Phosphorothioate Oligomers Research Articles

Understanding Antisense Oligonucleotide Efficiency in Inhibiting Prokaryotic Gene Expression.

Oligonucleotides offer a unique opportunity for sequence specific regulation of gene expression in bacteria. A fundamental question to address is the choice of oligonucleotide, given the large number of options available. Different modifications varying in RNA binding affinities and cellular uptake are available but no comprehensive comparisons have been performed. Herein, the efficiency of blocking expression of β-galactosidase (β-Gal) in E. coli was evaluated utilizing different antisense oligomers (ASOs). Fluorescein (FAM)-labeled oligomers were used to understand their differences in bacterial uptake. Flow cytometry analysis revealed significant differences in uptake, with high fluorescence seen in cells treated with FAM-labeled peptidic nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO) and phosphorothioate (PS) oligomers, and low fluorescence observed in cells treated with phosphodiester (PO) oligomers. Thermal denaturation (Tm) of oligomer:RNA duplexes and isothermal titration calorimetry (ITC) studies reveal that ASO binding to target RNA demonstrates a good correlation between Tm and Kd values. There was no correlation between Kd values and reduction of β-Gal activity in bacterial cells. However, cell-free translation assays demonstrated a direct relationship between Kd values and inhibition of gene expression by antisense oligomers, with tight binding oligomers such as LNA being the most efficient. Membrane active compounds such as polymyxin B and A22 further improved the cellular uptake of FAM-PNA and FAM-PS oligomers in wild-type E. coli cells. PNA and PMO were most effective in cellular uptake and reducing β-Gal activity as compared to oligomers with PS or those with PO linkages. Overall, cell uptake of the oligomers is shown as the key determinant in predicting their differences in bacterial antisense inhibition, and the RNA affinity is the key determinant in inhibition of gene expression in cell free systems.

Single Stranded Fully Modified-Phosphorothioate Oligonucleotides can Induce Structured Nuclear Inclusions, Alter Nuclear Protein Localization and Disturb the Transcriptome In Vitro.

Oligonucleotides and nucleic acid analogues that alter gene expression are now showing therapeutic promise in human disease. Whilst the modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, such modifications may also confer unexpected physicochemical and biological properties. Gapmer mixed-modified and DNA oligonucleotides on a phosphorothioate backbone can bind non-specifically to intracellular proteins to form a variety of toxic inclusions, driven by the phosphorothioate linkages, but also influenced by the oligonucleotide sequence. Recently, the non-antisense or other off-target effects of 2′ O- fully modified phosphorothioate linkage oligonucleotides are becoming better understood. Here, we report chemistry-specific effects of oligonucleotides composed of modified or unmodified bases, with phosphorothioate linkages, on subnuclear organelles and show altered distribution of nuclear proteins, the appearance of highly stable and strikingly structured nuclear inclusions, and disturbed RNA processing in primary human fibroblasts and other cultured cells. Phosphodiester, phosphorodiamidate morpholino oligomers, and annealed complimentary phosphorothioate oligomer duplexes elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of transfected fibroblasts exhibiting such disruption. Our data add to the growing body of evidence of off-target effects of some phosphorothioate nucleic acid drugs in primary cells and suggest alternative approaches to mitigate these effects.

Homopurine RP-stereodefined phosphorothioate analogs of DNA with hampered Watson-Crick base pairings form Hoogsteen paired parallel duplexes with (2'-OMe)-RNAs.

3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of 5'-O-DMT-N6-methyl-deoxyadenosine and 5'-O-DMT-N2,N2-dimethyl-O6-diphenylcarbamoyl-deoxyguanosine (OTP-NY, NY = DMT-m6dA or DMT-m,m2dGDPC) were synthesized, resolved onto pure P-diastereomers, and used in P-stereocontrolled synthesis of dinucleoside 3',5,-phosphorothioates NXPST (NX = m6dA or m,m2dG), in which the absolute configuration of the stereogenic phosphorus atom was established enzymatically. Diastereomerically pure OTP-NY and standard OTP-N (N = DMT-dABz or DMT-dGBz,DPC) were used in the synthesis of chimeric RP-stereodefined phosphorothioate oligomers ((RP-PS)-DN(NX)A) with hampered Watson-Crick base pairings. It was found that the m6dA units slightly reduce the thermodynamic stability of antiparallel duplexes formed with RNA and (2'-OMe)-RNA matrices, whereas m,m2dG units prevent their formation. The m6dA units stabilize (by up to 4.5 °C per modified unit) the parallel duplexes formed by (RP-PS)-DN(NX)A with Hoogsteen-paired (2'-OMe)-RNA templates compared to the analogous reference duplex containing only unmodified nucleobases. In contrast, the m,m2dG units destabilize such duplexes by up to 3 °C per modified unit. Both units prevent the formation of the corresponding parallel triplexes.

Abasic phosphorothioate oligomers inhibit HIV-1 reverse transcription and block virus transmission across polarized ectocervical organ cultures.

In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2' deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.

Peptide Conjugation of 2′-O-methyl Phosphorothioate Antisense Oligonucleotides Enhances Cardiac Uptake and Exon Skipping in mdx Mice

Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy that is currently being tested in various clinical trials. This approach is based on restoring the open reading frame of dystrophin transcripts resulting in shorter but partially functional dystrophin proteins as found in patients with Becker muscular dystrophy. After systemic administration, a large proportion of AONs ends up in the liver and kidneys. Therefore, enhancing AON uptake by skeletal and cardiac muscle would improve the AONs' therapeutic effect. For phosphorodiamidate morpholino oligomer, AONs use nonspecific positively charged cell penetrating peptides to enhance efficacy. However, this is challenging for negatively charged 2'-O-methyl phosphorothioate oligomer. Therefore, we screened a 7-mer phage display peptide library to identify muscle and heart homing peptides in vivo in the mdx mouse model and found a promising candidate peptide capable of binding muscle cells in vitro and in vivo. Upon systemic administration in dystrophic mdx mice, conjugation of a 2'-O-methyl phosphorothioate AON to this peptide indeed improved uptake in skeletal and cardiac muscle, and resulted in higher exon skipping levels with a significant difference in heart and diaphragm. Based on these results, peptide conjugation represents an interesting strategy to enhance the therapeutic effect of exon skipping with 2'-O-methyl phosphorothioate AONs for Duchenne muscular dystrophy.

Post-transcriptional silencing of Notch2 mRNA in chronic lymhocytic leukemic cells of B-CLL patients

Environmental and genomic stresses induce different pathological conditions and one of them is blood cancer. This escalating load of disease with a constant threat to life requires an intensive comprehensive response. For our understanding about the cancer treatment capabilities, novel medicinal platforms should be strived to explore among the existing conventional and molecular approaches that have already been proven to be successful in fighting against genetic diseases. Several DNA therapeutics previously studied are currently in clinical settings. RNA interfering antisense oligonucleotide (AS-ODN) is the most experimentally advanced molecular therapeutic which has the potential to modify the gene activity resulting in the down regulation of particular protein. In this study, we focused on the inhibition of Notch2 function in B-cell chronic lymphocytic leukemia (B-CLL) by AS-ODN (phosphorothioate oligomers) targeted to the initiation codon region of the Notch2 mRNA. We investigated the in vitro ability of four such oligomers to reduce the expression of Notch2 gene in peripheral blood mononuclear cells from B-CLL patients. Our findings implicate that AS-ODNs specifically designed for the region of 314-333 neucleotides (AS1) of Notch2 inhibits its gene expression better than other AS-ODNs designed for other regions and respond in a dose dependent manner. The results of cell proliferation assay for the evaluation of AS1 in gene silencing, infer that the number of cells were reduced to 80% (P<0.001). Our results implicate that using the AS-ODNs against specific Notch2 nucleotide sequence can be used as future therapeutic agent with the ability of Notch2 down regulation, which is the root problem in the pathogenicity of B-CLL.

ChemInform Abstract: Allyl Group as a Protecting Group for Internucleotide Phosphate and Thiophosphate Linkages in Oligonucleotide Synthesis: Facile Oxidation and Deprotection Conditions.

[reaction: see text] The allyl group, which serves as a protecting group for an internucleotide bond for both phosphates and phosphorothioates, can be easily removed by good nucleophiles under weakly basic or neutral conditions. For a practical synthesis on solid support, camphorsulfonyloxaziridine was used as the oxidizing agent for synthesizing DNA, while the Beaucage reagent was used for preparing phosphorothioate oligomers. Both types of oligonucleotides were easily deprotected by concentrated ammonium hydroxide containing 2% mercaptoethanol.

Cytoplasmic and Nuclear Accumulation of Antisense Oligomers Incubated as Streptavidin Delivery Nanoparticles

The cytoplasmic accumulation and nuclear targeting of antisense oligomers were evaluated in cell culture following incubation with the cell penetrating peptide tat or polyargenine as a two-component delivery nanoparticle using streptavidin as linker. The fluorescent labeled uniform phosphodiester (PO), phosphorothioate (PS) and MORF oligomers were antisense to the mdr1 mRNA and were studied in KB-G2 (Pgp++) and KB-31 (Pgp+/-) cells. Fluorescence microscopy showed much higher cellular accumulation using the antisense compared to the sense PS/tat nanoparticle. The distribution in cells receiving either the PS or MORF, but not the PO, antisense oligomers in all of their forms showed definite migration to the nucleus. Incubations in KB-G2 cells of all three antisense oligomers with streptavidin as the onecomponent or including tat as the two-component nanoparticles led to increases in Rhodamine 123 accumulation by 200∼250% compared to free oligomers, almost certainly due to antisense interference with Pgp function. Thus presence of the streptavidin linker had no obvious detrimental influence on the functions of the carriers or the antisense oligomers. Since nuclear migration appears to be a characteristic property of oligomers, delivery via the nanoparticle may be a useful method of increasing the accumulation into the nucleus of antisense oligomers radiolabeled for Auger emittingbased radiotherapy.

Antisense Strategies for Oncogene Inactivation

Antisense oligonucleotides have been evaluated as antineoplastic agents in a series of clinical trials, with mixed results. However, phase III trials incorporating G3139, a phosphorothioate oligomer targeted to the initiation codon region of the bcl-2 mRNA, have recently been completed in advanced melanoma, myeloma, and chronic lymphocytic leukemia (CLL). This article discusses the mechanism of the antisense effect and its dependence on the cellular internalization of oligonucleotides and the activity of RNase H. It also describes the properties, specific and nonspecific, of phosphorothioate oligonucleotides, the predominant species in current clinical trials, and discusses pharmacokinetic data obtained from earlier phase I and II trials employing these molecules. While the application of antisense technology to the treatment of human cancer is conceptually straightforward, in practice there are many complicated, mechanistically based questions that must be considered.

Cytosolic delivery of macromolecules: I. Synthesis and characterization of pH-sensitive acyloxyalkylimidazoles

A series of 1-(acyloxyalkyl)imidazoles (AAI) were synthesized by nucleophilic substitution of chloroalkyl esters of fatty acids with imidazole. The former was prepared from fatty acid chloride and an aldehyde. When incorporated into liposomes, these lipids show an apparent p K a value ranging from 5.12 for 1-(palmitoyloxymethyl)imidazole (PMI) to 5.29 for 1-[(α-myristoyloxy)ethyl]imidazole (α-MEI) as determined by a fluorescence assay. When the imidazole moiety was protonated, the lipids were surface-active, as demonstrated by hemolytic activity towards red blood cells. As expected, AAI were hydrolyzed in serum as well as in cell homogenate. They were significantly less toxic than biochemically stable N-dodecylimidazole (NDI) towards Chinese hamster ovary (CHO) and RAW 264.7 (RAW) cells as determined by MTT assay. When fed to RAW cells, fluorescein-labeled oligonucleotides encapsulated in liposomes containing 20 mol% 1-(stearoyloxymethyl)imidazole (SMI) resulted in punctate as well as partially diffuse fluorescence. In a functional assay involving down-regulation of luciferase in CV-1 cells, neutral liposomes containing imidazole lipids showed suboptimal delivery of antisense phosphorothioate oligomers. Taken together, the results suggest that AAI are of potential use in developing nontoxic, pH-sensitive liposomes. However, these liposomal formulations need to be optimized to achieve higher concentrations of pH-sensitive detergents within the endosome to facilitate efficient cytosolic release of liposome-entrapped contents.

Antisense inhibition of Flk-1 by oligonucleotides composed of 2'-deoxy-2'-fluoro-beta-D-arabino- and 2'-deoxy-nucleosides.

The design of new antisense oligomers with improved binding affinity for targeted RNA, while still activating RNase H, is a major research area in medicinal chemistry. RNase H recognizes the RNA-DNA duplex and cleaves the complementary mRNA strand, providing the main mechanism by which antisense oligomers elicit their activities. It has been shown that configuration inversion at the C2' position of the DNA sugar moiety (arabinonucleic acid, ANA), combined with the substitution of the 2'OH group by a fluorine atom (2'F-ANA) increases the oligomer's binding affinity for targeted RNA. In the present study, we evaluated the antisense activity of mixed-backbone phosphorothioate oligomers composed of 2'-deoxy-2'-fluoro-beta-D-arabinose and 2'-deoxyribose sugars (S-2'F-ANA-DNA chimeras). We determined their abilities to inhibit the protein expression and phosphorylation of Flk-1, a vascular endothelial growth factor receptor (VEGF), and VEGF biological effects on endothelial cell proliferation, migration, and platelet-activating factor synthesis. Treatment of endothelial cells with chimeric oligonucleotides reduced Flk-1 protein expression and phosphorylation more efficiently than with phosphorothioate antisenses (S-DNA). Nonetheless, these two classes of antisenses inhibited VEGF activities equally. Herein, we also demonstrated the capacity of the chimeric oligomers to elicit RNase H activity and their improved binding affinity for complementary RNA as compared with S-DNA.

Evaluating the Specificity of Antisense Oligonucleotide Conjugates: A DNA ARRAY ANALYSIS

Antisense oligonucleotides are potentially powerful tools for selective control of cellular and viral gene expression. Crucial to successful application of this approach is the specificity of the oligonucleotide for the chosen RNA target. Here we apply DNA array technology to examine the specificity of antisense oligonucleotide treatments. The molecules used in these studies consisted of phosphorothioate oligomers linked to the Antennapedia (Ant) delivery peptide. The antisense oligonucleotide component was complementary to a site flanking the AUG of the MDR1 message, which codes for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Using a DNA array of 2059 genes, we analyzed cellular responses to molecules comprised of Ant peptide-oligonucleotide conjugates, as well as to the Ant peptide alone. Besides the expected reduction in MDR1 message level, 37 other genes (approximately 2% of those tested) showed changes of comparable magnitude. The validity of the array results was confirmed for selected genes using Northern blots to assess messenger RNA levels. These results suggest that studies using antisense oligonucleotide technology to modulate gene expression need to be interpreted with caution.

Selective binding of trisamine-modified phosphorothioate antisense DNA to target mRNA improves antisense activity and reduces toxicity

Antisense activity in living cells has been thought to occur via a mechanism involving both DNA-mediated hybridization arrest of target mRNA and RNase H-mediated mRNA digestion. Therefore an ideal antisense agent should be permeable to the cell and possess capacities (1) to form a thermally stable duplex in vivo with its target, (2) to discriminate between mRNAs with different degrees of complementarity, and (3) to form antisense/RNA complexes that are susceptible to RNase H hydrolysis. A trisamine-modified deoxyuridine derivative of a novel phosphorothioate DNA 15-mer that meets all these criteria is described here. Compared with the unmodified phosphorothioate oligomer, the phosphorothioate derivative exhibits a higher antisense activity as well as reduced cytotoxicity in cells infected with HIV-1. Our data suggest that the melting temperature ( T m) between antisense DNA and the target mRNA is not only one of the factors contributing to this derivative's improved antisense activity. Also important are an enhanced ability to discriminate between sequences and an increased susceptibility of the DNA/mRNA complex to RNase H hydrolysis. These results will be useful in designing more active, clinically useful antisense drugs.

Protamine-fragment peptides fused to an SV40 nuclear localization signal deliver oligonucleotides that produce antisense effects in prostate and bladder carcinoma cells.

The development of antisense technology has focused on improving methods for oligonucleotide delivery into cells. In the present work, we describe a novel strategy for oligonucleotide delivery based on a bifunctional peptide composed of a C-terminal protamine-fragment that contains a DNA-binding domain and an N-terminal nuclear localization signal sequence derived from the SV40 large-T antigen (The sequences of two of the peptides are R6WGR6-PKKKRKV [s-protamine-NLS] and R4SR6FGR6VWR4-PKKKRKV [l-protamine-NLS]). We demonstrated, by intrinsic fluorescence quenching, that peptides of this class form complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20-mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA and G3139, an 18-mer phosphorothioate targeted to the first six codons of the human bcl-2 open reading frame, and complexed them with either of two peptides (s- or l-protamine-NLS). These peptides bind to and deliver antisense oligonucleotides to the nucleus of T24 bladder and PC3 prostate cancer cells, as demonstrated by confocal microscopy. Furthermore, as shown by Western and Northern blotting, the peptide-oligonucleotide complexes produced excellent downregulation of the expression of the complementary mRNAs, which in turn resulted in downregulation of protein expression. However, under certain circumstances (predominantly in PC3 cells), incubation of the cells with chloroquine was required to produce antisense activity. Using this strategy, PKC-alpha protein and mRNA expression in T24 and PC3 cells and bcl-2 expression in PC3 cells was reduced by approximately 75 +/- 10% at a minimum concentration of oligomer of 0.25 microM, in combination with 12-15 microM peptide. On the basis of our results, we conclude that arginine-rich peptides of this class may be potentially useful delivery vehicles for the cellular delivery of antisense oligonucleotides. This new strategy may have several advantages over other methods of oligonucleotide delivery and may complement already existing lipid-based technologies.

In vitro evidence for an intracellular site of angiotensin action.

To differentiate the relative effects of nuclear and cell surface angiotensin II (Ang II) receptors, we mutated the angiotensinogen cDNA by removing the signal sequence-encoding region to produce a nonsecreted form of angiotensinogen [Ang(-S)Exp]. Rat hepatoma cells (which produce renin and angiotensin-converting enzyme mRNAs) were stably transfected with Ang(-S)Exp/pSVL (or a corresponding control) expression plasmid, and mitotic indices were measured for stably transfected cell lines. Experimental clonal cell lines demonstrate an average of 33+/-4.4% (P<0.001) increase in percentage-labeled nuclei compared with control cell lines. The mitogenic effect is blocked by 10(-6) mol/L losartan and by 1 micromol/L renin antisense phosphorothioate oligomers but not by 10(-6) mol/L candesartan. In addition, phenylarsine oxide, which blocks angiotensin receptor internalization, abolishes the losartan inhibitory effect, suggesting that after cell-surface receptor-mediated endocytosis, losartan blocks Ang II nuclear receptors. PDGF mRNA levels are elevated 2.2-fold in Ang(-S)Exp transfected cell lines; addition of anti-PDGF antibodies to the culture medium partially blocks the mitogenic effect of Ang(-S)Exp, while anti-Ang II antibodies have no effect. These results suggest that the Ang(-S)Exp growth effect is due, in part, to autocrine/paracrine stimulation by secreted PDGF after Ang II/Ang II receptor intracellular interactions. We further demonstrate that these cells produce the alternative renin transcript, renin 1A, which apparently lacks a signal sequence and is maintained intracellularly. Collectively, these studies of cultured cells suggest that some cell types may possess components of the renin-angiotensin system that permit intracellular processing of angiotensinogen to Ang II and that Ang II generated intracellularly may be mitogenic.

Quantitative nuclear and cytoplasmic localization of antisense oligonucleotides by capillary electrophoresis with laser-induced fluorescence detection.

We demonstrate the use of simple extraction procedures to separate nuclear and cytoplasmic material from cell extracts, which have been scrape-loaded with a 2-O-methyl phosphorothioate antisense oligonucleotide. Separation and quantitation of the fluorescein-labeled antisense and the flourescein isothiocyanate (FITC)-dextran (molecular weight 40000) as an internal standard is done using capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). The bulky FITC-dextran is unable to penetrate the nuclear membrane thereby making it a quantitative indicator of any overlap between the nuclear and cytoplasmic materials during separation of the two phases. Using this procedure, the fluorescein-labeled phosphorothioate oligomer was quantitated at 4.1 x 10(-13) and 3.4x 10(-14) mol antisense/microg-total cellular protein in the nuclear and cytoplasmic extracts respectively following scrape-load delivery of the phosphorothioate to a batch of confluent HeLa cells at a concentration of 0.5 microM (5 x 10(-10) total moles of oligomer). Additionally, gene expression was monitored by measurement of the luciferase reporter protein activity. Scrape-load, spontaneous and liposomal delivery were investigated and compared for subcellular distribution of the oligomer and subsequent gene expression.

Effect of P-chirality of internucleotide bonds on B-Z conversion of stereodefined self-complementary phosphorothioate oligonucleotides of the [PS]-d(CG)4 and [PS]-d(GC)4 series.

Diastereomerically pure, partially modified (in selected positions) or fully modified phosphorothioate oligomers of the [PS]-d(CG)(4) and [PS]-d(GC)(4) series were investigated with respect to their ability to adopt the left-handed conformation at high sodium chloride concentration. NaCl induces the B-Z transition of [All-S(P)R(P)-PS]-d(CG)(4) with a midpoint of transition at ca. 2 M, which is approximately 1 M less than for unmodified d(CG)(4). Also, [All-R(P)S(P)-PS]-d(GC)(4) at 5 M NaCl converts to the Z form to the extent of ca. 55%, while the unmodified d(GC)(4) counterpart does not convert at all. This enhanced ability of stereodefined phosphorothioate oligomers to adopt the Z conformation is discussed in terms of already known structural factors (hydrogen bonding and water bridges) facilitating the B-Z transition, identified for unmodified d(CG)(n) oligonucleotides. By CD spectroscopy, the [All-S(P)-PS]-d(CG)(4) oligomer at a NaCl concentration higher than 0.01 M adopts a unique conformation as assessed from the presence of an additional negative band centered at 282 nm.

Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5'-protected thiol function and a 3'-amino group.

A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphor-othioate oligonucleotides substituted with a protected thiol function at their 5'-ends and an amino group at their 3'-ends in good yield (up to 72 OD units/micromol for a 19mer phosphorothioate). Syntheses of 3'-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphor-amidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2'-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This proced-ure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3'-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5'-terminal S -acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3'-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligo-nucleotide; the 5'-dithio-pyridyl group was then -quantitatively reduced to give a free thiol group which was then substituted by reaction with an N alpha-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide-oligonucleotide conjugate.

Allyl group as a protecting group for internucleotide phosphate and thiophosphate linkages in oligonucleotide synthesis: facile oxidation and deprotection conditions.

[reaction: see text] The allyl group, which serves as a protecting group for an internucleotide bond for both phosphates and phosphorothioates, can be easily removed by good nucleophiles under weakly basic or neutral conditions. For a practical synthesis on solid support, camphorsulfonyloxaziridine was used as the oxidizing agent for synthesizing DNA, while the Beaucage reagent was used for preparing phosphorothioate oligomers. Both types of oligonucleotides were easily deprotected by concentrated ammonium hydroxide containing 2% mercaptoethanol.

Sequence effects on energetic and structural properties of phosphorothioate DNA: a molecular modelling study.

Phosphorothioate (PS) oligonucleotides constitute a new class of potent drugs, resulting from the replacement of one anionic oxygen of the phosphodiester backbone by one sulphur atom. This replacement confers chirality to the phosphorus atom (PSS or PSR) and alters the energetic, structural and biological properties of B-DNA. These properties were assessed by molecular mechanics calculations on a set of regular sequences, d(YR)8.d(YR)8 and d(RR)8.d(YY)8 (R, purine; Y, pyrimidine). Results indicated: (i) destabilisation of both the PS(R)and the PSS oligomers, the loss of total energy being mainly due to a variation in the electrostatic term; (ii) an additional chirality effect, due to van der Waals and backbone angle energies, larger for PSS oligomers than for PSR oligomers; (iii) a clear sequence effect on stability, particularly from the base immediately preceding the PS group. Even though the PS group alters the stability of oligomers, it does not significantly modify the conformation. Altogether, our molecular modelling data parallel the available experimental data. Our results reveal that sequence effects on the energetic properties of PS oligomers are local and additive. Therefore, studies of the set of the 10 unique double-stranded modified dinucleotide steps included in regular oligomers could be used to predict the behaviour of any double-stranded PS-DNA.