Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages.

Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.

Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 'Slicer' independent and can be mediated by Ago1.

It is known that siRNAs are capable of reducing expression of non-target genes due to the interaction of the siRNA guide strand with a partially complementary site on the 'off-target' mRNA. In the current study, we show that reduction of cellular Ago2 levels has no effect on off-target reduction of endogenous genes and that off-target degradation of mRNA can occur even in an Ago2 knockout cell line. Using antisense mediated reduction of Ago proteins and chemically modified cleavage- and binding-deficient siRNAs, we demonstrate that siRNA mediated off-target reduction is Ago2 cleavage independent, but does require siRNA interaction with either Ago1 or Ago2 and the RISC-loading complex. We also show that depletion of P-body associated proteins results in a reduction of off-target siRNA-mediated degradation of mRNA. Finally, we present data suggesting that a significant portion of on-target siRNA activity is also Ago2 cleavage independent, however, this activity does not appear to be P-body associated.

Binding and cleavage specificities of human Argonaute2.

The endonuclease Argonaute2 (Ago2) mediates the degradation of the target mRNA within the RNA-induced silencing complex. We determined the binding and cleavage properties of recombinant human Ago2. Human Ago2 was unable to cleave preformed RNA duplexes and exhibited weaker binding affinity for RNA duplexes compared with the single strand RNA. The enzyme exhibited greater RNase H activity in the presence of Mn2+ compared with Mg2+. Human Ago2 exhibited weaker binding affinities and reduced cleavage activities for antisense RNAs with either a 5'-terminal hydroxyl or abasic nucleotide. Binding kinetics suggest that the 5'-terminal heterocycle base nucleates the interaction between the enzyme and the antisense RNA, and the 5'-phosphate stabilizes the interaction. Mn2+ ameliorated the effects of the 5'-terminal hydroxyl or abasic nucleotide on Ago2 cleavage activity and binding affinity. Nucleotide substitutions at the 3' terminus of the antisense RNA had no effect on human Ago2 cleavage activity, whereas 2'-methoxyethyl substitutions at position 2 reduced binding and cleavage activity and 12-14 reduced the cleavage activity. RNase protection assays indicated that human Ago2 interacts with the first 14 nucleotides at the 5'-pole of the antisense RNA. Human Ago2 preloaded with the antisense RNA exhibited greater binding affinities for longer sense RNAs suggesting that the enzyme interacts with regions in the sense RNA outside the site for antisense hybridization. Finally, transiently expressed human Ago2 immunoprecipitated from HeLa cells contained the double strand RNA-binding protein human immunodeficiency virus, type 1, trans-activating response RNA-binding protein, and deletion mutants of Ago2 showed that trans-activating response RNA-binding protein interacts with the PIWI domain of the enzyme.

Reduced levels of Ago2 expression result in increased siRNA competition in mammalian cells.

Administration of small interfering RNAs (siRNAs) leads to degradation of specific mRNAs utilizing the cellular RNA interference (RNAi) machinery. It has been demonstrated that co-administration of siRNAs may lead to attenuation of activity of one of the siRNAs. Utilizing antisense and siRNA-mediated RNA-induced silencing complex (RISC) gene reduction we show that siRNA competition is correlated with differences in the cellular expression levels of Ago2, while levels of other RISC proteins have no effect on competition. We also show that under certain conditions siRNA competition rather than reduction of cellular RISC levels may be responsible for apparent reduction in siRNA activity. Furthermore, exploiting siRNA competition, we show that the RISC pathway loads and results in detectable cleavage of the target RNA in approximately 2 h after transfection. The RISC pathway is also capable of being reloaded even in the absence of new protein synthesis. RISC reloading and subsequent induction of detectable cleavage of a new target RNA, requires about 9-12 h following the initial transfection.

Human Dicer binds short single-strand and double-strand RNA with high affinity and interacts with different regions of the nucleic acids.

Human Dicer is an integral component of the RNA interference pathway. Dicer processes premicro-RNA and double-strand RNA to, respectively, mature micro-RNA and short interfering RNA (siRNA) and transfers the processed products to the RNA-induced silencing complex. To better understand the factors that are important for the binding, translocation, and selective recognition of the siRNA strands, we determined the binding affinities of human Dicer for processed products (siRNA) and short single-strand RNAs (ssRNA). siRNAs and ssRNAs competitively inhibited human Dicer activity, suggesting that they are interacting with the active site of the enzyme. The dissociation constants (Kd) for unmodified siRNAs were 5-11-fold weaker compared with a 27-nucleotide double-strand RNA substrate. Chemically modified siRNAs exhibited binding affinities for Dicer comparable with the substrate. 3'-dinucleotide overhangs in the siRNA affected the binding affinity of human Dicer for the siRNA and biased strand loading into RNA-induced silencing complex. The Kd values for the ssRNAs ranged from 3- to 40-fold weaker than the Kd for the substrate. Sequence composition of the 3'-terminal nucleotides of the ssRNAs exhibited the greatest effect on Dicer binding. Dicer cleaved substrates containing short siRNA-like double-strand regions and extended 3' or 5' ssRNA overhangs in the adjacent ssRNA regions. Remarkably, cleavage sites were observed consistent with the enzyme entering the substrate from the extended 3' ssRNA terminus. These data suggest that the siRNAs and ssRNAs interact predominantly with the PAZ domain of the enzyme. Finally, the tightest binding siRNAs were also more potent inhibitors of gene expression.

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.

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.

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.

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.

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.