Stanley T. Crooke

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.

Combinatorial Drug Discovery: Which Methods Will Produce the Greatest Value?

Combinatorial strategies are important new approaches to drug discovery, and it seems quite likely that they will result in the discovery of interesting potential pharmaceutical. However, it is less clear whether combinatorial approaches will result in quantum advances in therapeutics. Nor is there general agreement about the factors most important in defining how combinatorial strategies will provide value to the discovery of lead and therapeutic compounds. In this review, we propose criteria that define the value of combinatorial strategies and categorize the various approaches by: (a) the type of chemical space to be searched, (b) the tactics employed to synthesize and screen libraries, and (c) the structures of individual molecules in libraries. We evaluate the strengths and weaknesses of the various strategies and suggest milestones that can help to track their success.

The Effect of Mitomycin C on Superficial Bladder Cancer

A course of intravesical mitomycin C, consisting of 8 weekly doses of 30 or 40 mg., was evaluated in 16 patients with superficial bladder cancer (stages O and A). Cystoscopically documented tumor was destroyed completely in 11 patients (69 per cent), while 3 patients exhibited partial tumor regression. Two patients had only multifocal, grade 3 carcinoma in situ and both had a complete response with negative biopsies and cytology at the 12-week evaluation. Toxicity was minimal. Further data, including longer followup, are needed to define the potentially promising role of this agent in the over-all management of superficial bladder cancer.

Low-Molecular-Weight Nuclear RNAs

[1, p. 285; 2-14]. Some RNAs smaller in size than these which contain dihydrouridine or dihydrothymidine have also been reported to be present in the nucleus, specifically in association with the chromatin fraction [15-20]. Although the functions of most of these LMWN RNAs are not defined, their specificity of localization in the nucleolus and the nuclear chromatin fraction along with the number of these molecules (which approximates that of the number of functional genes) has suggested they may have an initiating or structural role in gene readouts or a regulatory role in gene function [8, 10, 2123]. The recent findings which have indicated that some low-molecular-weight nucleolar RNAs have specific temporal associations with newly synthesized ribosomal precursors suggest the possibility that they may be involved in specific phases of synthesis or modification of the ribosomal precursors [24]. Number of low-molecular-weight nuclear RNAs.—In a search for a definite role for these molecules in cell function, one of the critical problems has been the clarification of the number of types of these RNA molecules. Although it is not yet possible to specify with precision the total number of low-molecular-weight RNA species in the various fractions of the nucleus, the minimal estimate is eleven (fig. 1), and the maximum is approximately forty-three [1, 25]. None of these values, however, exclude possible microheterogeneity of the various nuclear RNA species. Table 1 indicates that there are at least

Effect of leukotrienes, bradykinin and calcium ionophore (A 23187) on bovine endothelial cells: Release of prostacyclin

Incubation of the bovine endothelial cell line, CPAE, with the calcium ionophore (A23187), bradykinin (BK), leukotriene D4 (LTD4) or leukotriene C4 (LTC4) resulted in concentration dependent increases in prostacyclin release measured as 6-keto-prostaglandin F1 alpha. The kinetics of induction of prostacyclin synthesis differed among the agents studied. Statistically significant increases in prostacyclin were observed one minute after treatment, with A23187, at slightly longer times with bradykinin and after approximately three minutes with the leukotrienes. Two other leukotrienes were tested. Both leukotriene B4 and leukotriene E4 (LTE4) were inactive at concentrations up to 10 microM. The induction of prostacyclin synthesis by LTC4 and LTD4 was inhibited by cycloheximide and actinomycin-D. The effect of BK was inhibited by cycloheximide but not by actinomycin-D. Induction by A23187 was not inhibited by either actinomycin-D or cycloheximide. The results suggest that these agents induced the increases in prostacyclin synthesis by different mechanisms.

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.

Differential effects of manoalide on secreted and intracellular phospholipases

Manoalide, a novel nonsteroidal sesterterpenoid, is a potent inhibitor of phospholipase A2 isolated from bee and cobra venoms. This report compares the inhibition by manoalide of phospholipase A2 in crude cytosol fractions from four mammalian tissues with that of four purified extracellular phospholipase A2s. Phospholipase A2 isolated from bee venom (Apis mellifera) was the most sensitive to inactivation by manoalide (IC50 approximately equal to 0.12 microM). Extracellular phospholipase A2 from rattlesnake and cobra venom was intermediate in sensitivity to manoalide (IC50 values of 0.7 and 1.9 microM respectively). Porcine pancreatic phospholipase A2 was relatively resistant to inactivation by manoalide (IC50 approximately equal to 30 microM). The phospholipase A2 assayed in crude cytosol fractions from four mammalian tissues exhibited IC50 values of 30 microM or greater. Cytosolic proteins as well as bovine serum albumin and poly-L-lysine (Mr = 57,000) protected purified bee venom phospholipase A2 from inactivation by manoalide. In contrast, amino acids such as lysine and alanine failed to protect the purified enzyme from inactivation. Proteins and certain amino acids, such as lysine, formed a chromogenic product when incubated with manoalide. These data suggest that lysine is capable of reacting with manoalide, but only when it is present in macromolecules is it capable of protecting phospholipase A2 from inactivation by manoalide. Because cellular proteins protect PLA2 from inactivation by manoalide, high concentrations of manoalide must be applied topically to produce statistically significant inactivation of intracellular phospholipase A2. Finally, a chemical model is presented which explains the formation of a chromogenic product when manoalide is incubated with proteins and amino acids.

Cellular uptake and trafficking of antisense oligonucleotides

Antisense oligonucleotides (ASOs) modified with phosphorothioate (PS) linkages and different 2′ modifications can be used either as drugs (e.g., to treat homozygous familial hypercholesterolemia and spinal muscular atrophy) or as research tools to alter gene expression. PS-ASOs can enter cells without additional modification or formulation and can be designed to mediate sequence-specific cleavage of different types of RNA (including mRNA and non-coding RNA) targeted by endogenous RNase H1. Although PS-ASOs function in both the cytoplasm and nucleus, localization to different subcellular regions can affect their therapeutic potency. Cellular uptake and intracellular distribution of PS ASOs are mediated by protein interactions. The main proteins involved in these processes have been identified, and intracellular sites in which PS ASOs are active, or inactive, cataloged.

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.

Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames

Increasing the levels of therapeutic proteins in vivo remains challenging. Antisense oligonucleotides (ASOs) are often used to downregulate gene expression or to modify RNA splicing, but antisense technology has not previously been used to directly increase the production of selected proteins. Here we used a class of modified ASOs that bind to mRNA sequences in upstream open reading frames (uORFs) to specifically increase the amounts of protein translated from a downstream primary ORF (pORF). Using ASO treatment, we increased the amount of proteins expressed from four genes by 30–150% in a dose-dependent manner in both human and mouse cells. Notably, systemic treatment of mice with ASO resulted in an ∼80% protein increase of LRPPRC. The ASO-mediated increase in protein expression was sequence-specific, occurred at the level of translation and was dependent on helicase activity. We also found that the type of RNA modification and the position of modified nucleotides in ASOs affected translation of a pORF. ASOs are a useful class of therapeutic agents with broad utility.