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Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
2007 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 19, 5635-5646 p.Article in journal (Refereed) Published
Abstract [en]

The RNase H cleavage potential of the RNA strand basepaired with the complementary antisense oligonucleotides (AONs) containing NorthEast 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 (ΔTm = 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 Tm 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 Km and Vmax values obtained from the RNA concentration-dependent 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 (Kcat) 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/Km) 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 (Kcat/Km) 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.

Place, publisher, year, edition, pages
2007. Vol. 46, no 19, 5635-5646 p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-96312DOI: 10.1021/bi0620205ISI: 000246283600002PubMedID: 17411072OAI: oai:DiVA.org:uu-96312DiVA: diva2:170844
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2017-12-14
In thesis
1. Conformationally Constrained Nucleosides: Design, Synthesis, and Biochemical Evaluation of Modified Antisense Oligonucleotides
Open this publication in new window or tab >>Conformationally Constrained Nucleosides: Design, Synthesis, and Biochemical Evaluation of Modified Antisense Oligonucleotides
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is concerned with synthesis, structure and biochemical analysis of chemically modified oligonucleotides with potential therapeutic applications. The three types of chemical modifications described here are: (a) A North-East locked 1',2'-azetidine nucleoside (b) A North locked 2',4'-cyanomethylene bridged nucleoside and (c) A 2',4'-aza-ENA-T nucleoside. The synthesis of the 1',2'-azetidine fused nucleosides was described using two different approaches. A highly strained 2',4'-cyanomethylene locked nucleoside was synthesized but could not be converted to the phosphoramidite derivative due to instability during derivatization. The key cyclization step in the aza-ENA-T nucleoside synthesis gave rise to two separable diastereomers due to chirality at the exocyclic nitrogen. Conversion of diastereomer 55 to 56 occurred with a large free energy of activation (ΔG = 23.4 kcal mol-1 at 298 K in pyridine-d5). Of the two isomers the equatorial NH product was more stable than the axial one due to reduced 1,3 diaxial interactions. As a result, all NH axial product was converted to the equatorial isomer during subsequent steps in the synthesis. NMR and ab initio experiments confirmed the North-East structure of the 1',2'-azetidine locked nucleoside and North conformation of aza-ENA-T locked nucleosides with a chair conformation of the piperidine ring.

The amino modified nucleosides were incorporated into different positions of a 15mer oligonucleotide. The azetidine modified AONs did not form stable duplexes with complementary RNA (ΔTm ~-1 to -4 °C), but they performed better than previously synthesized isosequential 1',2'-oxetane modified oligonucleotides. The 2',4'-aza-ENA-T modified oligonucleotide, on the other hand, showed excellent target affinity with complementary RNA (ΔTm ~+4 °C). The azetidine and aza-ENA-T modified oligonucleotides showed significant stability in the presence of human serum and snake venom phosphodiesterase (3'-exonuclease) as compared to the unmodified native sequence. The singly modified 15mer oligonucleotides were also subjected to RNase H promoted digestion in order to evaluate their potential as effective antisense agents. The effective enzyme activity (kcat/Km) was found to be lower in the modified AONs due to reduced enzyme-substrate binding. However, the catalytic activity of RNase H with these modified-AON:RNA duplexes were higher than observed with the native duplex.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 60 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 354
Keyword
Organic chemistry, chemically modified oligonucleotides, azetidine, aza-ENA-T, cyanomethylene locked, free energy of activation, diaxial interactions, chair conformation, stable duplex, human serum, snake venom phosphodiesterase, antisense agents, RNase H, Organisk kemi
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-8266 (URN)978-91-554-6992-4 (ISBN)
Public defence
2007-11-09, B7:113a, BMC, Husargatan 3, Uppsala, 13:30 (English)
Opponent
Supervisors
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2012-08-03Bibliographically approved
2. Conformationally Constrained Nucleosides, Nucleotides and Oligonucleotides: Design, Synthesis and Properties
Open this publication in new window or tab >>Conformationally Constrained Nucleosides, Nucleotides and Oligonucleotides: Design, Synthesis and Properties
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is based on six original research publications describing synthesis, structure and physicochemical and biochemical analysis of chemically modified oligonucleotides (ONs) in terms of their potential diagnostic and therapeutic applications. Synthesis of two types of bicyclic conformationally constrained nucleosides, North-East locked 1',2'-azetidine and North locked 2',4'-aza-ENA, is described. Study of the molecular structures and dynamics of bicyclic nucleosides showed that depending upon the type of fused system they fall into two distinct categories with their respective internal dynamics and type of sugar conformation. The physicochemical properties of the nucleobases in the conformationally constrained nucleosides found to be depended on the site and ring-size of the fused system.

The incorporation of azetidine modified nucleotide units into 15mer ONs lowered the affinity toward the complementary RNA. However, they performed better than previously reported isosequential 1',2'-oxetane modified analogues. Whereas aza-ENA-T modification incorporated into ONs significantly enhanced affinity to the complementary RNA. To evaluate the antisense potential of azetidine-T and aza-ENA-T modified ONs, they were subjected to RNase H promoted cleavage as well as tested towards nucleolytic degradation. Kinetic experiments showed that modified ONs recruit RNase H, however with lower enzyme efficiency due to decreased enzyme-substrate binding affinity, but with enhanced turnover number. Both, azetidine-T and aza-ENA-T modified ONs demonstrated improved 3'-exonuclease stability in the presence of snake venom phosphodiesterase and human serum compared to the unmodified sequence.

Oligodeoxynucleotides (ODNs) containing pyrene-functionalized azetidine-T (Aze-pyr X) and aza-ENA-T (Aza-ENA-pyr Y) modifications showed different fluorescence properties. The X modified ODNs hybridized to the complementary DNA and RNA showed variable increase in the fluorescence intensity depending upon the nearest-neighbor at the 3'-end to X modification (dA > dG > dT > dC) with high fluorescence quantum yield. However, the Y modified ODNs showed a sensible enhancement of the fluorescence intensity only with complementary DNA. Also, the X modified ODN showed decrease (~37-fold) in the fluorescence intensity upon duplex formation with RNA containing a G nucleobase mismatch opposite to the modification site, whereas a ~3-fold increase was observed for the Y modified probe.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 71 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 440
Keyword
Bioorganic chemistry, antisense oligonucleotides, conformationally constrained nucleosides, azetidine, aza-ENA, target affinity, RNase H, exonuclease stability, pyrene-functionalized nucleotides, fluorescence, mismatch discrimination, Bioorganisk kemi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-8887 (URN)978-91-554-7219-1 (ISBN)
Public defence
2008-06-05, C10:301, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2008-05-14 Created: 2008-05-14 Last updated: 2010-03-03Bibliographically approved
3. Targeting RNA by the Antisense Approach and a Close Look at RNA Cleavage Reaction
Open this publication in new window or tab >>Targeting RNA by the Antisense Approach and a Close Look at RNA Cleavage Reaction
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis summarizes the results of studies on two aspects of nucleic acids. Chemically modified antisense oligonucleotides (AONs) have been evaluated with regards to their suitability for mRNA targeting in an antisense approach (Paper I – III). The chemically modified nucleotidic units 2'-O-Me-T, 2'-O-MOE-T, oxetane-T, LNA-T, azetidine-T, aza-ENA-T, carbocyclic-ENA-T and carbocyclic-LNA-T were incorporated into 15-mer AONs and targeted against a 15-mer RNA chosen from the coding region of SV-40 large T antigen. The comparative study showed that a single modified nucleotide in the AON with North-East locked sugar (oxetane-T and azetidine-T) lowered the affinity for the complementary RNA whereas North locked sugars (LNA-T, aza-ENA-T, carbocyclic-ENA-T, and carbocyclic-LNA-T) significantly improved the affinity. A comparative RNase H digestion study showed that modifications of the same type (North-East type or North type) in different sequences gave rise to similar cleavage patterns. Determination of the Michaelis-Menten parameters by kinetic experiments showed that the modified AONs recruit RNase H resulting in enhanced turnover numbers (kcat) although with weaker enzyme-substrate binding (1/Km) compared to the unmodified AON. The modified AONs were also evaluated with regards to resistance towards snake venom phosphodiesterase and human serum to estimate their stability toward exonucleases. The aza-ENA-T and carbocyclic-ENA-T modified AONs showed improved stability compared to all other modified AONs. In general, the modified AONs with North type nucleotides (except LNA-T) were found to be superior to the North-East type as they showed improved target affinity, comparable RNase H recruitment capability and improved exonuclease stability.

The second aspect studied in this thesis is based on physicochemical studies of short RNA molecules utilizing NMR based pH titration and alkaline hydrolysis reactions (Paper IV – V). The NMR based (1H and 31P) pH titration studies revealed the effect of guaninyl ion formation, propagated electrostatically through a single stranded chain in a sequence dependent manner. The non-identical electronic character of the internucleotidic phosphodiesters was further verified by alkaline hydrolysis experiments. The internucleotidic phosphodiesters, which were influenced by guaninyl ion formation, were hydrolyzed at a faster rate than those sequences where such guaninyl ion formation was prevented by replacing G with N1-Me-G.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 355
Keyword
Organic chemistry, mRNA targeting, antisense oligonucleotides, target affinity, RNase H, Michaelis-Menten kinetics, exo-nuclease stability, NMR, pH titration, alkaline hydrolysis, Organisk kemi
National Category
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-8272 (URN)978-91-554-6995-5 (ISBN)
Public defence
2007-11-08, B22, BMC, Box 576, SE-75123, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2012-08-03Bibliographically approved

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