Picomole amounts of oligodeoxynucleotides [polydeoxyadenylic acids, (dA)4&..s] were baseline resolved and analyzed in <8 min by high-performance capillary electrophoresis with polyacrylamide gels. In addition, fast analysis of a crude 70-mer oligodeoxynucleotide and a slab gel-purified 99-mer oligodeoxynucleotide was accomplished, demonstrating the ability of high-performance capillary electrophoresis to characterize rapidly synthesized oligonucleotides. Besides analytical separations, 800 ng of a primer (20-mer) was isolated in <20 min. The purified species was collected in water and subsequently used as a probe in a standard dot-blot analysis. The use of high-performance capillary electrophoresis for the analysis and purification of a variety of biopolymers is simple, rapid, and has the potential for automation. (7), was used at a wavelength of 260 nm. The tubing and the detector were cooled with a thermostated air bath. The power supply outlets were connected to platinum electrodes, 5, immersed in buffer reservoirs, 6 (for analytical runs), or in a microcentrifuge vial, 7 (for collection). An analog/digital interface, 8 (Nelson, Cupertino, CA), attached to a recorder, 9, and IBM PC/XT, 10, were used to record the results and process the data.Materials. The oligodeoxynucleotide (5' GCCACGTCCA-GATTTATCAG 3') in Fig. 4 was synthesized on a model 380A DNA synthesizer (Applied Biosystems, Foster City, CA) using the silica-based solid-phase method (8) and the proton-activated nucleoside phosphoramidate method with methoxyphosphoramidates (9). The side-chain protecting groups were liberated by treatment with ammonium hydroxide at 550C for 6 hr. After this step, the oligodeoxynucleotide was recovered by lyophilization and dissolved in water.Polydeoxyadenylic acid mixture (dA)40-60 was purchased from Pharmacia. The crude 70-mer and slab gel-purified 99-mer oligodeoxynucleotides were the gift of N. Bischoffer (Genentech, South San Francisco, CA Procedure. A modified version (10) of the polyacrylamide gel capillary column described in ref. 5 was filled with polyacrylamide gel (T = 7.5%, C = 3.3%) (11) in 7 M urea/0.1 mM Tris/0.25 mM borate, pH 8.3. The field applied to the gel was 400 V/cm. To remove impurities from the acrylamide gel, the capillary gel column was preelectrolyzed at 6 kV for 30-60 min. During electrophoresis, the capillary was maintained at room temperature. The samples were electrophoretically injected into the column by dipping the cathodic end of the capillary into an aqueous solution of the sample and applying a field of 400 V/cm for =10 sec.Abbreviations: HPCE, high-performance capillary electrophoresis. ITo whom reprint requests should be addressed. 9660The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N6-(A2-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to <1.5% of wild-type levels. The tRNA modification enzyme, A2-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells. A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation. (16,42).In contrast, there are a few examples of single mutations that abolish enzyme activities in different subcellular compartments, indicating that some of the analogous proteins may be the products of the same gene. Single nuclear mutations that affect tRNA modification in the cytoplasm and mitochondria of yeasts (18,25,39) fall into the latter category. The mutations trml, trm2, and mod5-1 abolish the dimethylguanosine, 5-methyluridine, and N6-(A2-isopentenyl)adenosine (i6A) modification, respectively, in all cellular tRNA in the yeast Saccharomyces cerevisiae. Although it is possible that these genes code for regulators which affect the activity of distinct mitochondrial and nuclear/cytoplasmic proteins, the most straightforward interpretation of these observations is that single nuclear genes code for products which can be targeted to the mitochondria and another cellular compartment.One way to distinguish between two hypothetical situations is to clone the TRMJ, TRM2, and MOD5 loci and utilize these sequences to study the nature of the gene products. A tRNA modification enzymes would allow the use of the cloned sequence to obtain information regarding the mechanism(s) by which a single gene could encode products capable of localizing to mitochondria as well as some other location.The recessive mutation mod5-I was detected as a lesion which reduced the efficiency of nonsense suppression by a tyrosine-inserting suppressor tRNA (21). Chromatographic analysis of nucleotides derived from tRNA of mod5-J mutant cells revealed that the amount of i6A is reduced to <1.5% of wild-type levels (21). The modified base, i6A, occurs in tRNAs at position 37 adjacent to the 3' end of the anticodon and is found only in tRNAs that recognize codons starting with U (7, 26). The mod5-J mutation does not interfere with tRNA processing or amino acid acceptance and has no effect on cell growth in various media at 28 and 37°C (21). The reduced efficiency of nonsense suppression caused by the mutation is presumably due to altered codon-anticodon interactions of the modification deficient tyrosyl tRNAUAA.

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Givosiran is a N-acetylgalactosamine (GalNAc)-conjugated RNA interference (RNAi) therapeutic that targets 5ʹ-aminolevulinate synthase 1 (ALAS1) messenger RNA (mRNA) in the liver and is currently marketed for the treatment of acute hepatic porphyria (AHP). Herein, nonclinical pharmacokinetic (PK) and absorption, distribution, metabolism, and excretion (ADME) properties of givosiran were characterized. Givosiran was completely absorbed after subcutaneous (SC) administration with relatively short plasma elimination t 1/2 (less than 4 hours).Plasma exposure increased approximately dose proportionally with no accumulation after repeat doses. Plasma protein binding (PPB) was concentration dependent across all species tested and was around 90% at clinically relevant concentration in human. Givosiran predominantly distributed to the liver by asialoglycoprotein receptor (ASGPR)-mediated uptake, and the elimination t 1/2 in the liver was significantly longer (~1 week). Givosiran was metabolized by nucleases, not cytochrome P450 (CYP) isozymes, across species with no human unique metabolites. Givosiran metabolized to form one primary active metabolite with the loss of 1 nucleotide from the 3ʹ end of antisense strand, AS(N-1)3ʹ givosiran which was equipotent to givosiran. Renal and fecal excretion were minor routes of elimination of givosiran as approximately 10% and 16% of the dose was recovered intact in excreta of rats and monkeys, respectively. Givosiran is not a substrate, inhibitor, or inducer of CYP isozymes, and it is not a substrate or inhibitor of uptake and most efflux transporters. Thus, givosiran has a low potential of mediating drug-drug interactions involving CYP isozymes and drug transporters.