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Walt F Lima

Publications and source records attributed to Walt F Lima.

9 recordsLinked to original sources
PubMed2006

Human RNase H1 discriminates between subtle variations in the structure of the heteroduplex substrate.

In a previous study, we demonstrated that the sugar conformation and helical geometry of the heteroduplex substrate at the catalytic site of human RNase H1 directs the selective recognition of the substrate by the enzyme (J Biol Chem 279: 36317-36326, 2004). In this study, we systematically introduced 2'-methoxyethoxy (MOE) nucleotides into the antisense oligodeoxyribonucleotide (ASO) of the heteroduplex to alter the helical geometry of the substrate. The MOE substitutions at the 3' and 5' poles of the ASO resulted in fewer cleavage sites and slower cleavage rates compared with the unmodified substrates. Furthermore, a greater reduction in cleavage activity was observed for MOE substitutions at the 5' pole of the ASO. The 3'- and 5'-most cleavage sites were positioned two and four to five base pairs, respectively, from the nearest MOE residues, suggesting a conformational transmission of the MOE/RNA helical geometry into the DNA/RNA portion of the heteroduplex. Similar conformational transmission was observed for Okazaki-like substrates containing deoxyribonucleotide substitutions at the 3' pole of the oligoribonucleotide. Finally, the heteroduplex substrates exhibited preferred cleavage sites that were cleaved 2- to 3-fold faster than other sites in the substrate, and these sites exhibited the greatest influence on the initial cleavage rates. The data presented here offer further insights into the role substrate structure plays in directing human RNase H1 activity as well as the design of effective ASOs.

Base Sequence↗
PubMed2006

The positional influence of the helical geometry of the heteroduplex substrate on human RNase H1 catalysis.

In a companion study published in this issue (p. 83), we showed that chimeric substrates containing 2'-methoxyethyl (MOE) nucleotides inhibited human RNase H1 activity. In this study, we prepared chimeric substrates containing a central DNA region with flanking northern-biased MOE nucleotides hybridized to complementary RNA. Conformationally biased and flexible modified nucleotides were positioned at the junctions between the DNA and MOE residues of the chimeric substrates to modulate the effects of the MOE residues on human RNase H1 activity. The strong northern-biased locked-nucleic acid modification exacerbated the negative effects of the MOE modifications resulting in slower human RNase H1 cleavage rates. Enhanced cleavage rates were observed for the eastern-biased 2'-ara-fluorothymidine and bulge inducing N-methylthymidine modifications positioned at the 5'-DNA/3'-MOE junction as well as the southern-biased 2'-methylthiothymidine and conformationally flexible tetrafluoroindole (TFI) modifications positioned at the 5'-MOE/3'-DNA junction. The heterocycle of the ribonucleotide opposing the TFI deoxyribonucleotide had no effect on the human RNase H1 activity, whereas nucleotide substitutions adjacent the TFI significantly affected the cleavage rate. Mismatch base pair(s) exhibited similar effects on human RNase H1 activity as the TFI modifications. The effects of the TFI modification and mismatch base pair(s) on human RNase H1 activity were influenced by the position of the modification relative to the nucleotides interacting with the catalytic site of the enzyme rather than the juxtaposition of the modification to the MOE residues. Finally, these results provide a method for enhancing the human RNase H1 activity of chimeric antisense oligonucleotides (ASO) as well as the design of more potent ASO drugs.

Base Sequence↗
PubMed2006

Antisense oligonucleotides: efficient synthesis of 2'-O-methoxyethyl phosphorothioate oligonucleotides using 4,5-dicyanoimidazole. Are these oligonucleotides comparable to those synthesized using 1H-tetrazole as coupling activator?

Multiple 2'-O-methoxyethyl modified phosphorothioate oligonucleotides of 18-20-mer in length were synthesized at various scales using 4,5-dicyanoimidazole (DCI) as coupling activator. Extensive synthetic, analytical (using ion-pair LC-MS), and in vivo pharmacological, toxicological studies showed that oligonucleotides made with DCI and 1H-tetrazole are chemically and biologically equivalent. This extensive study will help the oligonucleotide therapeutic industry to move from using a potentially explosive activator (1H-tetrazole) to a safe activator (DCI).

Animals↗
PubMed2004

Structural requirements at the catalytic site of the heteroduplex substrate for human RNase H1 catalysis.

Human RNase H1 cleaves RNA exclusively in an RNA/DNA duplex; neither double-strand DNA nor double-strand 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.

Binding Sites↗
PubMed2004

Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs.

Although ribonuclease H activity has long been implicated as a molecular mechanism by which DNA-like oligonucleotides induce degradation of target RNAs, definitive proof that one or more RNase H is responsible is lacking. To date, two RNase H enzymes (H1 and H2) have been cloned and shown to be expressed in human cells and tissues. To determine the role of RNase H1 in the mechanism of action of DNA-like antisense drugs, we varied the levels of the enzyme in human cells and mouse liver and determined the correlation of those levels with the effects of a number of DNA-like antisense drugs. Our results demonstrate that in human cells RNase H1 is responsible for most of the activity of DNA-like antisense drugs. Further, we show that there are several additional previously undescribed RNases H in human cells that may participate in the effects of DNA-like antisense oligonucleotides.

Adenoviridae↗
PubMed2003

Antisense phosphorothioate oligodeoxyribonucleotide targeted against ICAM-1: synthetic and biological characterization of a process-related impurity formed during oligonucleotide synthesis.

A phosphorothioate-linked oligonucleotide bearing a 3'-terminal phosphorothioate monoester has been synthesized and characterized. This oligonucleotide has been identified as a process-related impurity formed during synthesis of ISIS 2302. Biological properties of the compound have been determined. Based on these data, it can be concluded that this species (3'-TPT) has biological properties similar to parent drug.

Animals↗
PubMed2003

Human RNase H1 uses one tryptophan and two lysines to position the enzyme at the 3'-DNA/5'-RNA terminus of the heteroduplex substrate.

In a previous study, we showed that the RNA-binding domain of human RNase H1 is responsible for the positional preference for cleavage exhibited by the enzyme (Wu, H., Lima, W. F., and Crooke, S. T. (2001) J. Biol. Chem. 276, 23547-23553). Here, we identify the substituents on the heteroduplex substrate and the amino acid residues within the RNA-binding domain of human RNase H1 involved in positioning of the enzyme. The human RNase H1 cleavage patterns observed for heteroduplexes with various 3'-DNA/5'-RNA and 5'-DNA/3'-RNA termini indicate that the 5'-most cleavage site on the oligoribonucleotide is positioned 7 bp from the first 3'-DNA/5'-RNA base pair. The presence or absence of phosphate or hydroxyl groups at either the 3'-DNA or 5'-RNA terminus had no effect on the human RNase H1 cleavage pattern. Substitution of the 3'-deoxynucleotide with a ribonucleotide, 2'-methoxyethyl nucleotide, or mismatched deoxyribonucleotide resulted in the ablation of the 5'-most cleavage site on the oligoribonucleotide. Mutants in which Trp43 and Lys59-Lys60 of the RNA-binding domain were substituted with alanine showed a loss of the positional preference for cleavage. Comparison of the kcat, Km, and Kd for the alanine-substituted mutants with those for human RNase H1 suggests that Lys59 and Lys60 are involved in binding to the heteroduplex and that Trp43 is responsible for properly positioning the enzyme on the substrate for catalysis. These data suggest that Trp43, Lys59, and Lys60 constitute an extended nucleic binding surface for the RNA-binding domain of human RNase H1, with the entire interaction taking place at the 3'-DNA/5'-RNA pole of the heteroduplex. These results offer further insights into the interaction between human RNase H1 and the heteroduplex substrate as well as approaches to enhance the design of effective antisense oligonucleotides.

Alanine↗
PubMed2002

Human RNase H1 activity is regulated by a unique redox switch formed between adjacent cysteines.

Human RNase H1 is active only under reduced conditions. Oxidation as well as N-ethylmaleimide (NEM) treatment of human RNase H1 ablates the cleavage activity. The oxidized and NEM alkylated forms of human RNase H1 exhibited binding affinities for the heteroduplex substrate comparable with the reduced form of the enzyme. Mutants of human RNase H1 in which the cysteines were either deleted or substituted with alanine exhibited cleavage rates comparable with the reduced form of the enzyme, suggesting that the cysteine residues were not required for catalysis. The cysteine residues responsible for the observed redox-dependent activity of human RNase H1 were determined by site-directed mutagenesis to involve Cys(147) and Cys(148). The redox states of the Cys(147) and Cys(148) residues were determined by digesting the reduced, oxidized, and NEM-treated forms of human RNase H1 with trypsin and analyzing the cysteine containing tryptic fragments by micro high performance liquid chromatography-electrospray ionization-Fourier transform ion cyclotron mass spectrometry. The tryptic fragment Asp(131)-Arg(153) containing Cys(147) and Cys(148) was identified. The mass spectra for the Asp(131)-Arg(153) peptides from the oxidized and reduced forms of human RNase H1 in the presence and absence of NEM showed peptide masses consistent with the formation of a disulfide bond between Cys(147) and Cys(148). These data show that the formation of a disulfide bond between adjacent Cys(147) and Cys(148) residues results in an inactive enzyme conformation and provides further insights into the interaction between human RNase H1 and the heteroduplex substrate.

Amino Acid Sequence↗