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Gerald F Joyce

Publications and source records attributed to Gerald F Joyce.

At least 19 recordsLinked to original sources
PubMed2006

Microfluidic serial dilution circuit.

In vitro evolution of RNA molecules requires a method for executing many consecutive serial dilutions. To solve this problem, a microfluidic circuit has been fabricated in a three-layer glass-PDMS-glass device. The 400-nL serial dilution circuit contains five integrated membrane valves: three two-way valves arranged in a loop to drive cyclic mixing of the diluent and carryover, and two bus valves to control fluidic access to the circuit through input and output channels. By varying the valve placement in the circuit, carryover fractions from 0.04 to 0.2 were obtained. Each dilution process, which is composed of a diluent flush cycle followed by a mixing cycle, is carried out with no pipeting, and a sample volume of 400 nL is sufficient for conducting an arbitrary number of serial dilutions. Mixing is precisely controlled by changing the cyclic pumping rate, with a minimum mixing time of 22 s. This microfluidic circuit is generally applicable for integrating automated serial dilution and sample preparation in almost any microfluidic architecture.

Dimethylpolysiloxanes↗
PubMed2006

Conversion of a ribozyme to a deoxyribozyme through in vitro evolution.

An RNA ligase ribozyme was converted to a corresponding deoxyribozyme through in vitro evolution. The ribozyme was prepared as a DNA molecule of the same sequence, and had no detectable activity. A population of randomized variants of this DNA was constructed and evolved to perform RNA ligation at a rate similar to that of the starting ribozyme. When the deoxyribozyme was prepared as an RNA molecule of the same sequence, it had no detectable activity. Thus, the evolutionary transition from an RNA to a DNA enzyme represents a switch, rather than a broadening, of the chemical basis for catalytic function. This transfer of both information and function is relevant to the transition between two different genetic systems based on nucleic acid-like molecules, as postulated to have occurred during the early history of life on Earth.

Base Sequence↗
PubMed2005

The promise and peril of continuous in vitro evolution.

Experimental evolution methods can be used to address and illuminate issues central to the understanding of evolutionary theory. One of the most powerful of these methods involves the in vitro evolution of nucleic acid enzymes, taking advantage of the direct relationship between the genotype of a nucleic acid sequence and the phenotype of its associated catalytic function. This review and commentary focuses on the past, present, and future potential of systems for the continuous in vitro evolution of nucleic acid enzymes as tools for modeling evolutionary processes in biology. It offers a candid appraisal of both the strengths and the limitations of these systems.

Directed Molecular Evolution↗
PubMed2004

Selective derivatization and sequestration of ribose from a prebiotic mix.

Observations regarding the catalytic potential of RNA and the role of RNA in biology have formed the basis for the "RNA world" hypothesis, which suggests that a genetic system based on self-replicating polyribonucleotides preceded modern biology. However, attempts to devise a realistic prebiotic synthesis of nucleic acids from simple starting materials have been plagued by problems of poor chemical selectivity, lack of stereo- and regiospecificity, and similar rates of formation and degradation of some of the key intermediates. For example, ribose would have been only a small component of a highly complex mix of sugars resulting from the condensation of formaldehyde in a prebiotic world. In addition, ribose is more reactive and degrades more rapidly compared with most other monosaccharides. This study demonstrates an approach for the preferential sequestration of ribose relative to other sugars that takes advantage of its greater reactivity. Cyanamide reacts especially rapidly with ribose to form a stable bicyclic adduct. This product crystallizes spontaneously in aqueous solution, whereas the corresponding products derived from threose, galactose, glucose, mannose, and each of the other pentoses do not. Furthermore, when employing a racemic mixture of d- and l-ribose, enantiomerically twinned crystals are formed that contain discrete homochiral domains.

Cyanamide↗
PubMed2004

A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron.

Molecular self-assembly offers a means of spontaneously forming complex and well-defined structures from simple components. The specific bonding between DNA base pairs has been used in this way to create DNA-based nanostructures and to direct the assembly of material on the subnanometre to micrometre scale. In principle, large-scale clonal production of suitable DNA sequences and the directed evolution of sequence lineages towards optimized behaviour can be realized through exponential DNA amplification by polymerases. But known examples of three-dimensional geometric DNA objects are not amenable to cloning because they contain topologies that prevent copying by polymerases. Here we report the design and synthesis of a 1,669-nucleotide, single-stranded DNA molecule that is readily amplified by polymerases and that, in the presence of five 40-mer synthetic oligodeoxynucleotides, folds into an octahedron structure by a simple denaturation-renaturation procedure. We use cryo-electron microscopy to show that the DNA strands fold successfully, with 12 struts or edges joined at six four-way junctions to form hollow octahedra approximately 22 nanometres in diameter. Because the base-pair sequence of individual struts is not repeated in a given octahedron, each strut is uniquely addressable by the appropriate sequence-specific DNA binder.

Base Pairing↗
PubMed2004

Minimal self-replicating systems.

Minimal self-replicating systems typically consist of three components: a product molecule, and two substrate molecules that become joined to form another product molecule. An important characteristic of self-replicating systems is the ability of the product to catalyze the formation of additional product, resulting in autocatalytic behavior. Recent advances in the area of self-replication have led to improved efficiency of autocatalysis, both by increasing the fraction of product molecules that can participate in further rounds of replication, and by improving the efficiency of the catalysts themselves. This review analyzes chemical self-replicating systems that have been developed to date and discusses ongoing challenges in this area of research.

Catalysis↗
PubMed2004

Cross-catalytic replication of an RNA ligase ribozyme.

A self-replicating RNA ligase ribozyme was converted to a cross-catalytic format whereby two ribozymes direct each other's synthesis from a total of four component substrates. Each ribozyme binds two RNA substrates and catalyzes their ligation to form the opposing ribozyme. The two ribozymes are not perfectly complementary, as is the case for replicating nucleic acid genomes in biology. Rather, the ribozymes contain both template elements, which are complementary, and catalytic elements, which are identical. The specificity of the template interactions allows the cross-catalytic pathway to dominate over all other reaction pathways. As the concentration of the two ribozymes increases, the rate of formation of additional ribozyme molecules increases, consistent with the overall autocatalytic behavior of the reaction system.

Base Sequence↗
PubMed2004

Directed evolution of nucleic acid enzymes.

Just as Darwinian evolution in nature has led to the development of many sophisticated enzymes, Darwinian evolution in vitro has proven to be a powerful approach for obtaining similar results in the laboratory. This review focuses on the development of nucleic acid enzymes starting from a population of random-sequence RNA or DNA molecules. In order to illustrate the principles and practice of in vitro evolution, two especially well-studied categories of catalytic nucleic acid are considered: RNA enzymes that catalyze the template-directed ligation of RNA and DNA enzymes that catalyze the cleavage of RNA. The former reaction, which involves attack of a 2'- or 3'-hydroxyl on the alpha-phosphate of a 5'-triphosphate, is more difficult. It requires a comparatively larger catalytic motif, containing more nucleotides than can be sampled exhaustively within a starting population of random-sequence RNAs. The latter reaction involves deprotonation of the 2'-hydroxyl adjacent to the cleavage site, resulting in cleaved products that bear a 2',3'-cyclic phosphate and 5'-hydroxyl. The difficulty of this reaction, and therefore the complexity of the corresponding DNA enzyme, depends on whether a catalytic cofactor, such as a divalent metal cation or small molecule, is present in the reaction mixture.

DNA, Catalytic↗
PubMed2003

A modular, bifunctional RNA that integrates itself into a target RNA.

Nature often combines independent functional domains to achieve complex function, but this approach has not been extensively explored with artificial enzymes. Here, a group I ribozyme, which can act as an endoribonuclease, was partnered with the R3C ribozyme, which catalyzes the ligation of RNA molecules. The conjoined ribozymes have the potential to perform successive RNA cleavage and joining reactions, resulting in their mutual integration into a target RNA substrate. When simply joined together, however, the ribozymes were unable to achieve this outcome because of inefficient transfer of the substrate between the two catalytic subunits. In vitro evolution was used to optimize the behavior of the conjoined ribozymes, resulting in bifunctional molecules with substantially improved integration activity. The ligase subunit of these molecules was unchanged, whereas the group I subunit acquired several mutations, mostly in peripheral regions. The generation and study of this bifunctional assembly helps shed light on the evolution of modular enzymes and the obstacles that must be overcome in bringing together independent functional domains. These molecules also may be useful as tools for the insertional mutagenesis of target mRNAs.

Base Sequence↗
PubMed2003

Self-replication.

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DNA Replication↗
PubMed2003

Perfectly complementary nucleic acid enzymes.

The ability to maximize the use of available nucleic acid sequence space would have been crucial during the presumed RNA world and confers selective advantage in many contemporary organisms. One way to access sequence space at a higher density would be to make use of both strands of a duplex nucleic acid for the production of functional molecules. As a demonstration of this possibility, two pairs of nucleic acid enzymes were engineered to be perfect complements, each with the capacity to adopt a distinct structure and catalyze a particular chemical transformation. Both members of each pair of enzymes exhibited nearly the same level of activity as the canonical form of the corresponding catalytic motif. The ability to generate functional nucleic acids encoded by both strands of a duplex has implications for the evolution of catalytic nucleic acids and the prospects for realizing maximum functionality from a given genetic sequence.

Base Sequence↗
PubMed2003

Continuous in vitro evolution of ribozymes that operate under conditions of extreme pH.

Continuous in vitro evolution methods were used to study the behavior of an evolving population of RNA ligase ribozymes in response to selection pressures involving conditions of extreme pH. The starting population consisted of randomized variants of a ribozyme that had been optimized for activity at pH 8.5. The ribozymes were subjected to repeated rounds of selective amplification under progressively more acidic or more alkaline conditions. The two final evolved populations of ribozymes were able to operate at either pH 5.8 or pH 9.8, respectively. Representative individuals from the two final populations were isolated and characterized. The low-pH ribozyme exhibited a 10-fold increase in catalytic rate at pH 5.8 compared to the starting molecule. The high-pH ribozyme retained its structural integrity and activity at pH 9.8, whereas the starting molecule was denatured under this condition. These findings demonstrate that a population of functional macromolecules can adapt to stringent environmental conditions through the acquisition of relatively few mutations. The results establish continuous in vitro evolution as a useful model system for exploring the evolution of enzymatic function in extreme environments.

Base Sequence↗
PubMed2003

In search of an RNA replicase ribozyme.

The theory that an RNA world played a pivotal role in life's evolutionary past has prompted investigations into the scope of RNA catalysis. These efforts have attempted to demonstrate the plausibility of an RNA-based genetic system, which would require RNA molecules that catalyze their own replication. The mechanistic features of modern protein polymerases have been used to guide the laboratory evolution of catalytic RNAs (ribozymes) that exhibit polymerase-like activity. Ribozymes have been developed that recognize a primer-template complex in a general way and catalyze the template-directed polymerization of mononucleotides. These experiments demonstrate that RNA replicase behavior is likely within the catalytic repertoire of RNA, although many obstacles remain to be overcome in order to demonstrate that RNA can catalyze its own replication in a manner that could have sustained a genetic system on the early Earth.

Base Sequence↗
PubMed2002

RNA-cleaving DNA enzymes with altered regio- or enantioselectivity.

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5'-phosphodiester following a D-ribonucleotide or a 3',5'-phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5'-phosphodiester exhibits a k(cat) of approximately 0.01 min(-1) and catalytic efficiency, k(cat)/K(m), of approximately 10(8) M(-1) min(-1). The enzyme that cleaves an L-ribonucleotide is about 10-fold slower and has a catalytic efficiency of approximately 4 x 10(5) M(-1) min(-1). Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 degrees C. In a comparison of each enzyme's activity with either its corresponding substrate that contains an unnatural ribonucleotide or a substrate that instead contains a standard ribonucleotide, the 2',5'-phosphodiester-cleaving DNA enzyme exhibited a regioselectivity of 6000-fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 40-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.

DNA, Catalytic↗
PubMed2002

A self-replicating ligase ribozyme.

A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 min(-1), whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 x 10(-11) M.min(-1). Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication.

Base Sequence↗
PubMed2002

The antiquity of RNA-based evolution.

All life that is known to exist on Earth today and all life for which there is evidence in the geological record seems to be of the same form--one based on DNA genomes and protein enzymes. Yet there are strong reasons to conclude that DNA- and protein-based life was preceded by a simpler life form based primarily on RNA. This earlier era is referred to as the 'RNA world', during which the genetic information resided in the sequence of RNA molecules and the phenotype derived from the catalytic properties of RNA.

Biological Evolution↗