Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides

Phosphorothioate deoxyribonucleotides (PS-DNA) are among the most widely used antisense inhibitors. PS-DNA exhibits desirable properties such as enhanced nuclease resistance, improved bioavailability, and the ability to induce RNase H mediated degradation of target RNA. Unfortunately, PS-DNA possesses a relatively low binding affinity for target RNA that impacts on its potency in antisense applications. We recently showed that phosphodiester-linked oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (FANA) exhibit both high binding affinity for target RNA and the ability to elicit RNase H degradation of target RNA [Damha et al. (1998) J. Am. Chem. Soc. 120, 12976]. In the present study, we evaluated the antisense activity of phosphorothioate-linked FANA oligonucleotides (PS-FANA). Oligonucleotides comprised entirely of PS-FANA were somewhat less efficient in directing RNase H cleavage of target RNA as compared to their phosphorothioate-linked DNA counterparts, and showed only weak antisense inhibition of cellular target expression. However, mixed-backbone oligomers comprised of PS-FANA flanking a central core of PS-DNA were found to possess potent antisense activity, inhibiting specific cellular gene expression with EC(50) values of less than 5 nM. This inhibition was a true antisense effect, as indicated by the dose-dependent decrease in both target protein and target mRNA. Furthermore, the appearance of mRNA fragments was consistent with RNase H mediated cleavage of the mRNA target. We also compared a series of PS-[FANA-DNA-FANA] mixed-backbone oligomers of varying PS-DNA core sizes with the corresponding 2'-O-methyl oligonucleotide chimeras, i.e., PS-[2'meRNA-DNA-2'meRNA]. Both types of oligomers showed very similar binding affinities toward target RNA. However, the antisense potency of the 2'-O-methyl chimeric compounds was dramatically attenuated with decreasing DNA core size, whereas that of the 2'-fluoroarabino compounds was essentially unaffected. Indeed, a PS-FANA oligomer containing a single deoxyribonucleotide residue core retained significant antisense activity. These findings correlated exactly with the ability of the various chimeric antisense molecules to elicit RNase H degradation of the target RNA in vitro, and suggest that this mode of inhibition is likely the most important determinant for potent antisense activity.     

Synthesis and properties of oligonucleotides involving a perylene unit linked to a 2'-deoxyribose residue

We report here the synthesis and binding properties of oligonucleotides involving a perylene unit linked to the anomeric position of a 2'-deoxyribose residue. Both anomers were separated and incorporated separately at either the 5'-end or the internal position of a pyrimidine sequence. In any case the presence of the perylene unit stabilizes the complexes formed with either the single or the double-stranded target.     

The mitochondrial DNA polymerase beta from Crithidia fasciculata has 5'-deoxyribose phosphate (dRP) lyase activity but is deficient in the release of dRP

DNA polymerase beta (pol beta) has long been described as a nuclear enzyme involved in DNA repair. A pol beta from the trypanosomatid parasite Crithidia fasciculata, however, is the first example of a mitochondrial enzyme of this type. The mammalian nuclear enzyme functions not only as a nucleotidyl transferase but also has a dRP lyase activity that cleaves 5'-deoxyribose phosphate (dRP) groups from DNA, thus contributing to two consecutive steps of the base excision repair pathway. We find that the mitochondrial pol beta also has dRP lyase activity. Interestingly, the K(m) of this enzyme for a dRP-containing substrate is similar to that for the rat enzyme, but its k(cat) is very low. This difference is due to a deficiency of the mitochondrial enzyme in the release of dRP from the enzyme following its cleavage from the DNA.     

BCL2-CISD2

CISD2, an ER BCL2-associated autophagy regulator also known as NAF-1, is responsible for the human degenerative disorder Wolfram Syndrome 2. In order to interrogate the physiological role of CISD2 we generated and characterized the Cisd2 gene deletion in mice. Cisd2 null mice manifest significant degeneration in skeletal muscle tissues, which is accompanied with augmented autophagy, dysregulated Ca²⁺ homeostasis and elongated mitochondria. Our findings describe a novel role for BCL2-CISD2 in the homeostatic maintenance of skeletal muscle. It remains to be elucidated how and if the antagonism of the BECN1 autophagy-initiating complex and modulation of ER Ca²⁺ homeostasis by BCL2-CISD2 are interconnected.     

Synthesis of [11-2H2], [8-2H2], [7-2H2], [6-2H2], [5-2H2], [4-2H2] and [3-2H2] cis-9-octadecenoates

Deuterated oleates have been synthesized by semihydrogenation of acetylenic intermediates. [11-²H₂]Oleate was prepared by two-carbon chain extension of the C₁₆ alcohol obtained from [1-²H₂]octyl bromide and 7-octyn-1-ol. [8-²H₂] and [7-²H₂]oleates were both prepared from dimethyl suberate, tetradeutero intermediate C₁₆ alcohols were synthesized from [1,8-²H₄] and [2,7-²H₄]octane diols by monobromination, conversion to deuterated 9-decyn-1-ols and reaction with octyl bromide. Oxidation gave [8-²H₂]-9-octadecynoate and [2,7-²H₂]-9-octadecynoate, after semihydrogenation of the latter, deuterons at C-2 were removed by exchange with aqueous alkali. [6-²H₂] and [5-²H₂]oleates were obtained from methyl 5-tetradecynoate, semihydrogenation, deuterium exchange at C-2 and two malonate extensions gave [6-²H₂]oleate; reduction with lithium aluminum deuteride, two malonate extensions and semihydrogenation gave the [5-²H₂] ester. [4-²H₂] and [3-²H₂]oleates were both obtained from methyl 7-cis-hexadecenoate, exchange of the α protons and chain extension gave the [4-²H₂] ester and reduction with lithium aluminum deuteride and chain extension gave the [3-²H₂] ester.     

Synthesis and physicochemical properties of 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl oligonucleotides

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Stereospecific formation of alpha-hydroxycarboxylato oxo peroxo complexes of vanadium(V). Crystal structure of (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O

An overview of structurally characterized alpha-hydroxycarboxylatodioxo- and alpha-hydroxycarboxylatooxoperoxovanadates(V) is presented and the geometric parameters of the V2O2 bridging core are discussed. The first case of a stereospecific formation of oxoperoxovanadates(V) is reported: The crystal structures of the isomeric compounds (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O (lact = C3H4O3(2-), the anion of the lactic acid) differ mainly in the arrangement of the V2O2 core and in mutual orientation of the V=O bonds. The complexes with achiral ligands adopt the same structural type as the complexes formed from a racemic mixture of a chiral ligand, while the structure obtained using an enantiopure L,L-hydroxycarboxylate is different.     

Stereoselective Synthesis of 2-Amino-2-deoxy-d-arabinose and 2-Deoxy-d-ribose

An efficient method for the stereoselective synthesis of 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose is described.

The key step in this method was accomplished by the nucleophilic addition of methyl isocyanoacetate to 2,3-O-isopropylidene-d-glyceraldehyde with high erythro-selectivity (nearly 100%).

Subsequent intermolecular cyclization predominantly gave the desired oxazoline derivative (trans-form), in which two new chiral centers were formed. The oxazoline derivative was efficiently converted to both 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose.     

Convenient synthesis of 2'-deoxy-2-fluoroadenosine from 2-fluoroadenine

A convenient synthesis of 2'-deoxy-2-fluoroadenosine from commercially available 2-fluoroadenine is described. The coupling reaction of silylated 2-fluoroadenine with phenyl 3,5-bis[O-(t-butyldimethylsilyl)]-2-deoxy-1-thio-D-erythro-pentofuranoside gave the corresponding 2-fluoro-2'-deoxyadenosine derivative (alpha/beta = 1:1) in good yield. The alpha- and beta-anomers were separated by chromatography, and then desilylated to give compounds 1a and 1b.     

A concise synthesis of 4-nitrophenyl 2-azido-2-deoxy- and 2-acetamido-2-deoxy-D-mannopyranosides

4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha- and beta-D-mannopyranosides were prepared from methyl 4,6-O-benzylidene-alpha-D-glucopyranoside and 1,3,4,6-tetra-O-acetyl-alpha-D-glucopyranose, respectively. Chemoselective reduction of both azides with hydrogen sulfide readily afforded 4-nitrophenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D- and -beta-D-mannopyranosides in higher yields than reduction with triphenylphosphine or a polymer-supported triarylphosphine. Subsequent de-O-acetylation yielded 4-nitrophenyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside and 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-mannopyranoside in 20% and 44% overall yields, respectively.     

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.