Gerald F. Joyce

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Name: Joyce, Gerald

Organization: Departments of Chemistry and Molecular Biology and The Skaggs Institute for Chemical Biology , USA

Department: Departments of Chemistry and Molecular Biology and The Skaggs Institute for Chemical Biology

Title: Professor(PhD)

Chemicals
References

Amplification of RNA by an RNA polymerase ribozyme

Co-reporter:David P. Horning

PNAS 2016 Volume 113 (Issue 35 ) pp:9786-9791

Publication Date(Web):2016-08-30

DOI:10.1073/pnas.1610103113

In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life—the replication of genetic information and its conversion into functional molecules—can now be accomplished with RNA in the complete absence of proteins.

Specific Inhibition of MicroRNA Processing Using l-RNA Aptamers

Co-reporter:Jonathan T. Sczepanski

Journal of the American Chemical Society 2015 Volume 137(Issue 51) pp:16032-16037

Publication Date(Web):December 10, 2015

DOI:10.1021/jacs.5b06696

In vitro selection was used to obtain l-RNA aptamers that bind the distal stem-loop of various precursor microRNAs (pre-miRs). These l-aptamers, termed “aptamiRs”, bind their corresponding pre-miR target through highly specific tertiary interactions rather than Watson–Crick pairing. Formation of a pre-miR–aptamiR complex inhibits Dicer-mediated processing of the pre-miR, which is required to form the mature functional microRNA. One of the aptamiRs, which was selected to bind oncogenic pre-miR-155, inhibits Dicer processing under simulated physiological conditions, with an IC50 of 87 nM. Given that l-RNAs are intrinsically resistant to nuclease degradation, these results suggest that aptamiRs might be pursued as a new class of miR inhibitors.

An L-RNA Aptamer that Binds and Inhibits RNase

Co-reporter:Charles Olea Jr., Joachim Weidmann, Philip E. Dawson, Gerald F. Joyce

Chemistry & Biology 2015 Volume 22(Issue 11) pp:1437-1441

Publication Date(Web):19 November 2015

DOI:10.1016/j.chembiol.2015.09.017

•L-RNA aptamers were selected that bind and inhibit barnase RNase•This required synthesizing the full-length synthetic D-enantiomer of barnase•The L-RNA aptamers are competitive inhibitors of degradation of D-RNA substrates•The first time a mirror-image aptamer has been raised against an entire enzymeL-RNA aptamers were developed that bind to barnase RNase and thereby inhibit the function of the enzyme. These aptamers were obtained by first carrying out in vitro selection of D-RNAs that bind to the full-length synthetic D-enantiomer of barnase, then reversing the mirror and preparing L-RNAs of identical sequence that similarly bind to natural L-barnase. The resulting L-aptamers bind L-barnase with an affinity of ∼100 nM and function as competitive inhibitors of enzyme cleavage of D-RNA substrates. L-RNA aptamers are resistant to degradation by ribonucleases, thus enabling them to function in biological samples, most notably for applications in molecular diagnostics and therapeutics. In addition to the irony of using RNA to inhibit RNase, L-RNA aptamers such as those described here could be used to measure the concentration or inhibit the function of RNase in the laboratory or in biological systems.Figure optionsDownload full-size imageDownload high-quality image (187 K)Download as PowerPoint slide

Reflections of a Darwinian Engineer

Co-reporter:Gerald F. Joyce

Journal of Molecular Evolution 2015 Volume 81( Issue 5-6) pp:146-149

Publication Date(Web):2015 December

DOI:10.1007/s00239-015-9724-6

The Expanding View of RNA and DNA Function

Co-reporter:Ronald R. Breaker, Gerald F. Joyce

Chemistry & Biology 2014 Volume 21(Issue 9) pp:1059-1065

Publication Date(Web):18 September 2014

DOI:10.1016/j.chembiol.2014.07.008

RNA and DNA are simple linear polymers consisting of only four major types of subunits, and yet these molecules carry out a remarkable diversity of functions in cells and in the laboratory. Each newly discovered function of natural or engineered nucleic acids enforces the view that prior assessments of nucleic acid function were far too narrow and that many more exciting findings are yet to come. This Perspective highlights just a few of the numerous discoveries over the past 20 years pertaining to nucleic acid function, focusing on those that have been of particular interest to chemical biologists. History suggests that there will continue to be many opportunities to engage chemical biologists in the discovery, creation, and manipulation of nucleic acid function in the years to come.

Highly Efficient Self-Replicating RNA Enzymes

Co-reporter:Michael P. Robertson, Gerald F. Joyce

Chemistry & Biology 2014 Volume 21(Issue 2) pp:238-245

Publication Date(Web):20 February 2014

DOI:10.1016/j.chembiol.2013.12.004

•An optimized self-replicating RNA enzyme was obtained by directed evolution•The enzyme undergoes sustained exponential growth with a doubling time of 5 min•Amplification of 10100-fold was achieved over a period of 37.5 hr•The optimized enzyme will enable more complex synthetic genetic systemsAn RNA enzyme has been developed that catalyzes the joining of oligonucleotide substrates to form additional copies of itself, undergoing self-replication with exponential growth. The enzyme also can cross-replicate with a partner enzyme, resulting in their mutual exponential growth and enabling self-sustained Darwinian evolution. The opportunity for inventive evolution within this synthetic genetic system depends on the diversity of the evolving population, which is limited by the catalytic efficiency of the enzyme. Directed evolution was used to improve the efficiency of the enzyme and increase its exponential growth rate to 0.14 min−1, corresponding to a doubling time of 5 min. This is close to the limit of 0.21 min−1 imposed by the rate of product release, but sufficient to enable more than 80 logs of growth per day.Figure optionsDownload full-size imageDownload high-quality image (130 K)Download as PowerPoint slide

Limits of Neutral Drift: Lessons From the In Vitro Evolution of Two Ribozymes

Co-reporter:Katherine L. Petrie

Journal of Molecular Evolution 2014 Volume 79( Issue 3-4) pp:75-90

Publication Date(Web):2014 October

DOI:10.1007/s00239-014-9642-z

The relative contributions of adaptive selection and neutral drift to genetic change are unknown but likely depend on the inherent abundance of functional genotypes in sequence space and how accessible those genotypes are to one another. To better understand the relative roles of selection and drift in evolution, local fitness landscapes for two different RNA ligase ribozymes were examined using a continuous in vitro evolution system under conditions that foster the capacity for neutral drift to mediate genetic change. The exploration of sequence space was accelerated by increasing the mutation rate using mutagenic nucleotide analogs. Drift was encouraged by carrying out evolution within millions of separate compartments to exploit the founder effect. Deep sequencing of individuals from the evolved populations revealed that the distribution of genotypes did not escape the starting local fitness peak, remaining clustered around the sequence used to initiate evolution. This is consistent with a fitness landscape where high-fitness genotypes are sparse and well isolated, and suggests, at least in this context, that neutral drift alone is not a primary driver of genetic change. Neutral drift does, however, provide a repository of genetic variation upon which adaptive selection can act.

Binding of a Structured d-RNA Molecule by an l-RNA Aptamer

Co-reporter:Jonathan T. Sczepanski

Journal of the American Chemical Society 2013 Volume 135(Issue 36) pp:13290-13293

Publication Date(Web):August 26, 2013

DOI:10.1021/ja406634g

An l-RNA aptamer was developed that binds the natural d-form of the HIV-1 trans-activation responsive (TAR) RNA. The aptamer initially was obtained as a d-aptamer against l-TAR RNA through in vitro selection. Then the corresponding l-aptamer was prepared by chemical synthesis and used to bind the desired target. The l-aptamer binds d-TAR RNA with a Kd of 100 nM. It binds d-TAR exclusively at the six-nucleotide distal loop, but does so through tertiary interactions rather than simple Watson–Crick pairing. This complex is the first example of two nucleic acids molecules of opposing chirality that interact through a mode of binding other than primary structure. Binding of the l-aptamer to d-TAR RNA inhibits formation of the Tat-TAR ribonucleoprotein complex that is essential for TAR function. This suggests that l-aptamers, which are intrinsically resistant to degradation by ribonucleases, might be pursued as an alternative to antisense oligonucleotides to target structured RNAs of biological or therapeutic interest.

Kinetic Properties of an RNA Enzyme That Undergoes Self-Sustained Exponential Amplification

Co-reporter:Antonio C. Ferretti and Gerald F. Joyce

Biochemistry 2013 Volume 52(Issue 7) pp:

Publication Date(Web):January 23, 2013

DOI:10.1021/bi301646n

A special class of biochemical reactions involves a set of enzymes that generate additional copies of themselves and transfer heritable information from parent to progeny molecules, thus providing the basis for genetics and Darwinian evolution. Such a process has been realized with a pair of self-replicating RNA enzymes that undergo exponential amplification at a constant temperature. Exponential growth requires that the rate of production of new enzymes be directly proportional to the existing concentration of enzymes, which is the case for this system and provides a doubling time of ∼20 min. However, the catalytic rate of the underlying enzymes is ∼100-fold faster than the observed rate of replication. As in biological replication, other aspects of the system limit the generation time, chiefly the propensity of the substrate molecules to form nonproductive complexes that limit their availability for replication. An analysis of this and other kinetic properties of the self-replicating RNA enzymes reveals how exponential amplification is achieved and how the rate of amplification might be increased.

Ligand-Dependent Exponential Amplification of a Self-Replicating l-RNA Enzyme

Co-reporter:Charles Olea, Jr., David P. Horning, and Gerald F. Joyce

Journal of the American Chemical Society 2012 Volume 134(Issue 19) pp:8050-8053

Publication Date(Web):May 2, 2012

DOI:10.1021/ja302197x

A nuclease-resistant RNA enzyme, constructed entirely from l-ribonucleotides, was shown to undergo ligand-dependent, self-sustained replication with exponential growth. The catalytic motif is based on a previously described RNA ligase that can undergo either self- or cross-replication but had been limited in its application to ligand sensing due to its susceptibility to degradation by ribonucleases. The self-replicating RNA enzyme and its RNA substrates were prepared synthetically from either d- or l-nucleoside phosphoramidites. The d and l reaction systems undergo isothermal, ligand-dependent exponential amplification in the same manner, but only the l system is impervious to ribonucleases and can operate, for example, in the presence of human serum. This system has potential for the quantitative detection of various ligands that are present within biological or environmental samples. In addition, this work provides the first demonstration of the self-sustained exponential amplification of nonbiological molecules.

Synthetic Evolving Systems that Implement a User-Specified Genetic Code of Arbitrary Design

Co-reporter:Jonathan T. Sczepanski, Gerald F. Joyce

Chemistry & Biology 2012 Volume 19(Issue 10) pp:1324-1332

Publication Date(Web):26 October 2012

DOI:10.1016/j.chembiol.2012.08.017

A synthetic genetic system, based on cross-replicating RNA enzymes, provides a means to evaluate alternative genetic codes that relate heritable information to corresponding molecular function. A special implementation of encoded combinatorial chemistry was used to construct complex populations of cross-replicating RNA enzymes in accordance with a user-specified code that relates genotype and phenotype on a molecule-by-molecule basis. The replicating enzymes were made to undergo self-sustained Darwinian evolution, resulting in the emergence of the most advantageous variants. These included both highly active enzymes that sustained the population as a whole and poorly active enzymes that survived as parasites of the active molecules. This evolutionary outcome was a consequence of the information capacity and fidelity of the genetic code, suggesting how these parameters should be adjusted to implement codes tailored to particular applications.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (163 K)Download as PowerPoint slideHighlights► Populations of replicating RNAs were constructed by encoded combinatorial chemistry ► The RNAs evolve based on a heritable genotype and corresponding catalytic phenotype ► Genotype and phenotype are related by a user-specified genetic code ► The evolved enzymes included both highly active catalysts and molecular parasites

An Isothermal System that Couples Ligand-Dependent Catalysis to Ligand-Independent Exponential Amplification

Co-reporter:Bianca J. Lam

Journal of the American Chemical Society 2011 Volume 133(Issue 9) pp:3191-3197

Publication Date(Web):February 15, 2011

DOI:10.1021/ja111136d

A system was devised that enables quantitative, ligand-dependent exponential amplification for various ligands that can be recognized by an RNA aptamer. The aptamer is linked to an RNA enzyme that catalyzes the joining of two oligonucleotide substrates. The product of this reaction is another RNA enzyme that undergoes self-sustained replication at constant temperature, increasing in copy number exponentially. The concentration of the ligand determines the amount of time required for the replication products to reach a threshold concentration. A standardized plot of time to threshold versus ligand concentration can be used to determine the concentration of ligand in an unknown sample. This system is analogous to quantitative polymerase chain reaction (PCR), linking rare recognition events to subsequent exponential amplification, but unlike PCR can be applied to the quantitative detection of non-nucleic acid ligands.

Microfluidic Compartmentalized Directed Evolution

Co-reporter:Brian M. Paegel, Gerald F. Joyce

Chemistry & Biology 2010 Volume 17(Issue 7) pp:717-724

Publication Date(Web):30 July 2010

DOI:10.1016/j.chembiol.2010.05.021

Directed evolution studies often make use of water-in-oil compartments, which conventionally are prepared by bulk emulsification, a crude process that generates nonuniform droplets and can damage biochemical reagents. A microfluidic emulsification circuit was devised that generates uniform water-in-oil droplets (21.9 ± 0.8 μm radius) with high throughput (107–108 droplets per hour). The circuit contains a radial array of aqueous flow nozzles that intersect a surrounding oil flow channel. This device was used to evolve RNA enzymes with RNA ligase activity, selecting enzymes that could resist inhibition by neomycin. Each molecule in the population had the opportunity to undergo 108-fold selective amplification within its respective compartment. Then the progeny RNAs were harvested and used to seed new compartments. During five rounds of this procedure, the enzymes acquired mutations that conferred resistance to neomycin and caused some enzymes to become dependent on neomycin for optimal activity.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (326 K)Download as PowerPoint slideHighlights► A microfluidic device was developed that generates highly uniform water-in-oil emulsions for use in directed evolution experiments ► The device generates tens of millions of droplets per hour that vary in diameter by less than 4% ► The device was used to evolve RNA enzymes with RNA ligase activity, selecting enzymes that could resist inhibition by neomycin ► The evolved enzymes acquired resistance to neomycin and some became dependent on neomycin for optimal activity

Niche partitioning in the coevolution of 2 distinct RNA enzymes

Co-reporter:Sarah B. Voytek

PNAS 2009 Volume 106 (Issue 19 ) pp:7780-7785

Publication Date(Web):2009-05-12

DOI:10.1073/pnas.0903397106

Organisms that compete for limited resources within a common environment may evolve traits that allow them to exploit distinct ecological niches, thus enabling multiple species to coexist within the same habitat. The process of niche partitioning now has been captured at the molecular level, employing the method of continuous in vitro evolution. Mixed populations of 2 different “species” of RNA enzymes were made to compete for limited amounts of one or more substrates, with utilization of the substrate being necessary for amplification of the RNA. Evolution in the presence of a single substrate led to the extinction of one or the other enzyme, whereas evolution in the presence of 5 alternative substrates led to the accumulation of mutations that allowed each enzyme to exploit a different preferred resource. The evolved enzymes were capable of sustained coevolution within a common environment, exemplifying the emergence of stable ecological niche behavior in a model system. Biochemical characterization of the 2 evolved enzymes revealed marked differences in their kinetic properties and adaptive strategies. One enzyme reacted with its preferred substrate ≈100-fold faster than the other, but the slower-reacting species produced 2- to 3-fold more progeny per reacted parent molecule. The in vitro coevolution of 2 or more species of RNA enzymes will make possible further studies in molecular ecology, including the exploration of more complex behaviors, such as predation or cooperation, under controlled laboratory conditions.

Co-reporter:Tracey A. Lincoln

Science 2009 Volume 323(Issue 5918) pp:

Publication Date(Web):

DOI:10.1126/science.1167856

Abstract

An RNA enzyme that catalyzes the RNA-templated joining of RNA was converted to a format whereby two enzymes catalyze each other's synthesis from a total of four oligonucleotide substrates. These cross-replicating RNA enzymes undergo self-sustained exponential amplification in the absence of proteins or other biological materials. Amplification occurs with a doubling time of about 1 hour and can be continued indefinitely. Populations of various cross-replicating enzymes were constructed and allowed to compete for a common pool of substrates, during which recombinant replicators arose and grew to dominate the population. These replicating RNA enzymes can serve as an experimental model of a genetic system. Many such model systems could be constructed, allowing different selective outcomes to be related to the underlying properties of the genetic system.

Emergence of a fast-reacting ribozyme that is capable of undergoing continuous evolution

Co-reporter:Sarah B. Voytek

PNAS 2007 104 (39 ) pp:15288-15293

Publication Date(Web):2007-09-25

DOI:10.1073/pnas.0707490104

It is possible to evolve RNA enzymes in a continuous manner by employing a simple serial-transfer procedure. This method was previously applied only to descendants of one unusually fast-reacting RNA enzyme with RNA ligase activity. The present study establishes a second continuously evolving RNA enzyme, also with RNA ligase activity, but with a completely independent evolutionary origin. Critical to achieving the fast catalytic rate necessary for continuous evolution, development of this enzyme entailed the addition and evolutionary maturation of a 35-nucleotide accessory domain and the application of highly stringent selection pressure, with reaction times as short as 15 ms. Once established, continuous evolution was carried out for 80 successive transfers, maintaining the population against an overall dilution of 10207-fold. The resulting RNA enzymes exhibited ≈105-fold improvement in catalytic efficiency, compared with the starting molecules, and became dependent on a structural feature of the substrate that previously conferred no selective advantage. This adaptation was eliminated by deleting the substrate feature and then carrying out 20 additional transfers of continuous evolution, which resulted in molecules with even greater catalytic activity. Now that two distinct species of continuously evolving enzymes have been established, it is possible to conduct molecular ecology experiments in which the two are made to compete for limited resources within a common environment.

Forty Years of In Vitro Evolution

Co-reporter:Gerald F. Joyce

Angewandte Chemie International Edition 2007 Volume 46(Issue 34) pp:

Publication Date(Web):16 JUL 2007

DOI:10.1002/anie.200701369

It has been 40 years since Spiegelman and co-workers demonstrated how RNA molecules can be evolved in the test tube. This result established Darwinian evolution as a chemical process and paved the way for the many directed evolution experiments that followed. Chemists can benefit from reflecting on Spiegelman's studies and the subsequent advances, which have taken the field to the brink of the generation of life itself in the laboratory. This Review summarizes the concepts and methods for the directed evolution of RNA molecules in vitro.

Vierzig Jahre Evolution im Reagenzglas

Co-reporter:Gerald F. Joyce

Angewandte Chemie 2007 Volume 119(Issue 34) pp:

Publication Date(Web):16 JUL 2007

DOI:10.1002/ange.200701369

Vierzig Jahre ist es her, dass Spiegelman und Mitarbeiter demonstrierten, wie RNA-Moleküle in vitro (“im Reagenzglas”) evolviert werden können. Damit war einwandfrei festgestellt, dass die Darwinsche Evolution ein chemischer Prozess ist, und der Weg für zahlreiche Folgeexperimente zur gerichteten Evolution war geebnet. Auch die Chemie fand Inspiration in Spiegelmans bahnbrechenden Arbeiten und den daraus hervorgegangenen Entwicklungen, die das Gebiet bis dicht an die Erschaffung von Leben im Labor geführt haben. Dieser Aufsatz gibt einen Überblick über die Konzepte und Methoden zur gerichteten Evolution von RNA-Molekülen im Reagenzglas.

Conversion of a Ribozyme to a Deoxyribozyme through In Vitro Evolution

Co-reporter:Natasha Paul, Greg Springsteen, Gerald F. Joyce

Chemistry & Biology 2006 Volume 13(Issue 3) pp:329-338

Publication Date(Web):March 2006

DOI:10.1016/j.chembiol.2006.01.007

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.

A DNA-Templated Aldol Reaction as a Model for the Formation of Pentose Sugars in the RNA World

Co-reporter:Michael Oberhuber,Gerald F. Joyce

Angewandte Chemie International Edition 2005 44(46) pp:7580-7583

Publication Date(Web):

DOI:10.1002/anie.200503387

A DNA-Templated Aldol Reaction as a Model for the Formation of Pentose Sugars in the RNA World

Co-reporter:Michael Oberhuber Dr. Dr.

Angewandte Chemie 2005 Volume 117(Issue 46) pp:

Publication Date(Web):20 OCT 2005

DOI:10.1002/ange.200503387

Zucker am Stiel: Zwei Oligonucleotide, eines mit Glyceraldehyd (blau) derivatisiert, das andere mit Glycolaldehyd (rot), werden von einem komplementären DNA-Templat gebunden, das die Bildung von Pentosezuckern durch eine gekreuzte Aldolreaktion dirigiert. Die Reaktion dient als Modell für die RNA-katalysierte Synthese von Ribose, die für einen hypothetischen auf RNA basierenden Metabolismus in der Frühzeit des Lebens auf der Erde von Bedeutung ist.

A ribozyme composed of only two different nucleotides

Co-reporter:John S. Reader and Gerald F. Joyce

Nature 2002 420(6917) pp:841

Publication Date(Web):

DOI:10.1038/nature01185

A self-replicating ligase ribozyme

Co-reporter:Natasha Paul

PNAS 2002 Volume 99 (Issue 20 ) pp:12733-12740

Publication Date(Web):2002-10-01

DOI:10.1073/pnas.202471099

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 × 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.

In Vitro Evolution of a Ribozyme that Contains 5-Bromouridine

Co-reporter:Xiaochang Dai;Gerald F. Joyce

Helvetica Chimica Acta 2000 Volume 83(Issue 8) pp:1701-1710

Publication Date(Web):15 AUG 2000

DOI:10.1002/1522-2675(20000809)83:8<1701::AID-HLCA1701>3.0.CO;2-1

The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reaction compared to the behavior of the unmodified ribozyme. A population of 10¹³ variant ribozymes was constructed, each containing 5-bromouridine in place of uridine. Five successive `generations' of in vitro evolution were carried out, selecting for improved phosphoester transferase activity. The evolved molecules exhibited a 27-fold increase in catalytic efficiency compared to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutations were found to be responsible for this altered behavior. These mutations enhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out their catalytic function.

A DNA enzyme with N-glycosylase activity

Co-reporter:Terry L. Sheppard;Phillip Ordoukhanian

PNAS 2000 Volume 97 (Issue 14 ) pp:7802-7807

Publication Date(Web):2000-07-05

DOI:10.1073/pnas.97.14.7802

In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 106-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

Nucleoglycoconjugates: Design and Synthesis of a New Class of DNA–Carbohydrate Conjugates

Co-reporter:Terry L. Sheppard Dr.;Chi-Huey Wong

Angewandte Chemie 2000 Volume 112(Issue 20) pp:

Publication Date(Web):13 OCT 2000

DOI:10.1002/1521-3757(20001016)112:20<3806::AID-ANGE3806>3.0.CO;2-Y

A ribozyme that lacks cytidine

Co-reporter:Jeff Rogers and Gerald F. Joyce

Nature 1999 402(6759) pp:323

Publication Date(Web):

DOI:10.1038/46335

The RNA-world hypothesis proposes that, before the advent of DNA and protein, life was based on RNA, with RNA serving as both the repository of genetic information and the chief agent of catalytic function1. An argument against an RNA world is that the components of RNA lack the chemical diversity necessary to sustain life. Unlike proteins, which contain 20 different amino-acid subunits, nucleic acids are composed of only four subunits which have very similar chemical properties. Yet RNA is capable of a broad range of catalytic functions2, 3, 4, 5, 6, 7. Here we show that even three nucleic-acid subunits are sufficient to provide a substantial increase in the catalytic rate. Starting from a molecule that contained roughly equal proportions of all four nucleosides, we used in vitro evolution to obtain an RNA ligase ribozyme that lacks cytidine. This ribozyme folds into a defined structure and has a catalytic rate that is about 105-fold faster than the uncatalysed rate of template-directed RNA ligation.

Crystal structure of an 82-nucleotide RNA−DNA complex formed by the 10-23 DNA enzyme

Co-reporter:

Nature Structural and Molecular Biology 1999 6(2) pp:151-156

Publication Date(Web):

DOI:10.1038/5839

The structure of a large nucleic acid complex formed by the 10−23 DNA enzyme bound to an RNA substrate was determined by X−ray diffraction at 3.0 Å resolution. The 82−nucleotide complex contains two strands of DNA and two strands of RNA that form five double−helical domains. The spatial arrangement of these helices is maintained by two four−way junctions that exhibit extensive base−stacking interactions and sharp turns of the phosphodiester backbone stabilized by metal ions coordinated to nucleotides at these junctions. Although it is unlikely that the structure corresponds to the catalytically active conformation of the enzyme, it represents a novel nucleic acid fold with implications for the Holliday junction structure.