New methylene-bridged hexopyranosyl nucleoside modified oligonucleotides (BHNA): synthesis and biochemical studies (original) (raw)
Related papers
Biochemistry, 2007
The RNase H cleavage potential of the RNA strand basepaired with the complementary antisense oligonucleotides (AONs) containing North-East conformationally constrained 1′,2′-methylene-bridged (azetidine-T and oxetane-T) nucleosides, North-constrained 2′,4′-ethylene-bridged (aza-ENA-T) nucleoside, and 2′-alkoxy modified nucleosides (2′-O-Me-T and 2′-O-MOE-T modifications) have been evaluated and compared under identical conditions. When compared to the native AON, the aza-ENA-T modified AON/RNA hybrid duplexes showed an increase of melting temperature (∆T m ) 2.5-4°C per modification), depending on the positions of the modified residues. The azetidine-T modified AONs showed a drop of 4-5.5°C per modification with respect to the native AON/RNA hybrid, whereas the isosequential oxetane-T modified counterpart, showed a drop of ∼5-6°C per modification. The 2′-O-Me-T and 2′-O-MOE-T modifications, on the other hand, showed an increased of T m by 0.5°C per modification in their AON/ RNA hybrids. All of the partially modified AON/RNA hybrid duplexes were found to be good substrates for the RNase H mediated cleavage. The K m and V max values obtained from the RNA concentrationdependent kinetics of RNase H promoted cleavage reaction for all AON/RNA duplexes with identical modification site were compared with those of the reference native AON/RNA hybrid duplex. The catalytic activities (K cat ) of RNase H were found to be greater (∼1.4-2.6-fold) for all modified AON/RNA hybrids compared to those for the native AON/RNA duplex. However, the RNase H binding affinity (1/K m ) showed a decrease (∼1.7-8.3-fold) for all modified AON/RNA hybrids. This resulted in less effective (∼1.1-3.2-fold) enzyme activity (K cat /K m ) for all modified AON/RNA duplexes with respect to the native counterpart. A stretch of five to seven nucleotides in the RNA strand (from the site of modifications in the complementary modified AON strand) was found to be resistant to RNase H digestion (giving a footprint) in the modified AON/RNA duplex. Thus, (i) the AON modification with azetidine-T created a resistant region of five to six nucleotides, (ii) modification with 2′-O-Me-T created a resistant stretch of six nucleotides, (iii) modification with aza-ENA-T created a resistant region of five to seven nucleotide residues, whereas (iv) modification with 2′-O-MOE-T created a resistant stretch of seven nucleotide residues. This shows the variable effect of the microstructure perturbation in the modified AON/RNA heteroduplex depending upon the chemical nature as well as the site of modifications in the AON strand. On the other hand, the enhanced blood serum as well as the 3′-exonuclease stability (using snake venom phosphodiesterase, SVPDE) showed the effect of the tight conformational constraint in the AON with aza-ENA-T modifications in that the 3′-exonuclease preferentially hydrolyzed the 3′-phosphodiester bond one nucleotide away (n + 1) from the modification site (n) compared to all other modified AONs, which were 3′exonuclease cleaved at the 3′-phosphodiester of the modification site (n). The aza-ENA-T modification in the AONs made the 5′-residual oligonucleotides (including the n + 1 nucleotide) highly resistant in the blood serum (remaining after 48 h) compared to the native AON (fully degraded in 2 h). On the other hand, the 5′-residual oligonucleotides (including the n nucleotide) in azetidine-T, 2′-O-Me-T, and 2′-O-MOE-T modified AONs were more stable compared to that of the native counterpart but more easily degradable than that of aza-ENA-T containing AONs.
Nucleic Acids Research, 1993
Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA-DNA or RNA-DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not.
Nucleic Acids Research, 2001
The hybridising potential of anhydrohexitol nucleoside analogues (HNAs) is well documented, but tedious synthesis of the monomers hampers their development. In a search for better analogues, the synthesis of two new methylated anhydrohexitol congeners 1 and 2 was accomplished and the physico-chemical properties of their respective oligomers were evaluated. Generally, oligonucleotides (ONs) containing the 3′-O-methyl derivative 1 showed a small increase in thermal stability towards complementary sequences as compared to HNA. Compared to the altritol modification, 3′-O-methylation seems to cause a small decrease in thermal stability of duplexes, especially when targeting RNA. These results suggest the possibility of derivatisation of the 3′-hydroxyl group of altritol-containing congeners without significantly affecting the thermal stability of the duplexes. The methyl glycosidic analogues 2 likewise increased the affinity for RNA in comparison with well-known HNA, while at the same time being economically more favorable monomers. However, homopolymers of 2 displayed self-pairing, but not so homopolymers of 1. Upon incorporation of the hexitols within RNA sequences in an effort to induce a beneficial pre-organised structure, the positive effect of the 3′-O-methyl derivative 1 proved larger than that of 2.
Biochemistry, 2002
A novel 2′-modification, 2′-O-[2-(methylthio)ethyl] or 2′-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2′-O-[2-(methoxy)ethyl] 2′-O-MOE modification. As expected, the 2′-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2′-O-MOE modified oligonucleotides. The 2′-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2′-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2′-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2′-O-MTE modified oligonucleotides to exonuclease degradation.
Tetrahedron Letters, 1994
Oligonucleotides containing noiel N24midazolyl~ro ylguanine and N2-Imidazolylpropyl-2-amiadenine moieties wem synthesked and studxxl F or their hybridization and biophysical propertk. Intemstingly, these hetcmcyle modified oli on~leotides showed a remakable enhancement of heteroduplcx binding affdty when hybn '&ed to complementary DNA. Modified oligonucleotides are of interest as antiscnse therapeutic agents.1 Although this relatively new drug discovery concept is presently thought to be of gzeat therapeutic potential. a number of problems have yet to be sokd.1 For example. the precise event(s) that terminates or in some way interferes with an essential RNA function after sequcncc-specifii binding of an oEgonucleutide to a target RNA is unclear_ Antisense effects are bust to result as a consequence of simply binding of the olig~~l~ti& to targekd RNA or binding and subsequent cleavage of targeted RNA by endogenous RNase ~1.2 Both mechanisms require nucleasc resistant-oligonucelotides which effectively bind to target RNA. Unfortunately, first generation modifxations such aa phosphorottdoates, methylphosphanates, and phoaphoramidates, although providing n&ease resistance, compromised heteroduplex binding affinity.1 The RNase H mode of action requires that the heteroduplex formed between the RNA target and the DNA be bound and cleaved by the enzyme. However, all reported modificati~ of the sugar-phosphate backbone, with the exception of phosphorothioates and phosphorodithioates, obliterate the RNase H terminating event, 1~3 We m pursuing several amhes to develop antisense oligonucleotides with precise terminating events. One is based on the concept that antisense oligonuckotides containing ~~~~-rn~fi~ti~, rather than ~~-p~~ rn~~~, can be resistant to nucleolytic &gradation, yet on h~~tion to target RNA provides a heteroduplex &at supports RNase H-mediated cleavage.7 Another approach is directed to the development of sequence-specific chemical cleavers of RNA. This concept requires the attachment of pendent groups with acid/base properties to oligonucleotides; when hybridized to RNA, the pendent groups of the oligonucleotides would be accessible, via the minor groove, to the a'-hydroxyl and ~~~Of~~~A Obviously, pendent groups designed to support eitherenzymatic orchemical cleavage of RNA must be compatible with the requisite hybridization step. We have focused on pendcnt groups on the N2-position of guanine and 2-aminoadenine as these groups should protrude into the minor groove of a DNA-RNA
Nucleic Acids Research, 1998
Here we report that the poor binding of methylphosphonate oligodeoxynucleosides (MP-ODNs) to their nucleic acid targets can be improved by additional inversion of the anomeric configuration (from β to α) in the sugar moieties to give a new class of analogs, MP α-oligonucleosides. MP α-dT 12 and MP 5′ α-d(TCTTAA-CCCACA) 3′ were synthesized and their ability to form hybrids with complementary single stranded (ss)DNA and ssRNA, as well as with double stranded (ds)DNA, was evaluated. The thermal stability of hybrids formed with MP α-analogs was compared with the affinity of phosphodiester (PO) and phosphorothioate (PS) βand α-oligomers for their targets. Non-ionic MP α-oligonucleosides bound to their complementary DNA and RNA strands more tightly than their homologues with natural β-anomeric configuration did. With DNA target, MP α-oligomers formed duplexes more stable than the corresponding natural PO β-oligomer did. MP α-heteropolymer hybridized to RNA target better than PS β-oligonucleotide did but the hybrid was less stable (∆T m -0.5_C per mod.) than the hybrid formed with the natural PO β-oligomer. Only MP α-dT 12 bound to dsDNA target at low salt concentration (0.1 M NaCl).
Chemically modified oligonucleotides with efficient RNase H response
Bioorganic & Medicinal Chemistry Letters, 2008
Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage.
Synthesis and properties of 2′-O-neopentyl modified oligonucleotides
Organic & Biomolecular Chemistry, 2013
a 2'-O-Neopentyldeoxyuridine (Un) was synthesized and incorporated into a series of oligodeoxyribonucleotides. Single and triple incorporations in various arrangements were performed. The Watson and Crick pairing properties with complementary DNA and RNA were investigated by UV melting curves, CD spectroscopy, and molecular dynamic simulations. The results were compared to those obtained with DNA-DNA and DNA-RNA duplexes involving dU at the same positions. Oligonucleotides containing Un clearly demonstrated their ability to form duplexes with both complementary DNA and RNA but with higher stabilities for the DNA-RNA duplexes similar to the one of the parent DNA-RNA duplex. Investigations into the thermodynamic properties of these 17-base-pair duplexes revealed ΔG values (37°C) that are in line with the measured T m values for both the DNA-DNA and DNA-RNA duplexes. CD spectroscopic structural investigations indicated that the conformations of the DNA-DNA and DNA-RNA duplexes involving Un are similar to those of the dT-rA and dU-rA containing duplexes. Only small changes in intensities and weak blue shifts were observed when three Uns were incorporated into the duplexes. The results of the molecular dynamic simulations showed, for the six duplexes involving the modified nucleoside Un, calculated curvatures similar to those of the corresponding unmodified duplexes without base-pair disruption. The neopentyl group is able to be accommodated in the minor grooves of both the DNA-DNA and RNA-DNA duplexes. However, molecular dynamic simulations indicated that the Uns adopt a C2'-exo sugar pucker conformation close to an A-helix type without perturbing the C2'endo sugar pucker conformations of their 2'-deoxynucleoside neighbours. These results confirm the potential of 2'-O-neopentyldeoxyuridine as a nucleoside surrogate for oligonucleotide based therapeutic strategies.