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Biomedical subjects

G F Joyce

Publications and source records attributed to G F Joyce.

At least 37 records · Page 2Linked to original sources
PubMed1999

Variation in inpatient resource use in the treatment of HIV: do the privately insured receive more care?

OBJECTIVE: To estimate the impact of insurance status on inpatient resource use after adjusting for health upon admission and site of care. DESIGN: Detailed patient information linked to billing records from the AIDS Cost and Service Utilization Survey (ACSUS), a longitudinal analysis of inpatient and outpatient care between March 1991 and August 1992. SETTING: Hospitalizations of human immunodeficiency virus (HIV) patients from 10 US cities with high incidence of AIDS. PATIENTS: One thousand, nine hundred and forty nine adolescents and adults at various stages of HIV. MAIN OUTCOME MEASURES: We estimate inpatient charges, payments and length of stay as a function of patient, and provider and reimbursement characteristics for more than 1,500 hospitalizations to HIV patients. We control for patient characteristics and underlying risk factors including disease stage, CD4 percentage, mode of transmission, discharge status, type of admission, and region. We use hospital-fixed effects to control for unmeasured differences across facilities. RESULTS: Unadjusted means indicate that uninsured patients or patients covered by public insurance have significantly lower charges and payments than privately insured patients with similar medical conditions. We find that those differences are substantially reduced after controlling for the hospital in which care is received. Further, we find little evidence that "underinsured" patients are discharged sooner on average. CONCLUSIONS: Inpatient resource use is affected by both the hospital in which care is received and the type of patient admitted. Failure to control for unmeasured differences across hospitals is likely to overstate the impact of insurance substantially.

Adolescent↗
PubMed1999

Crystallization of the 10-23 DNA enzyme using a combinatorial screen of paired oligonucleotides.

One of the most difficult steps in the X-ray crystallography of nucleic acids is obtaining crystals that diffract to high resolution. The choice of the nucleotide sequence has proven to be more important in producing high-quality crystals than the composition of the crystallization solution. This manuscript describes a systematic procedure for identifying the optimal sizes of a multi-stranded nucleic acid complex which provide high-quality crystals. This approach was used to crystallize the in vitro evolved 10-23 DNA enzyme complexed with its RNA substrate. In less than two months, 81 different enzyme-substrate complexes were generated by combinatorial mixing and annealing of complementary oligonucleotides which differed in length, resulting in duplexes of varying length, with or without nucleotide overhangs. Each of these complexes was screened against a standard set of 48 crystallization conditions and evaluated for crystal formation. The screen resulted in over 40 crystal forms, the best of which diffracted to 2.8 A resolution when exposed to a synchrotron X-ray source.

Combinatorial Chemistry Techniques↗
PubMed1998

Mechanism and utility of an RNA-cleaving DNA enzyme.

We previously reported the in vitro selection of a general-purpose RNA-cleaving DNA enzyme that exhibits a catalytic efficiency (kcat/KM) exceeding that of any other known nucleic acid enzyme [Santoro, S. W. and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4262-4266]. This enzyme contains approximately 30 deoxynucleotides and can cleave almost any RNA substrate under simulated physiological conditions, recognizing the substrate through two Watson-Crick binding domains. The kinetics of cleavage under conditions of varying pH, choice of divalent metal cofactor, and divalent metal concentration are consistent with a chemical mechanism involving metal-assisted deprotonation of a 2'-hydroxyl of the substrate, leading to substrate cleavage. Kinetic measurements reveal that the enzyme strongly prefers cleavage of the substrate over ligation of the two cleavage products and that the enzyme's catalytic efficiency is limited by the rate of substrate binding. The enzyme displays a high level of substrate specificity, discriminating against RNAs that contain a single base mismatch within either of the two substrate-recognition domains. With appropriate design of the substrate-recognition domains, the enzyme exhibits a potent combination of high substrate sequence specificity and selectivity, high catalytic efficiency, and rapid catalytic turnover.

Base Composition↗
PubMed1997

A general purpose RNA-cleaving DNA enzyme.

An in vitro selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson-Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (kcat/Km) of approximately 10(9) M-1.min-1 under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg2+ ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1 gag/pol, env, vpr, tat, and nef mRNAs.

DNA, Catalytic↗
PubMed1997

Continuous in vitro evolution of catalytic function.

A population of RNA molecules that catalyze the template-directed ligation of RNA substrates was made to evolve in a continuous manner in the test tube. A simple serial transfer procedure was used to achieve approximately 300 successive rounds of catalysis and selective amplification in 52 hours. During this time, the population size was maintained against an overall dilution of 3 x 10(298). Both the catalytic rate and amplification rate of the RNAs improved substantially as a consequence of mutations that accumulated during the evolution process. Continuous in vitro evolution makes it possible to maintain laboratory "cultures" of catalytic molecules that can be perpetuated indefinitely.

Base Sequence↗
PubMed1997

Direct observation of aminoglycoside-RNA interactions by surface plasmon resonance.

The specificity of neomycin B and related aminoglycoside antibiotics in their interaction with the Rev responsive element (RRE) of HIV-1 mRNA has been studied by directly observing the aminoglycoside-RNA complexes using surface plasmon resonance. Several different RNA sequences, each with a biotin tag, have been prepared using T7 RNA polymerase-catalyzed transcription of synthetic DNA templates and have been immobilized on a streptavidin-coated surface for the binding study. The results indicate that neomycin B is not specific for the G-rich bubble region in RRE. Rather, it appears to interact with three different sites, each with a submicromolar dissociation constant, within the 67-nucleotide domain II of RRE. Further analysis of neomycin B binding with three short synthetic RNA hairpins showed binding with submicromolar affinity and 1:1 stoichiometry in each case. This suggests that neomycin B may generally bind with this affinity to regular A-form RNA or hairpin loops. The approach described here is generally useful for understanding the fundamental interactions involved in the specific recognition of nucleic acids by small molecules which is the basis of rational drug design.

Anti-Bacterial Agents↗
PubMed1997

A simple procedure for constructing 5'-amino-terminated oligodeoxynucleotides in aqueous solution.

A rapid method for the synthesis of oligodeoxynucleotides (ODNs) terminated by 5'-amino-5'-deoxythymidine is described. A 3'-phosphorylated ODN (the donor) is incubated in aqueous solution with 5'-amino- 5'-deoxythymidine in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), extending the donor by one residue via a phosphoramidate bond. Template- directed ligation of the extended donor and an acceptor ODN, followed by acid hydrolysis, yields the acceptor ODN extended by a single 5'-amino-5'-deoxythymidine residue at its 5'terminus.

Base Sequence↗
PubMed1996

Specialization of the DNA-cleaving activity of a group I ribozyme through in vitro evolution.

In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group I ribozyme to obtain individuals with a 10(5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approximately 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiments confirmed the selective advantage of two covarying mutations within the catalytic core of the ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average of 28 mutations relative to the wild-type that are responsible for the altered phenotype.

Animals↗
PubMed1996

Targeting sites within HIV-1 cDNA with a DNA-cleaving ribozyme.

A variant of the Tetrahymena ribozyme that efficiently cleaves single-stranded DNA under simulated physiological conditions [Tsang, J., & Joyce, G. F. (1994) Biochemistry 33, 5966-5973] was evaluated as a potential therapeutic agent on the basis of its ability to cleave synthetic oligonucleotide substrates corresponding to conserved target sites within HIV-I cDNA. In order to increase the sequence selectivity of the ribozyme, its substrate recognition domain was extended from 6 to 12 nucleotides, allowing base pairing with substrate nucleotides that lie both upstream and downstream of the cleavage site. The sequence of the extended recognition domain could be changed to allow cleavage of a variety of different DNA targets. The ribozyme exhibited a high degree of sequence specificity, discriminating by a factor of 10(2) to more than 10(4) against substrates that form a single-base mismatch with the ribozyme's recognition domain. Mismatches that occurred close to the cleavage site led to a greater decrease in activity compared to those that occurred farther away.

Animals↗
PubMed1996

Building the RNA world. Ribozymes.

The isolation of an RNA enzyme with RNA replicase activity, at present only a hypothetical molecule, is considerably closer following the recent demonstration of RNA-catalyzed polymerization of nucleoside triphosphates.

Biopolymers↗
PubMed1996

Template-directed ligation of peptides to oligonucleotides.

BACKGROUND: Oligonucleotide-peptide conjugates have several applications, including their potential use as therapeutic agents. We developed a strategy for the chemical ligation of unprotected peptides to oligonucleotides in aqueous solution. The two compounds are joined via a stable amide bond in a template-directed reaction. RESULTS: Peptides, ending in a carboxy-terminal thioester, were converted to thioester-linked oligonucleotide-peptide intermediates. The oligonucleotide portion of the intermediate binds to a complementary oligonucleotide template, placing the peptide in close proximity to an adjacent template-bound oligonucleotide that terminates in a 3' amine. The ensuing reaction results in the efficient formation of an amide-linked oligonucleotide-peptide conjugate. CONCLUSIONS: An oligonucleotide template can be used to direct the ligation of peptides to oligonucleotides via a highly stable amide linkage. The ligation reaction is sequence-specific, allowing the simultaneous ligation of multiple oligonucleotide-peptide pairs.

Amides↗
PubMed1995

Cleavage of an amide bond by a ribozyme.

A variant form of a group I ribozyme, optimized by in vitro evolution for its ability to catalyze magnesium-dependent phosphoester transfer reactions involving DNA substrates, also catalyzes the cleavage of an unactivated alkyl amide when that linkage is presented in the context of an oligodeoxynucleotide analog. Substrates containing an amide bond that joins either two DNA oligos, or a DNA oligo and a short peptide, are cleaved in a magnesium-dependent fashion to generate the expected products. The first-order rate constant, kcat, is 0.1 x 10(-5) min-1 to 1 x 10(-5) min-1 for the DNA-flanked substrates, which corresponds to a rate acceleration of more than 10(3) as compared with the uncatalyzed reaction.

Amides↗
PubMed1995

Self-incorporation of coenzymes by ribozymes.

RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNA-catalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD+ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the "RNA world," when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.

Animals↗