucleoside by X-ray analysis. Tricyclodeoxynucleoside building blocks of all four bases were used for the synthesis of fully modified mixed-base oligonucleotides. Their Watson-Crick pairing properties with complementary DNA, RNA, and with itself were investigated by UV melting curves, CD spectroscopy, and molecular modeling. Tricyclo-DNA was found to be a very stable Watson-Crick base-pairing system. A UV melting curve analysis of the decamers tcd(pcgtgacagtt) and tcd(paactgtcacg) showed increased thermal stabilities of up to DeltaT(m)/mod. = +1.2 degrees C with complementary DNA and +2.4 degrees C with complementary RNA. With itself, tricyclo-DNA showed an increase in stability of +3.1 degrees C/base pair relative to DNA. Investigations into the thermodynamic properties of these decamers revealed an entropic stabilization and an enthalpic destabilization for the tricyclo-DNA/DNA duplexes. CD spectroscopic structural investigations indicated that tricyclo-DNA containing duplexes preferrably exist in an A-conformation, a fact which is in agreement with results from molecular modeling.

Enhancement of innate immunity against Mycobacterium avium infection by immunostimulatory DNA is mediated by indoleamine 2,3-dioxygenase.

Bacterial DNA and its synthetic immunostimulatory oligodeoxynucleotide analogs (ISS-ODN) activate innate immunity and promote Th1 and cytotoxic T-lymphocyte immune responses. Based on these activities, we investigated whether ISS-ODN could modify the course of Mycobacterium avium infection. M. avium growth in vitro was significantly inhibited by ISS-ODN treatment of human and mouse macrophages, and M. avium growth in vivo was similarly inhibited in C57BL/6 mice treated with ISS-ODN. This protective effect of ISS-ODN was largely independent of tumor necrosis factor alpha (TNF-alpha), interleukin 12 (IL-12), nitric oxide, NADPH oxidase, alpha/beta interferon (IFN-alpha/beta), and IFN-gamma. In contrast, we found that the induction of indoleamine 2,3-dioxygenase (IDO) was required for the antimycobacterial effect of ISS-ODN. To evaluate the potential for synergism between ISS-ODN and other antimycobacterial agents, treatment with a combination of ISS-ODN and clarithromycin (CLA) was tested in vitro and in vivo. ISS-ODN significantly enhanced the therapeutic effect of CLA in both human and mouse macrophages and in C57BL/6 mice. This study newly identifies IDO as being involved in the antimicrobial activity of ISS-ODN and suggests the usefulness of ISS-ODN when used in combination with conventional chemotherapy for microbial infections.

A combination treatment of c-myc antisense DNA with all-trans-retinoic acid inhibits cell proliferation by downregulating c-myc expression in small cell lung cancer.

The dysregulation of both c-myc expression and retinoid signaling pathways commonly occurs in small cell lung cancers (SCLC), frequently accompanying tumor relapse, and contributing to the poor prognosis of patients with SCLC. In this study, we investigated whether c-myc antisense oligodeoxynucleoside phosphorothioate (OPT) covering the translational initiation site of c-myc mRNA used in combination with all-trans-retinoic acid (RA) would be more effective than either agent alone in inhibiting the growth of an SCLC cell line, NCI-H82, overexpressing c-myc with amplification of this gene, and whether this combination could be an experimental therapeutic tool against SCLC. c-myc antisense OPT decreased c-myc expression in Northern and Western blot analyses, thus inducing 40% and 20% cell growth inhibition compared with scrambled and sense OPT and with scrambled four guanosine-containing OPT (p < 0.01, and p < 0.01, respectively). All-trans-RA also inhibited cell proliferation at the rate of 40% by downregulating c-myc expression. Having obtained these results, we tested the hymothesis that c-myc antisense OPT in combination with all-trans-RA may further reduce c-myc expression and lead to improved cell growth control. This combination showed a greater inhibition of cell proliferation than either agent given alone (p < 0.01) (60% inhibition of cell growth compared with treatment of control scrambled or sense OPT alone, p < 0.01) through enhanced downregulation of c-myc expression. In conclusion, c-myc antisense DNA in combination with other modalities for c-myc downregulation may represent an attractive gene regulation-based therapy of SCLC in the future. Further efforts, however, using new oligodeoxynucleotide analogs, specific interventions for DNA delivery into cells, and more potent therapeutic agents are required to increase the potentiation of c-myc downregulation and cell growth inhibition.

Chimeric oligodeoxynucleotide analogs: Chemical synthesis, purification, and molecular and cellular biology protocols

Publisher Summary The consequences of forming an intracellular antisense oligodeoxynucleotide (ON)-RNA heteroduplex may include inhibition of splicing, or other RNA-processing events, and inhibition of translation. Perhaps the most widely anticipated result is cleavage of the RNA, within the heteroduplex region, resulting from the action of the apparently ubiquitous endogenous enzyme ribonuclease H (RNase H). RNases H have been convincingly demonstrated to mediate many of the antisense effects observed in in vitro systems, following ON microinjection into Xenopus oocytes, eggs, and embryos, and following delivery of antisense effectors into human cells in culture. Clearly, if the transcript becomes degraded through the action of RNase H, then translation of the cognate protein is impossible. This chapter focuses on chimeric ON constructed with terminal methylphosphonodiester sections and a central phosphodiester region. Procedures for the synthesis, fluorescent labeling, purification, and use of chimeric ON are provided. All of the procedures described have been found to be robust and most are generally applicable to a wide range of antisense ON structures.

Synthesis and enzymatic stability of oligonucleotides consisting of isonucleosides

Abstract

In vitro and in vivo activities of oligodeoxynucleotide-based thrombin inhibitors containing neutral formacetal linkages.

A series of 15-mer oligodeoxynucleotide analogues were synthesized, and their thrombin inhibitory activities in vitro and in vivo were evaluated. These oligodeoxynucleotide analogues share the same sequence (GGTTGGTGTGGTTGG) but have one or more phosphodiester linkages replaced by a neutral formacetal group. The results obtained from monosubstitutions show that no single phosphodiester group is critical for the thrombin inhibitory activity, suggesting that the interaction between the oligodeoxynucleotide and thrombin is based on a multiple-site charge-charge interaction. Analysis of the effects of different phosphodiester replacements indicates that the backside and left side of the chairlike structure formed by the molecule may be involved in binding with thrombin, presumably by having direct contacts with the anion-binding exosite of the enzyme. For the oligodeoxynucleotides containing two noncontiguous formacetal groups, the effect of the disubstitution is the sum of the effects obtained from the corresponding two monosubstitutions. Infusion of an oligodeoxynucleotide containing four formacetal groups into monkeys showed an increased in vivo anticoagulant effect and an extended in vivo half-life compared to the unmodified oligodeoxynucleotide.

Synthesis and binding properties of oligodeoxynucleotides containing phenylphosphon(othio)ate linkages

A method for the synthesis of chimeric oligodeoxynucleotides comprised of phosphodiester and phenylphosphonate [3′-O-P(=O)(C 6H 5)-O-5′] or phenylphosphono-thioate [3′-O-P(=S)(C 6H 5)-O-5′] linkages has been developed. Synthesis was performed using suitably protected nucleoside phenylphosphonamidites as building blocks following an adjusted solid-phase phosphoramidite synthesis protocol. The new oligodeoxy-nucleotide analogues were characterized by electrospray ionization- and matrix-assisted laser desorption mass spectrometry, as well as by 31P NMR spectroscopy. Additionally, their binding properties to complementary oligodeoxynucleotides has been studied.

Replacement of the Phosphodiester Backbone of Oligodeoxynucleotide Analogues by Non-Ionic Phosphoramidate (P-NH2)

The synthesis and the biophysical properties of oligodeoxyribonucleotide analogues containing phosphoramidate (P-NH2) internucleoside linkages and β or a deoxynucleoside units is reported.

Automated synthesis of branched oligodeoxynucleotide analogues using arabino-uridine as branching nucleotide

Fully automated synthesis of branched oligodeoxynucleotides (branched ODNs) has been accomplished using 2′-O-levulinyl- or 5′-O-levulinyl-protected arabino-uridine derivatives as branching monomers. Selective removal of the levulinyl groups is accomplished using 0.5 M hydrazine hydrate in a pyridine–acetic acid–water (4∶3∶0.35, v/v/v) buffer. The affinity of the branched ODNs towards complementary DNA has been evaluated at 260 nm and 284 nm during thermal denaturing experiments. Enhanced affinity of a branched ODN compared with the corresponding linear reference is attributed to bimolecular triple helix formation.

Synthesis and properties of α- and β-oligodeoxynucleotides containing α- and [formula omitted

Synthesis of the α- and β-anomer of 2′-OMe-araT (α- and β-1-(2-O-methyl-d-arabinofuranosyl)thymine) and their incorporation into oligodeoxynucleotide (ODN) analogues is described. Condensation of the key arabinofuranose derivative with silylated thymine afforded the α-anomer and the β-anomer which were converted into the respective phosphoramidite building blocks. Automated synthesis of β-ODNs containing β-2′-OMe-araT (by use of standard β-amidites and phosphoramidite building block 9b) and α-ODNs containing α-2′-OMe-araT (by use of α-T-amidite and phosphoramidite building block 9a) allowed evaluation of their properties. With regard to 3′-exonucleolytic degradation, 3′-end incorporation of either β- or α-2′-OMe-araT resulted in considerable stabilization compared to unmodified β-ODNs. Thermal stabilities of duplexes formed between modified ODNs and both unmodified DNA and RNA were evaluated and compared to unmodified controls. In all experiments stable duplexes were formed, but whereas β-ODNs containing β-2′-OMe-araT showed moderately lowered thermal stabilities towards both DNA and RNA, α-ODNs containing α-2′-OMe-araT exhibited significantly increased melting points (compared to β-ODN controls) when complexed with RNA. These results illustrate the potential of using arabino-configurated nucleosides as modified monomers in biologically active ODN-analogues, either as, e.g., 2′-O-alkylated or 2′-O-functionalized derivatives.

Incorporation of amide linked thymidine dimers into oligodeoxynucleotides

The synthesis of thymidine dimers in which the phosphodiester linkage has been replaced by an amide linkage containing diamines (homopiperazine, 2,5- trans-dimethylpiperazine, N, N-dimethylethylenediamine and N, N-diethylethylenediamine) and their incorporation into oligodeoxynucleotides is described. The thermal and nucleolytic stability of the resulting oligodeoxynucleotide analogues are reported.

Separation of “Uncharged” Oligodeoxynucleotide Analogs by Anion-Exchange Chromatography at High pH

Ion-exchange chromatography is a well-established method for the analysis and purification of phosphodiester-linked oligonucleotides. If elution is carried out under alkaline conditions, the secondary structure of G- and C-rich oligomers is disrupted. Furthermore, elution times become more sensitive to the G and T content of the oligomer, because G and T are deprotonated at pH 10. In recent work on peptide-nucleic acids (PNAs) we noted that mixtures of PNA oligomers G(sub 4), G(Sub 6), G(sub 8), and G(sub 10) are readily separated by elution at pH 12 on an RPC-5 column. Here we show that this separation method is more generally applicable.

ChemInform Abstract: Synthesis of Novel 3′‐C‐(Hydroxymethyl)thymidines and Oligodeoxynucleotide Analogues Containing Compressed 3′‐C‐ Hydroxymethyl‐Linked Phosphodiester Backbones.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.