The Influence of Antisense Oligonucleotide-induced RNA Structure on Escherichia coli RNase H1 Activity

Lima, Walt F.; Mohan, V.; Crooke, Stanley T.

doi:10.1074/jbc.272.29.18191

Cited by 53 publications

(28 citation statements)

References 27 publications

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“…The K m valves for all three substrates were substantially lower than those of E. coli RNase H1 (Table III) (18,19). As previously reported for E. coli RNase H1, the K m for a phosphorothioate-containing duplex was lower than that of a phosphodiester duplex.…”

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confidence: 55%

Properties of Cloned and Expressed Human RNase H1

Lima

Crooke

1999

Journal of Biological Chemistry

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We have characterized cloned His-tag human RNase H1. The activity of the enzyme exhibited a bell-shaped response to divalent cations and pH. The optimum conditions for catalysis consisted of 1 mM Mg 2؉ and pH 7-8. In the presence of Mg 2؉ , Mn 2؉ was inhibitory. Human RNase H1 shares many enzymatic properties with Escherichia coli RNase H1. The human enzyme cleaves RNA in a DNA-RNA duplex resulting in products with 5-phosphate and 3-hydroxy termini, can cleave overhanging single strand RNA adjacent to a DNA-RNA duplex, and is unable to cleave substrates in which either the RNA or DNA strand has 2 modifications at the cleavage site. Human RNase H1 binds selectively to "A-form"-type duplexes with approximately 10 -20-fold greater affinity than that observed for E. coli RNase H1. The human enzyme displays a greater initial rate of cleavage of a heteroduplex-containing RNA-phosphorothioate DNA than an RNA-DNA duplex. Unlike the E. coli enzyme, human RNase H1 displays a strong positional preference for cleavage, i.e. it cleaves between 8 and 12 nucleotides from the 5-RNA-3-DNA terminus of the duplex. Within the preferred cleavage site, the enzyme displays modest sequence preference with GU being a preferred dinucleotide. The enzyme is inhibited by single-strand phosphorothioate oligonucleotides and displays no evidence of processivity. The minimum RNA-DNA duplex length that supports cleavage is 6 base pairs, and the minimum RNA-DNA "gap size" that supports cleavage is 5 base pairs.

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confidence: 55%

Properties of Cloned and Expressed Human RNase H1

Lima

Crooke

1999

Journal of Biological Chemistry

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158

140

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“…Furthermore, RNase H activity is also often implicated in antisense oligodeoxynucleotide-mediated degradation of RNA. In a previous study examining E. coli RNase H1 activity on antisense oligonucleotide-induced RNA pseudo-half-knot structures we have shown that cleavage of the structured RNA is profoundly affected by the binding directionality of the enzyme (25). Taken together, these studies suggest that the binding polarity exhibited by RNase H has important implications both biologically and pharmacologically.…”

Section: Rnase H Cleavage Of Single Strand Rna 27514mentioning

confidence: 77%

Cleavage of Single Strand RNA Adjacent to RNA-DNA Duplex Regions by Escherichia coli RNase H1

Lima

Crooke

1997

Journal of Biological Chemistry

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RNase H1 from Escherichia coli cleaves single strand RNA extending 3 from an RNA-DNA duplex. Substrates consisting of a 25-mer RNA annealed to complementary DNA ranging in length from 9 -17 nucleotides were designed to create overhanging single strand RNA regions extending 5 and 3 from the RNA-DNA duplex. Digestion of single strand RNA was observed exclusively within the 3 overhang region and not the 5 overhang region. RNase H digestion of the 3 overhang region resulted in digestion products with 5-phosphate and 3-hydroxyl termini. The number of single strand RNA residues cleaved by RNase H is influenced by the sequence of the single strand RNA immediately adjacent to the RNA-DNA duplex and appears to be a function of the stacking properties of the RNA residues adjacent to the RNA-DNA duplex. RNase H digestion of the 3 overhang region was not observed for a substrate that contained a 2-methoxy antisense strand. The introduction of 3 deoxynucleotides at the 5 terminus of the 2-methoxy antisense oligonucleotide resulted in cleavage. These results offer additional insights into the binding directionality of RNase H with respect to the heteroduplex substrate.RNase H hydrolyzes RNA in an RNA-DNA duplex (1). RNase H activity appears to be ubiquitous in eukaryotes and bacteria (2, 3). This family of enzymes functions as endonucleases exhibiting limited sequence specificity and requiring divalent cations to produce 5Ј-phosphate and 3Ј-hydroxyl termini (4).RNase H1 from Escherichia coli is perhaps the best characterized isotype of this family of enzymes. The substrate requirements for RNase H1 have been studied extensively. The dissociation constants for various duplexes and E. coli RNase H1 indicate that this enzyme is a double strand RNA-binding protein that cleaves RNA only in an RNA-DNA duplex (5). Furthermore, the cleavage specificity of the enzyme appears to be due to the catalytic process and not the binding interaction between RNase H and the substrate duplex. For example, although RNase H exhibited a binding affinity for substrates containing 2Ј-methoxy modifications approximately equal to the unmodified RNA-DNA heteroduplex, RNase H activity was ablated by these modifications. Kinetic analyses of substrates containing RNA duplexed with chimeric oligonucleotides composed of a mixture of deoxy-and 2Ј-modified nucleotides demonstrated that the rate of cleavage was a function of the length of the deoxy portion of the chimeric oligonucleotide, i.e. the cleavage rate decreased with a diminishing number of contiguous deoxynucleotides, and complete loss in activity was observed for chimeric oligonucleotides containing less than 4 contiguous deoxynucleotides (6, 7). In all cases E. coli RNase H1 cleavage of the RNA was observed to occur exclusively within regions base-paired to DNA.In this communication we report that E. coli RNase H1 can cleave single strand RNA extending 3Ј from an RNA-DNA duplex. The fact that the products are 5Ј-phosphate and 3Ј-hydroxyl suggests that the enzyme cleaves single and double strand regi...

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“…Many of the conformationally constrained nucleotides have been shown to enhance the stability of the modified AON/RNA heteroduplex, but they failed to show any RNase H recruiting capability (1, 6, 20, 23). Increase in the affinity of AON toward target RNA without inducing RNase H activity does not give expected results for developing new antisense drugs (41). It has emerged that for a modified AON/RNA heteroduplex to become a substrate for RNase H the functional groups present in the minor groove of the duplex should not inhibit binding of the enzyme.…”

Section: Discussionmentioning

confidence: 99%

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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The Influence of Antisense Oligonucleotide-induced RNA Structure on Escherichia coli RNase H1 Activity

Cited by 53 publications

References 27 publications

Properties of Cloned and Expressed Human RNase H1

Properties of Cloned and Expressed Human RNase H1

Cleavage of Single Strand RNA Adjacent to RNA-DNA Duplex Regions by Escherichia coli RNase H1

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

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