Conformationally Constrained 2‘-<i>N</i>,4‘-<i>C</i>-Ethylene-Bridged Thymidine (Aza-ENA-T):  Synthesis, Structure, Physical, and Biochemical Studies of Aza-ENA-T-Modified Oligonucleotides

Varghese, Oommen P.; Barman, Jharna; Pathmasiri, Wimal; Plashkevych, Oleksandr; Honcharenko, Dmytro; Chattopadhyaya, J.

doi:10.1021/ja0634977

Cited by 67 publications

(95 citation statements)

References 74 publications

(146 reference statements)

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“…hybrid duplexes with respect to the unmodified hybrid duplex was not changed, which was evident from the CD comparison (29,30,32). We conclude that the resistant stretch of five ribonucleotides in the chimeric azetidine-T modified AONs 9-10/RNA and aza-ENA-T modified AON 3/RNA duplexes is created, as for isosequential oxetane-T AONs chimeras (30), as a result of microstructure perturbation in those resistant stretches of the modified heteroduplexes compared to the regular native AON/RNA duplex.…”

Section: Discussionmentioning

confidence: 67%

Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides

et al. 2007

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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...

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Section: Discussionmentioning

confidence: 67%

Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides

et al. 2007

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“…The oligos are named according to the nomenclature of previously published work (18) to allow easy comparison of results between individual studies. Base positions with mismatches to the target are underlined. for oligo sequences); hexitol nucleic acid (HNA) (26), altritol nucleic acid (ANA) (27), LNA (28), UNA (29), 2′-O-aminoethyl (EA) (30,31), 2′,4′-carbocyclic-ENA-LNA (CENA), 2′,4′-carbocyclic-LNA-locked nucleic acid (CLNA), 2′-deoxy-2′-N,4′-C-ethylene-locked nucleic acid (AENA) (32), 4′- C -hydroxymethyl-DNA (HM) (33) and 2′-O-methyl (OMe); (Table 1 and Figure 2B). We decided to preferentially modify the AS seed as this region guides initial miRNA–target interactions (34), is responsible for off-target effects (12) and is more tolerant to single mismatches (and thereby expectably chemical modification) towards target mRNAs as compared to the central and 3′-end region of the AS (35).…”

Section: Resultsmentioning

confidence: 99%

A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects

Bramsen¹,

Pakula²,

Hansen³

et al. 2010

Nucleic Acids Research

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Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2–8 of either siRNA strand counting from the 5′-end) and complementary sequences in the 3′UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA–target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low.

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“…89−92 Using the 2′-O-TEM based RNA synthesis strategy that has been developed in our lab, 93,94 7′-Me-carba-LNA and 8′R-Mecarba-ENA modified siRNAs have been synthesized to target the eGFP gene in HeLa cells. 89 α-L-LNA, aza-ENA, 28 UNA, 97 HNA, 98 ANA, 99 etc. This exhaustive study provides valuable guidance to chemically modify siRNAs as potential therapeutics.…”

Section: Rnai Potency Of Sirnas Containingmentioning

confidence: 99%

Intramolecular Free-Radical Cyclization Reactions on Pentose Sugars for the Synthesis of Carba-LNA and Carba-ENA and the Application of Their Modified Oligonucleotides as Potential RNA Targeted Therapeutics

Zhou

Chattopadhyaya

2012

Chem. Rev.

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Introduction 3808 2. Synthesis of Carba-ENA and Carba-LNA Nucleosides 3810 2.1. Synthesis of Carba-ENA through Ring Closure Metathesis 3810 2.2. Application of Intramolecular Free-Radical Cyclization on Constructing Bicyclic Nucleosides 3810 2.3. Synthesis of Carba-LNA and Carba-ENA through Intramolecular Free-Radical Addition to CC 3812 2.4. Synthesis of Carba-LNA and Carba-ENA through Intramolecular Free-Radical Addition to CN 3814 2.5. Synthesis of Carba-LNA through Intramolecular Free-Radical Addition to CC 3814 2.6. Synthesis of α-L-Carba-LNA Derivatives through Intramolecular Free-Radical Addition 3814 2.7. Regio-Selectivity and Stereoselectivity of the Free-Radical Cyclization 3815 3. Conformation of Carba-LNA and Carba-ENA Nucleosides 3818 4. Orientation of Carbocyclic Moieties of Carba-LNA and α-L-Carba-LNA in Duplex Form 3818 5. Thermal Stability of Modified AON/RNA Duplex 3820 5.1.

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Conformationally Constrained 2‘-N,4‘-C-Ethylene-Bridged Thymidine (Aza-ENA-T): Synthesis, Structure, Physical, and Biochemical Studies of Aza-ENA-T-Modified Oligonucleotides

Cited by 67 publications

References 74 publications

Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides

Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides

A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects

Intramolecular Free-Radical Cyclization Reactions on Pentose Sugars for the Synthesis of Carba-LNA and Carba-ENA and the Application of Their Modified Oligonucleotides as Potential RNA Targeted Therapeutics

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