Structural Requirements at the Catalytic Site of the Heteroduplex Substrate for Human RNase H1 Catalysis

Abstract

Human RNase H1 cleaves RNA exclusively in an RNA/ DNA duplex; neither double-strand DNA nor doublestrand RNA is a viable substrate. Previous studies suggest that the helical geometry and sugar conformation of the DNA and RNA may play a role in the selective recognition of the heteroduplex substrate by the enzyme. We systematically evaluated the influence of sugar conformation, minor groove bulk, and conformational flexibility of the heteroduplex on enzyme efficiency. Modified nucleotides were introduced into the oligodeoxyribonucleotide at the catalytic site of the heteroduplex and consisted of southern, northern, and eastern biased sugars with and without 2-substituents, non-hydrogen bonding base modifications, abasic deoxyribonucleotides, intranucleotide hydrocarbon linkers, and a ganciclovir-modified deoxyribonucleotide. Heteroduplexes containing modifications exhibiting strong northern or southern conformational biases with and without a bulky 2-substituent were cleaved at a significantly slower rate than the unmodified substrate. Modifications imparting the greatest degree of conformational flexibility were the poorest substrates, resulting in dramatically slower cleavage rates for the ribonucleotide opposing the modification and the surrounding ribonucleotides. Finally, modified heteroduplexes containing modifications predicted to mimic the sugar pucker and conformational flexibility of the deoxyribonucleotide exhibited cleavage rates comparable with those of the unmodified substrate. These data suggest that sugar conformation, minor groove width, and the relative positions of the intra-and internucleotide phosphates are the crucial determinants in the selective recognition of the heteroduplex substrate by human RNase H1 and offer immediate steps to improve the performance of DNA-like antisense oligonucleotides.RNase H hydrolyzes RNA in RNA-DNA hybrids (1). RNase H activity appears to be ubiquitous in eukaryotes and bacteria (2-7). Although RNases H constitute a family of proteins of varying molecular mass, the nucleolytic activity and substrate requirements appear to be similar for the various isotypes. For example, all RNases H studied to date function as endonucleases, exhibiting limited sequence specificity and requiring divalent cations (e.g. Mg 2ϩ and Mn 2ϩ ) to produce cleavage products with 5Ј-phosphate and 3Ј-hydroxyl termini (8). Recently, two human RNase H genes have been cloned and expressed (9 -11). RNase H1 is a 286-amino acid protein and is expressed ubiquitously in human cells and tissues (9). The amino acid sequence of human RNase H1 displays strong homology to RNase H1 from yeast, chicken, Escherichia coli, and mouse (9). The human RNase H2 enzyme is a 299-amino acid protein with a calculated mass of 33.4 kDa and has also been shown to be ubiquitously expressed in human cells and tissues (10).1 Human RNase H2 shares strong amino acid sequence homology with RNase H2 from Caenorhabditis elegans, yeast, and E. coli (10). Although the biological roles for the human enzymes are not fully under...

Antisense locked nucleic acids efficiently suppress BCR/ABL and induce cell growth decline and apoptosis in leukemic cells

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Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines

“…Although the enzyme binds RNA duplexes they are not recognized as substrates (46), presumably because a canonical A-form geometry precludes endonucleolytic cleavage of the RNA strand. Consistent with this observation, incorporation of m 5 s 2 U into the DNA strand of heteroduplexes dramatically reduced the cleavage rate at ribonucleotides opposite modified residues (47). Although this puts limitations on the use of this modification in antisense applications, where RNase H-mediated degradation of the target mRNA is considered important for efficacy, the properties of the s 2 U, m 5 s 2 U and m 5 s 2 Umoe modifications may render them of interest for use in siRNAs.…”

An Overview of Sugar‐Modified Oligonucleotides for Antisense Therapeutics

“…This observation suggested that the ANA/RNA duplex was a substrate for RNase H enzyme which was later demonstrated [50]. However, the efficiency was relatively poor compared to the native duplex and was attributed to the lower thermal stability of the duplex as a result of lesser amount of ANA/RNA duplex available for the enzyme to process.…”