Synthesis, Hybridization, and Nuclease Resistance Properties of 2'-O-AMINOOXYETHYL Modified Oligonucleotides (original) (raw)
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We have designed and synthesized mixed backbone oligonucleotides (MBOs) containing 2′-5′-ribo-and 3′-5′-deoxyribonucleotide segments. Thermal melting studies of the phosphodiester MBOs (three 2′-5′ linkages at each end) with the complementary 3′-5′-DNA and -RNA target strands suggest that 2′-5′-ribonucleoside incorporation into 3′-5′-oligodeoxyribonucleotides reduces binding to the target strands compared with an all 3′-5′-oligodeoxyribonucleotide of the same sequence and length. Increasing the number of 2′-5′ linkages (from six to nine) further reduces binding to the DNA target strand more than the RNA target strand [Kandimalla,E.R. and Agrawal,S. (1996) Nucleic Acids Symp. Ser., 35, 125-126]. Phosphorothioate (PS) analogs of MBOs destabilize the duplex with the DNA target strand more than the duplex with the RNA target strand. Circular dichroism studies indicate that the duplexes of MBOs with the DNA and RNA target strands have spectral characteristics of both A-and B-type conformations. Compared with the control oligonucleotide, MBOs exhibit moderately higher stability against snake venom phosphodiesterase, S1 nuclease and in fetal calf serum. Although 2′-5′ modification does not evoke RNase H activity, this modification does not effect the RNase H activation property of the 3′-5′-deoxyribonucleotide segment adjacent to the modification. In vitro studies with MBOs suggest that they have lesser effects on cell proliferation, clotting prolongation and hemolytic complement lysis than do control PS oligodeoxyribonucleotides. PS analogs of MBOs show HIV-1 inhibition comparable with that of a control PS oligodeoxyribonucleotide with all 3′-5′ linkages. The current results suggest that a limited number of 2′-5′ linkages could be used in conjunction with PS oligonucleotides to further modulate the properties of antisense oligonucleotides as therapeutic agents.
Facile preparation of nuclease resistant 3′ modified oligodeoxynucleotides
Nucleic Acids Research, 1993
An efficient chemical procedure for the immobilization of carboxylate containing conjugate groups onto controlled pore glass (CPG) is described. The derivatized supports were used in the automated synthesis of an oligodeoxynucleotide (20-mer ODN) containing a 3' phosphodiester linked hexanol, aminohexyl, acridine, or cholesterol group. The stability of the oligomer in a hepatoma cell culture was found to be prolonged two to three fold by the presence of any one of the 3' tails. By contrast, an aminohexyl group appended to the 5' terminus of the ODN only marginally improved its nuclease resistance. These data support the notion that antisense ODNs are primarily degraded by 3' exonucleases. Introduction of simple 3' tails which incorporate a normal phosphodiester linkage can increase ODN stability by interfering with these enzymes.
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.
2‘- O -[2-(Amino)-2-oxoethyl] Oligonucleotides
Organic Letters, 2003
Oligonucleotides with novel modifications, 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.
2′-O-Propargyl oligoribonucleotides: Synthesis and hybridisation
Tetrahedron, 1998
Fully modified oligonucleotide sequences containing 2'-O-propargylribonucleotides were synthesised on automated DNA-synthesisers using the phosphoramidite approach. A highly selective alkylation procedure was used to introduce the propargyl functionality, thereby enabling the synthesis of protected 2'-O-propargyl-3'-O-phosphoramidites, building blocks for the assembly of 2'-O-propargyl oligoribonucleotides. The suitability of phosphoramidite chemistry for the introduction of this modified nucleoside was proven using MALDI or ES mass spectrometry of the final oligomer. The 2'-O-propargyl oligoribonucleotides showed an increase in the Tm of duplexes with complementary RNA relative to the corresponding RNA homoduplex. These analogues should prove useful for a variety of antisense applications. © 1998 Elsevier Science Ltd. All rights reserved.
Journal of the Chemical Society, Perkin Transactions 2, 2001
Although the T m drops ∼6 ЊC/modification (note: T m loss is ∼10 ЊC/mismatch) in the oxetane, [1-(1Ј,3Ј-Oanhydro-β--psicofuranosyl)thymine, T], modified antisense (AON)-RNA heteroduplexes, the relative rates of the complementary RNA cleavage by RNase H remain the same as or comparable to that of the native counterpart. The RNA cleavage in the native hybrid duplex was 68 ± 3% (T m = 44 ЊC), whereas it was found to be 64 ± 10% for the single T modified AON-RNA duplex (T m = 39 ЊC), 56 ± 9% for the double T modified AON-RNA duplex (T m = 33 ЊC) and 60 ± 7% for the triple T modified AON-RNAs (T m = 26 ЊC). The oxetane modifications in AON reduce the endonuclease cleavage (DNase 1) significantly. One modification gives ∼2-fold protection and three modifications give ∼4-fold protection compared to that of the native. Introductions of both interior oxetane modifications in conjunction with the 3Ј-DPPZ (dipyridophenazine) group give the resulting AON-RNA hybrid an RNase H cleavage rate at least the same as that of the native counterpart, which, additionally, gives full stability against both exo-and endonucleases. The conformational transmission of the constrained 3Ј-endo sugar of the oxetane nucleotide in the AON strand is found to be transmitted up to a stretch of five nucleotides in the heteroduplex as is evident by the RNase H resistance to the cleavage of the complementary RNA strand, thereby showing that this five-nucleotide region most probably takes up a local RNA-RNA type conformation. This is the first report of an antisense oligonucleotide construct which fulfils three important criteria simultaneously: (1) the modified AON promotes the complementary RNA cleavage by RNase H at an efficiency comparable to that of the native counterpart, (2) the modified AON has substantially more endonuclease stability than that of the native AON, and finally, (3) the DPPZ group at the 3Ј-end provides the expected exonuclease stability. This also shows that the T m increase of the AON-RNA hybrid duplex is not mandatory for RNase H promoted destruction of the target RNA.
Nucleic Acids Research, 1993
6-Azathymidine, 6-aza-2'-deoxycytidine, 6-methyl-2'deoxyuridine, and 5,6-dimethyl-2'-deoxyuridine nucleosides have been converted to phosphoramidite synthons and incorporated into oligodeoxynucleotides (ODNs). ODNs containing from 1 to 5 of these modified pyrimidines were compared with known 2'-deoxyuridine, 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxycytidine, and 5-methyl-2'-deoxycytidine nucleoside modifications. Stability in 10% heat inactivated fetal calf serum, binding affinities to RNA and DNA complements, and ability to support RNase H degradation of targeted RNA in DNA-RNA heteroduplexes were measured to determine structure-activity relationships. 6-Azathymidine capped ODNs show an enhanced stability in serum (7to 12-fold increase over unmodified ODN) while maintaining hybridization properties similar to the unmodified ODNs. A 22-mer ODN having its eight thymine bases replaced by eight 6-azathymines or 5-bromouracils hybridized to a target RNA and did not inhibit RNase H mediated degradation.
Organic & Biomolecular Chemistry, 2002
Antisense oligonucleotides (AONs) with single and double oxetane C modifications [1Ј,2Ј-oxetane constrained cytidine, 1-(1Ј,3Ј-O-anhydro-β--psicofuranosyl)cytosine] have been evaluated, in comparison with the corresponding T-modified AONs, for their antisense potentials by targeting to a 15mer complementary RNA. Although the C modified mixmer AONs show ∼3 ЊC drop per modification in melting temperature (T m) of their hybrid AON-RNA duplexes, they are found to be good substrates for RNase H, in comparison with the native AON-RNA duplex. An AON with double C modifications along with 3Ј-DPPZ (dipyridophenazine) conjugation shows the T m of the hybrid duplexes as high as that of the native, and the RNase H activity as good as its unconjugated counterpart. A detailed Michaelis-Menten kinetic analysis of RNase H cleavage showed that the single and double C modified AON-RNA duplexes as well as double C modifications along with 3Ј-DPPZ have catalytic activities (k cat) close to the native. However, the RNase H binding affinity (1/K m) showed a slight decrease with increase in the number of modifications, which results in less effective enzyme activity (k cat /K m) for C modified AON-RNA duplexes. All oxetane modified AON-RNA hybrids showed a correlation of T m with the 1/K m , V max , or V max /K m. The C modified AONs (with 3Ј-DPPZ), as in the T counterpart, showed an enhanced tolerance towards the endonuclease and exonuclease degradation compared to the native (the oxetane-sugar and the DPPZ based AONs are non-toxic to K562 cell growth, ref. 18). Thus a balance has been found between exo and endonuclease stability visa -vis thermostability of the heteroduplex and the RNase H recruitment capability and cleavage with the oxetaneconstrained cytidine incorporated AONs as potential antisense candidates with a fully phosphate backbone for further biological assessment.