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Targeting RNA by the Antisense Approach and a Close Look at RNA Cleavage Reaction
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
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. , p. 58
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 355
Keywords [en]
Organic chemistry, mRNA targeting, antisense oligonucleotides, target affinity, RNase H, Michaelis-Menten kinetics, exo-nuclease stability, NMR, pH titration, alkaline hydrolysis
Keywords [sv]
Organisk kemi
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-8272ISBN: 978-91-554-6995-5 (print)OAI: oai:DiVA.org:uu-8272DiVA, id: diva2:170860
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
List of papers
1. Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides
Open this publication in new window or tab >>Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides
2007 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 19, p. 5635-5646Article 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.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-96312 (URN)10.1021/bi0620205 (DOI)000246283600002 ()17411072 (PubMedID)
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2017-12-14
2. Conformationally Constrained 2'-N,4'-C-Ethylene-Bridged Thymidine (Aza-ENA-T): Synthesis, Structure, Physical, and Biochemical Studies of Aza-ENA-T-Modified Oligonucleotides
Open this publication in new window or tab >>Conformationally Constrained 2'-N,4'-C-Ethylene-Bridged Thymidine (Aza-ENA-T): Synthesis, Structure, Physical, and Biochemical Studies of Aza-ENA-T-Modified Oligonucleotides
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2006 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, no 47, p. 15173-15187Article in journal (Refereed) Published
Abstract [en]

The 2'-deoxy-2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T) has been synthesized using a key cyclization step involving 2'-ara-trifluoromethylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-bis-OBn- protected diastereomerically pure aza-ENA-Ts (12a and 12b) with the fused piperidino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7 < P < 27 degrees, 44 degrees < phi(m) < 52 degrees). The origin of the chirality of two diastereomerically pure aza-ENA-Ts was found to be due to the endocyclic chiral 2'-nitrogen, which has axial N-H in 12b and equatorial N-H in 12a. The latter is thermodynamically preferred, while the former is kinetically preferred with E-a 25.4 kcal mol(-1), which is thus far the highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The 5'-O-DMTr-aza-ENA-T-3'-phosphoramidite was employed for solid-phase synthesis to give four different singly modified 15-mer antisense oligonucleotides (AONs). Their AON/RNA duplexes showed a T m increase of 2.5-4 degrees C per modification, depending upon the modification site in the AON. The relative rates of the RNase H1 cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different. The aza-ENA-T modifications also made the AONs very resistant to 3' degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h). Thus, the aza-ENA-T modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-96272 (URN)10.1021/ja0634977 (DOI)000242216100046 ()17117869 (PubMedID)
Available from: 2007-10-16 Created: 2007-10-16 Last updated: 2017-12-14
3. Five- and Six-Membered Conformationally Locked 2‘,4‘-Carbocyclic ribo-Thymidines: Synthesis, Structure, and Biochemical Studies
Open this publication in new window or tab >>Five- and Six-Membered Conformationally Locked 2‘,4‘-Carbocyclic ribo-Thymidines: Synthesis, Structure, and Biochemical Studies
Show others...
2007 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 129, no 26, p. 8362-8379Article in journal (Refereed) Published
Abstract [en]

Two unusual reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant double bond at C4' and the radical center at C2' of the ribofuranose ring of thymidine have been used as key steps to synthesize North-type conformationally constrained cis-fused bicyclic five-membered and six-membered carbocyclic analogues of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) in high yields. Their structures have been confirmed unambiguously by long range iH-13C NMR correlation (HMBC), TOCSY, COSY, and NOE experiments. The carbocyclic-LNA-T and carbocyclic-ENA-T were subsequently incorporated into the antisense oligonucleotides (AONs) to show that they enhance the Tm of the modified AON/RNA heteroduplexes by 3.5-5 °C and 1.5 °C/modification for carbocyclic-LNA-T and carbocyclic-ENA-T, respectively. Whereas the relative RNase H cleavage rates with carbocyclic-LNA-T, carbocyclic-ENA-T, aza-ENA-T, and LNA-T modified AON/RNA duplexes were found to be very similar to that of the native counterpart, irrespective of the type and the site modification in the AON strand, a single incorporation of carbocyclic-LNA and carbocyclic-ENA into AONs leads to very much more enhanced nuclease stability in the blood serum (stable >48 h) as compared to that of the native (fully degraded <3 h) and the LNA-modified AONs (fully degraded <9 h) and aza-ENA (≈85% stable in 48 h). Clearly, remarkably enhanced lifetimes of these carbocyclic-modified AONs in the blood serum may produce the highly desired pharmacokinetic properties because of their unique stability and consequently a net reduction of the required dosage. This unique quality as well as their efficient use as the AON in the RNase H-promoted cleavage of the target RNA makes our carbocyclic-LNA and carbocyclic-ENA modifications excellent candidates as potential antisense therapeutic agents.

National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-96273 (URN)10.1021/ja071106y (DOI)000247563700050 ()17552524 (PubMedID)
Available from: 2007-10-16 Created: 2007-10-16 Last updated: 2017-12-14Bibliographically approved
4. Significant pKa Perturbation of Nucleobases Is an Intrinsic Property of the Sequence Context in DNA and RNA
Open this publication in new window or tab >>Significant pKa Perturbation of Nucleobases Is an Intrinsic Property of the Sequence Context in DNA and RNA
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2004 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 28, p. 8674-8681Article in journal (Refereed) Published
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-96321 (URN)10.1021/ja048484c (DOI)
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2017-12-14Bibliographically approved
5. Non-identical electronic characters of the internucleotidic phosphates in RNA modulate the chemical reactivity of the phosphodiester bonds
Open this publication in new window or tab >>Non-identical electronic characters of the internucleotidic phosphates in RNA modulate the chemical reactivity of the phosphodiester bonds
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2006 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 4, no 5, p. 928-941Article in journal (Refereed) Published
Abstract [en]

We here show that the electronic properties and the chemical reactivities of the internucleotidic phosphates in the heptameric ssRNAs are dissimilar in a sequence-specific manner because of their non-identical microenvironments, in contrast with the corresponding isosequential ssDNAs. This has been evidenced by monitoring the delta H8(G) shifts upon pH-dependent ionization (pK(a1)) of the central 9-guaninyl (G) to the 9-guanylate ion (G(-)), and its electrostatic effect on each of the internucleotidic phosphate anions, as measured from the resultant delta P-31 shifts (pKa(2)) in the isosequential heptameric ssRNAs vis-`a-vis ssDNAs: [d/r( 5'-Cp(1)Ap(2)Q(1)p(3)Gp(4)Q(2)p(5)Ap(6)C-3'): Q(1) = Q(2) = A (5a/5b) or C (8a/8b), Q(1) = A, Q(2) = C (6a/6b), Q(1) = C, Q(2) = A (7a/7b)]. These oligos with single ionizable G in the centre are chosen because of the fact that the pseudoaromatic character of G can be easily modulated in a pH-dependent manner by its transformation to G(-) (the 2'-OH to 2-O- ionization effect is not detectable below pH 11.6 as evident from the N1-Me-G analog), thereby modulating/titrating the nature of the electrostatic interactions of G to G- with the phosphates, which therefore constitute simple models to interrogate how the variable pseudoaromatic characters of nucleobases under different sequence context (J. Am. Chem. Soc., 2004, 126, 8674-8681) can actually influence the reactivity of the internucleotide phosphates as a result of modulation of sequence context-specific electrostatic interactions. In order to better understand the impact of the electrostatic effect of the G to G- on the tunability of the electronic character of internucleotidic phosphates in the heptameric ssRNAs 5b, 6b, 7b and 8b, we have also performed their alkaline hydrolysis at pH 12.5 at 20 degrees C, and have identified the preferences of the cleavage sites at various phosphates, which are p(2), p(3) and p(4) (Fig. 3). The results of these alkaline hydrolysis studies have been compared with the hydrolysis of analogous N1-Me-G heptameric ssRNA sequences 5c, 7c and 8c under identical conditions in order to establish the role of the electrostatic effect of the 9-guanylate ion (and the 2'-OH to 2-O- ionization) on the internucleotidic phosphate. It turned out that the relative alkaline hydrolysis rate at those particular phosphates ( p2, p3 and p(4)) in the N1-Me-G heptamers was reduced from 16-78% compared to those in the native counterparts [Fig. 4, and ESI 2 (Fig. S11)]. Thus, these physico-chemical studies have shown that those p2, p3 and p4 phosphates in the native heptameric RNAs, which show pK(a2) as well as more deshielding ( owing to weaker P-31 screening) in the alkaline pH compared to those at the neutral pH, are more prone to the alkaline hydrolysis because of their relatively enhanced electrophilic character resulting from weaker P-31 screening. This screening effect originates as a result of the systematic charge repulsion effect between the electron cloud in the outermost orbitals of phosphorus and the central guanylate ion, leading to delocalization of the phosphorus pp charge into its d pi orbitals. It is thus likely that, just as in the non-enzymatic hydrolysis, the enzymatic hydrolysis of a specific phosphate in RNA by general base-catalyss in RNA-cleaving proteins (RNase A, RNA phosphodiesterase or nuclease) can potentially be electrostatically influenced by tuning the transient charge on the nucleobase in the steric proximity or as a result of specific sequence context owing to nearest-neighbor interactions.

National Category
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-96322 (URN)10.1039/B516733G (DOI)000235992700023 ()
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2017-12-14Bibliographically approved

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