US 20050176017 A1
Polynucleotides having allosteric properties that modify a function or configuration of the polynucleotide with a chemical effector and/or physical signal are employed primarily as biosensors and/or enzymes for diagnostic and catalytic purposes. In some preferred embodiments, the polynucleotides are DNA enzymes that are used in solution/suspension or attached to a solid support as biosensors to detect the presence or absence of a compound, its concentration, or physical change in a sample by observation of self-catalysis. Chemical effectors include organic compounds such as amino acids, amino acid derivatives, peptides, nucleosides, nucleotides, steroids, and mixtures of these with each other and with metal ions, cellular metabolites or blood components obtained from biological samples, steroids, pharmaceuticals, pesticides, herbicides, food toxins, and the like. Physical signals include radiation, temperature changes, and combinations thereof.
21. A polynucleotide of mixed RNA and DNA nucleotide composition comprising an allosteric site and an enzyme domain spatially distinct from said allosteric site, wherein reversible interaction of a chemical effector with the allosteric site on the polynucleotide reversibly alters the cleavage function or configuration of the polynucleotide, wherein the chemical effector is a metal ion or small molecule having a molecular weight of 300 Daltons or less.
22. The polynucleotide of mixed RNA and DNA nucleotide composition of
23. A polynucleotide of mixed RNA and DNA nucleotide composition comprising an allosteric site and an enzyme domain spatially distinct from said allosteric site, wherein the rate of catalysis of the enzyme domain is reversibly modulated by interaction with a chemical effector, wherein the chemical effector is a metal ion or small molecule having a molecular weight of 300 Daltons or less.
24. The polynucleotide of mixed RNA and DNA nucleotide composition of
25. The polynucleotide of mixed RNA and DNA composition of
26. The polynucleotide of mixed RNA and DNA composition of
27. The polynucleotide of mixed RNA and DNA nucleotide composition according to claims 21 or 23, wherein the chemical effector is a small molecule selected from the group consisting of amino acids, amino acid derivatives, peptides, nucleosides, nucleotides, and steroids.
28. The polynucleotide of mixed RNA and DNA nucleotide composition according to claims 21 or 23, wherein said composition comprises modified nucleotides.
29. A biosensor comprising the polynucleotides of mixed RNA and DNA nucleotide composition according to claims 21 or 23.
30. The biosensor of
31. A method for detecting the presence or absence of a compound or its concentration in a sample, comprising the step of:
contacting the sample with a polynucleotide of mixed RNA and DNA composition, said polynucleotide comprising an allosteric site and an enzyme domain spatially distinct from said allosteric site, wherein reversible interaction of the compound with the allosteric site on the polynucleotide alters the cleavage function or configuration of the polynucleotide relative to that of a control sample, wherein the chemical effector is a metal ion or small molecule having a molecular weight of 300 Daltons or less; and
further wherein an alteration in function or configuration of the polynucleotide indicates the presence or absence of a compound or its concentration in the sample.
32. The method of
33. The method of
34. A polynucleotide of mixed RNA and DNA composition, said polynucleotide comprising an allosteric site and an enzyme domain spatially distinct from said allosteric site, having three stem components, stem I, stem II and stem III, wherein stem I and stem III are polynucleotide sequences which together form the enzyme domain and stem II is a polynucleotide sequence which forms the allosteric site, wherein interaction of a chemical effector with the allosteric site reversibly alters the cleavage function or configuration of the polynucleotide, further wherein the chemical effector is a metal ion or a small molecule having a molecular weight of 300 Daltons or less.
35. The polynucleotide according to
36. A polynucleotide of mixed RNA and DNA composition, said polynucleotide comprising an allosteric site and an enzyme domain spatially distinct from said allosteric site, having three stem components, stem I, stem II and stem III, wherein stem I and stem III are polynucleotide sequences which together form the enzyme domain and stem II is a polynucleotide sequence which forms the allosteric site, wherein interaction of a chemical effector with the allosteric site reversibly modulates the rate of catalysis of the polynucleotide, further wherein the chemical effector is a metal ion or a small molecule having a molecular weight of 300 Daltons or less.
37. The polynucleotide of
38. The polynucleotide of
39. The polynucleotide of
40. A biosensor comprising the polynucleotide according to claims 34 or 36.
41. A method for detecting the presence or absence of a compound or its concentration in a sample comprising contacting the sample with a polynucleotide according to claims 34 or 36, whereby reversible interaction of the compound with the allosteric site alters the cleavage function or configuration of the polynucleotide relative to that of a control sample, and observing said alteration in the cleavage function or configuration of the polynucleotide, wherein the compound is a chemical effector that is a metal ion or small molecule having a molecular weight of 300 Daltons or less and further wherein an alteration in function or configuration of the polynucleotide indicates the presence or absence of a compound or its concentration in the sample.
42. The method of
43. The method of
44. A polynucleotide of
45. A biosensor of
This application is a continuation-in-part of co-pending U.S. application Ser. No. 09/331,809, filed Jun. 18, 1999 as a national phase entry of PCT/US97/24158, filed internationally Dec. 18, 1997 and claiming priority benefit of U.S. Provisional application Ser. No. 60/033,684, filed Dec. 19, 1996 and Ser. No. 60/055,039, filed Aug. 8, 1997.
The invention was made with partial government support under NIH grant GM59343. The government has certain rights in the invention.
1. Field of the Invention
This invention relates primarily to functional DNA polynucleotides that exhibit allosteric properties, and to catalytic RNA and DNA polynucleotides that have catalytic properties with rates that can be controlled by a chemical effector, a physical signal, or combinations thereof. Bioreactive allosteric polynucleotides of the invention are useful in a variety of applications, particularly as biosensors.
Biosensors are widely used in medicine, veterinary medicine, industry, and environmental science, especially for diagnostic purposes. Biosensors are typically composed of a biological compound (sensor material) that is coupled to a transducer, in order to produce a quantitative readout of the agent or conditions under analysis. Usually, the biological component of the biosensor is a macromolecule, often subject to a conformational change upon recognition and binding of its corresponding ligand. In nature, this effect may immediately initiate a signal process (e.g., ion channel function in nerve cells). Included in the group of ‘affinity sensors’ are lectins, antibodies, receptors, and oligonucleotides. In biosensors, ligand binding to the affinity sensor is detected by optoelectronic devices, potentiometric electrodes, field effect transistors (FETs), or the like.
Alternatively, the specificity and catalytic power of proteins have been harnessed to create biosensors that operate via enzyme function. Likewise, proteins have been used as immobilized catalysts for various industrial applications. The catalytic activity of purified enzymes or even whole organelles, microorganisms or tissues can be monitored by potentiometric or amperometric electrodes, FETs, or thermistors. The majority of biosensors that are commercially available are based on enzymes, of which the oxidoreductases and lyases are of great interest. It is nearly exclusively the reactants of the reactions catalyzed by these enzymes for which transducers are available. These transducers include potentiometric electrodes, FETs, pH- and O2-sensitive probes, and amperometric electrodes for H2O2 and redox mediators. For example, the oxidoreductases, a group of enzymes that catalyze the transfer of redox equivalents, can be monitored by detectors that are sensitive to H2O2 or O2 concentrations.
Enzymes are well-suited for application in sensing devices. The binding constants for many enzymes and receptors can be extremely low (e.g., avidin; Kd=1015 M) and the catalytic rates are on the order of a few thousand per second, but can reach 600,000 sec−1 (carboanhydrase) (45). Enzymes can be monitored as biosensors via their ability to convert substrate to product, and also be the ability of certain analytes to act as inhibitors of catalytic function.
Organic chemistry and biochemistry have reached a state of proficiency where new molecules can be made to simulate the function of protein receptors and enzymes. Macrocyclic rings, polymers for imprinting, and self-assembling monolayers are now being intensively investigated for their potential application in biosensors. In addition, the immune system of animals can be harnessed to create new ligand-binding proteins that can act as artificial biorecognition systems. Antibodies that have been made to bind transition-state analogues can also catalyze chemical reactions, thereby functioning as novel ‘artificial enzymes’ (36). The latter examples are an important route to the creation of biosensors that can be used to detect non-natural compounds, or that function under non-physiologic conditions.
2. Description of Related Art
In nature, RNA not only serves as components of the information transfer process, but also performs tasks that are typically accomplished by proteins, including molecular recognition and catalysis. A seemingly endless variety of aptamers, and even DNA aptamers can be created in vitro that bind various ligands with great affinity and specificity (17). Nucleic acids likely have an extensive and as yet untapped ability adopt specific conformations that can bind ligands and also to catalyze chemical transformations (16). The engineering of new RNA and DNA receptors and catalysts is primarily achieved via in vitro selection, a method by which trillions of different oligonucleotide sequences are screened for molecules that display the desired function. This method consists of repeated rounds of selection and amplification in a manner that simulates Darwinian evolution, but with molecules and not with living organisms (4). One drawback to the use of existing enzymes as biosensors is that one is limited to developing a sensor based on the properties of existing enzymes or receptors. A significant advantage can be gained if one could ‘tailor-make’ the sensor for a particular application. It would be desirable to employ nucleic acids to create entirely new biosensors that have properties and specificities that span beyond the range of capabilities of current biosensors.
In vitro selection has been the main vehicle for new ribozyme discoveries in recent years. The catalytic repertoire of ribozymes includes RNA and DNA phosphoester hydrolysis and transesterification, RNA ligation, RNA phosphorylation, alkylation, amide and ester bond formation, and amide cleavage reactions. Recent evidence has shown that biocatalysis is not solely the realm of RNA and proteins. DNA has been shown to form catalytic structures that efficiently cleave RNA (5,7), that ligate DNA (10), and that catalyze the metallation of porphyrin rings (24). As described herein, self-cleaving DNAs have been isolated from a random-sequence pool of molecules that operate via a redox mechanism, making possible the use of an artificial DNA enzyme in place of oxidoreductase enzymes in biosensors. In addition, these DNA enzymes or ‘deoxyribozymes’ are considerably more stable that either RNA or protein enzymes—an attractive feature for the sensor component of a biosensor device.
It is an object of the invention to provide examples of RNA and DNA sensing elements for use in biosensors, including polynucleotides attached to a solid support. Both RNA and DNA can be designed to bind a variety of ligands with considerable specificity and affinity. In addition, both RNA and DNA can be made to catalyze chemical transformations under user-defined conditions. A combination of rational design and combinatorial methods has been used to create prototype biosensors based on RNA and DNA.
These and other objects of the invention are accomplished by the present invention, which provides purified functional DNA polynucleotides that exhibit allosteric properties that modify a function or configuration of the polynucleotide with a chemical effector, a physical signal, or combinations thereof. The invention further provides purified functional polynucleotides having catalytic properties with rates that can be controlled by a chemical effector, a physical signal, or combinations thereof. Some embodiments are enzymes exhibiting allosteric properties that modify the rate of catalysis of the enzyme. In addition, the invention encompasses biosensors comprising bioreactive allosteric polynucleotides described herein.
Examples of chemical effectors include, but are not limited to, organic compounds such as amino acids, amino acid derivatives, peptides, nucleosides, nucleosides, nucleotides, steroids, and mixtures of organic compounds and metal ions. In some embodiments, the effectors are microbial or cellular metabolites or components of bodily fluids such as blood and urine obtained from biological samples. In other embodiments, the effectors are pharmaceuticals, pesticides, herbicides, and food toxins. Physical signals include, but are not limited to, radiation and temperature changes.
The invention also provides methods for determining the presence or absence of compounds, or compound concentrations in biological, industrial, and environmental samples, and physical changes in such samples using bioreactive allosteric polynucleotides of the invention and biosensors incorporating them.
Natural ribozymes and artificial ribozymes and deoxyribozymes that have been isolated by in vitro selection are not known to operate as allosteric ribozymes. This invention is based upon the finding that small-molecule effectors can bind to ribozyme and deoxyribozyme domains and modulate catalytic rate. As will be discussed more fully below, in the practice of the invention, an effector molecule or effect binds or affects an allosteric site that is spatially distinct from that of the enzyme or reporter domain. Allosteric polynucleotides of the invention can thus rapidly interconvert from an “off” state to an “on” state, or vice versa, reversibly, on a time scale that is relevant for their use as biosensors and bioswitches. For example, using rational design strategies, a ‘hammerhead’ self-cleaving ribozyme described herein was coupled to different aptamer domains to produce ribozymes whose rates can be specifically controlled by adenosine and it's 5′-phosphorylated derivatives. A number of other allosteric ribozymes have been created that are sensitive to a variety of other effectors, including drug compounds, biological metabolites, and toxic metals. It is possible to construct, using a mix of in vitro selection and rational design strategies, novel biosensors that rely on nucleic acid sensor elements. To achieve this, unique RNA or DNA sequences can be appended to ribozymes or deoxyribozymes, thereby creating new enzymes having catalytic rates that can be influenced by specific chemical effectors (e.g., molecules of diagnostic interest), physical signals, and combinations thereof.
About 50 years ago, it was observed with some polypeptide enzymes that catalytic plots of reaction velocity, V, versus substrate concentration [S], displayed sigmoidal plots, rather than hyperbolic plots predicted by the simple enzyme+substrate model of enzymatic action described by Michaelis-Menten in 1913. In 1965, Monod, et al., explained these findings by suggesting that enzymatic reaction rates were altered by regulatory domains (3a). In this classical model of “allostery”, enzymatic activity by “allosteric enzymes” is modulated by reversible binding to compounds, termed “effectors”, at specific sites other than the enzyme's substrate binding sites, which, accordingly, are called “allosteric” sites. At constant enzyme and substrate concentrations, binding of a negative “effector” reduces the reaction rate (“allosteric inhibition”), and binding of a positive “effector” increases the rate (“allosteric activation”). Allosteric inhibition may be achieved a number of ways, including reducing the binding affinity of the enzyme for its substrate (often reported as increases in Michaelis-Menton parameter Km) and/or by increasing the time required for each catalytic turnover (often reported as a decrease in Vmax). Conversely, allosteric activation may occur either by reduction in Km or by an increase in Vmax, or both.
Decades later it was found that polynucleotides could also catalyze chemical reactions, and in 1995, Porta and Lizardi described what they called the first “allosteric” ribozyme (32a). This was a hammerhead, self-cleaving ribozyme that could be rendered active by incubating it with a 35-nucleotide antisense DNA oligomer for several hours. Notwithstanding the terminology used in the paper, this was not a true allosteric effect. Antisense interactions such as that described between the oligonucleotide and the ribozyme are typically comprised of strong base pairing contacts that have slow kinetic interchange between bound and unbound states. There was no allosteric interconversion (from an “off” state to an “on” state, or vice versa) disclosed upon addition of the 35-mer to an ongoing reaction mixture. Instead, Porta and Lizardi described a ribozyme construct which had a folding pathway that could be dictated by the 35-mer, but not allosterically switched from active to inactive forms immediately upon addition or depletion of a small effector molecule to or from the reaction mixture. Hence, their need for long preincubation and incubation times, and a large oligonucleotide that could kinetically and thermodynamically lock the ribozyme into an active configuration.
In contrast, in the practice of the invention, purified functional DNA and/or polynucleotides that exhibit true allosteric properties that modify a function or configuration of the polynucleotide with a chemical effector, a physical signal, or combinations thereof, are constructed. The function of polynucleotides of the invention is not necessarily controlled by base pairing to an oligonucleotide, but, instead, by binding of a small molecule effector to an allosteric binding site, or interaction of a physical signal with an allosteric site, spatially distinct from the enzyme domain, such that the function of the polynucleotide is allosterically modulated. In some embodiments, the polynucleotide is an enzyme exhibiting allosteric properties that modify the rate of catalysis of the enzyme. The invention further provides functional RNA or DNA polynucleotides having catalytic properties with rates that can be positively and/or negatively controlled by a chemical effector, a physical signal, or combinations thereof. For example, where enzyme polynucleotides of the invention exhibit a reaction rate that is enhanced or inhibited by reversible binding to a chemical effector at an allosteric binding site spatially distinct from the substrate binding or self-cleaving site. In some embodiments, the polynucleotides contain from about 100 or fewer bases; others are much larger.
Allosteric polynucleotides of the invention are comprised of any natural, recombinant, or synthetic RNA, DNA and mixtures of RNA and DNA. As used herein, the terms “DNA” and “RNA” specifically include sequences that have RNA and/or DNA analogues. Analogues include chemically modified bases and unusual natural bases. Further encompassed by the invention are polynucleotides modified during or after preparation of the domains and constructs using standard means. DNA and/or RNA starting materials for the domains, and constructs and complexes containing them, may be isolated from whole organisms, tissues or tissue cultures; constructed from nucleotides and oligonucleotides using standard means; obtained commercially; selected from random and enriched in vitro or in vivo sequence pools; and combinations thereof.
Any element, ion, and/or molecule can be used as chemical effectors for interaction with the bioreactive allosteric polynucleotides of the invention. It is an advantage of the invention that the rational design strategies used to construct the polynucleotides (discussed more fully below) can be adapted to a great variety of effectors. A vast number of ligand-responsive ribozymes with dynamic structural characteristics can be generated in a massively parallel fashion (23b). Examples include, but are not limited to, organic compounds and mixtures of organic compounds and metal ions. Chemical effectors may be amino acids, amino acid derivatives, peptides (including peptide hormones), polypeptides, nucleosides, nucleotides, steroids, sugars or other carbohydrates, pharmaceuticals, and mixtures of any of these. Many are small; hence, peptides having 9 or fewer amino acid substitutents and disaccharides and trisaccharides are typical polypeptide and carbohydrate effectors. Illustrated hereafter are theophylline, ATP and modified ATP; 3-methylxanthine, cGMP, cCMP, cAMP, FMN, cobalt, cadmium, nickel, zinc, and manganese have also been shown to be effectors that modulate the reaction rates of polynucleotides of the invention (see, for example, various effectors described in 21a, 23a, 23b, 36a, 39a, 39b, 39c, and 39e). In many preferred embodiments, small molecule effectors, typically having a molecular weight of about 300 or less, are employed, including metal ions, amino acids, amino acid derivatives, nucleosides, nucleotides, simple sugars, and steroids. Effectors can be much larger in other embodiments; larger molecule effectors can have molecular weights ranging in the tens or thousands Da, and sometimes even larger; protein effectors, for example, can range up to 500,000, and sometimes several million, Da. In some embodiments, the chemical effectors are microbial or cellular metabolites or other biological samples. Components found in liquid biological samples such blood, serum, urine, semen, tears, and biopsy homogenates taken from patients for medical or veterinary diagnostic or therapeutic purposes are particularly preferred chemical effectors in some embodiments (36a). In industrial and environmental applications, the effectors are pesticides, herbicides, food toxins, product ingredients, reactants, and contaminants, drugs, and the like. Allosteric polynucleotides of the invention can be used to detect the presence or absence of compounds, as well as their concentration (36a).
Bioreactive polynucleotides of the invention exhibit allosteric properties that modify polymer function or configuration with a physical signal or a combination of a physical signal and a chemical effector in alternate embodiments. Physical signals include, but are not limited to, radiation (23a), temperature changes, movement, physical conformational changes in samples, and combinations thereof. Physical signals include, but are not limited to, tags, beacons, and the like allosteric reporters that respond to UV, IR, and/or visible light (23b, 44b). The effects are reversible. Chemical effectors binding to allosteric ribozymes and/or deoxyribozymes of the invention, for example, can enhance or inhibit the catalytic rate, or do both. It is an advantage of the invention that, because the molecules are truely allosteric, any type of allosteric interconversion is possible. Hence, a sample of allosteric polynucleotide enzymes can be fully active, partially active, or fully inactive. In other words, acting as a switch, they can be all “on” or all “off”, or exhibit any level of activity between “on” or “off”. (For a further discussion of switches, see Soukup and Breaker, 39c). Morever, because they are truly allosteric, the observable response time to an effector molecule or effect is immediate. The kinetics of allosteric polynucleotides are similar to what is observed with allosteric polypeptides. Illustrated hereafter are polynucleotides that react in less than 60 minutes, preferably inless than 6 minutes, and most preferably, in less than about a minute. (See, for example,
Many embodiments employ bioreactive allosteric polynucleotides of the invention as biosensors in solution or suspension or attached to a solid support such as that illustrated in
The initial studies described in the Examples that follow have involved the creation and characterization of novel RNA- and DNA-cleaving enzymes that function with specific cofactors, or that can be regulated by specific small-molecule chemical effectors, physical signals, or combinations thereof. It is clear that additional molecules with similar sensor and biocatalytic properties can be created by similar means, thereby expanding the applications of such molecules. The creation and characterization of a prototype biosensor for ATP is given herein. One construct (H3) in particular shows ATP concentration-dependent catalytic activity, indicating that this ribozyme could be adapted for use in reporting the concentration of this ligand in test solutions. Specifically, H3 RNA actively self-cleaves in concentrations of ATP that are below 1 micromolar, but is maximally inhibited (170-fold rate reduction) in the presence of 1 millimolar ATP (
New and highly-specific receptors can be made via in vitro selection or ‘SELEX’ (4,5) using simple chromatographic and nucleic acid amplification techniques (4, and illustrated in the Examples). RNA and DNA ‘aptamers’ produced in this way can act as efficient and selective receptors for small organic compounds, metal ions, and even large proteins. In a dramatic display of RNA receptor function, a series of RNA aptamers for theophilline have been isolated (35) that show ˜10,000-fold discrimination against caffeine, which differs from theophilline by a single methyl group.
One can isolate new classes of aptamers that are specific for innumerable compounds to create novel biosensors or even controllable therapeutic ribozymes for use in medical diagnostics, environmental analysis, etc. In the examples that follow, simple design strategies have been used to create conjoined aptamer-ribozyme complexes who's rates can be controlled by small effector molecules. Preliminary studies have already shown that theophilline-dependent ribozymes can be created through rational design. Theophilline, for example, is an important drug for the treatment of asthma and it's therapeutic effect is highly dependent on concentration. A biosensor for theophilline concentration would be of significant value. Further examination of this allosteric ribozyme and of other model ribozymes will help to lay the biochemical and structural foundations for the design of additional sensor molecules based on RNA and DNA.
It is an advantage of the invention that the discovery that DNA can function as an enzyme (5) has made practical the engineering of enzymes that are chemically more stable than either RNA or proteins. The half-life for the hydrolytic breakdown of a DNA phosphoester is 200 million years, making DNA the most stable of the three major biopolymers. These features of DNA, coupled with the fact that DNA also can be made to bind various ligand with great specificity and affinity, make this polymer an attractive medium for the creation of new industrial enzymes and as sensor elements for diagnostics. Also, modified DNAs can be made that are resistant to degradation by natural nucleases, making DNA analogues an attractive format for use in biological solutions. As illustrated hereafter, it has been found that DNA can be made to self-cleave in a metal ion-dependent fashion. The creation of these DNAs that catalyze their own cleavage in the presence of copper can now be used as a sensitive reporter of free copper concentration in solution. Another example given below is a polynucleotide reactive to histidine. Further engineering of such catalysts will yield allosteric DNA enzymes that can be used to detect a wide variety of ligands, or that report other reaction conditions such as the concentration of salts, pH, temperature, etc. In addition, these DNAs may be conducive to monitoring via amperometric H2O2 probes or by spectrophotometric analysis of the redox state of copper. Clearly, the diversity of signal read-out for both RNA and DNA sensors can be expanded.
Another feature of the invention is that use of polynucleotides as biosensors offer advantages over protein-based enzymes in a number of commercial and industrial processes. Problems such as protein stability, supply, substrate specificity and inflexible reaction conditions all limit the practical implementation of natural biocatalysts. As outlined above, however, DNA can be engineered to operate as a catalyst under defined reactions conditions. Moreover, catalysts made from DNA are expected to be much more stable and can be easily made by automated oligonucleotide synthesis. In addition, DNA catalysts are already selected for their ability to function on a solid support and are expected to retain their activity when immobilized.
The invention further encompasses the use of bioreactive allosteric polynucleotides attached to a solid support for use in catalytic processes. Immobilizing novel DNA enzymes will provide a new form of enzyme-coated surfaces for the efficient catalysis of chemical transformations in a continuous-flow reactor under both physiological and non-physiological conditions. The isolation of new DNA enzymes can be each tailor-made to efficiently catalyze specific chemical transformations under user-defined reaction conditions. The function of catalytic DNAs to create enzyme-coated surfaces that can be used in various catalytic processes is described herein and illustrated in
A variety of different chromatographic resins and coupling methods can be employed to immobilize DNA enzymes. For example, a simple non-covalent method that takes advantage of the strong binding affinity of streptavidin for biotin to carry out a model experiment is illustrated in
A prototype system for the large-scale processing of RNA substrates using an immobilized DNA enzyme is described herein. Product yields have been determined by analysis of 32P-labeled substrate and product molecules by polyacrylamide gel electrophoresis of eluant samples. Multiple turn-over of immobilized enzyme during tests of the reactive chromatographic surface has been observed (
The following examples are presented to further illustrate and explain the present invention and should not be taken as limiting in any regard.
As mentioned above, natural ribozymes (8) and ribozymes that have been isolated by in vitro selection are not known to operate as allosteric enzymes (6). This example illustrates allosteric ribozymes.
Using simple rational design concepts, aptamer domains with hammerhead self-cleaving ribozymes (13) were joined in a modular fashion, to create a series of catalytic RNAs that are amenable to both positive and negative allosteric control by small-molecule effectors. Initial efforts were focused on the 40-nucleotide ATP-binding aptamer, termed ‘ATP-40-1′, that was described by Sassanfar and Szostak (35). This motif shows a specific affinity for adenosine 5′ triphosphate (ATP; KD ˜10 μM) and adenosine, but has no detected affinity for a variety of ATP analogues including 2′-deoxyadenosine 5′ triphosphate (dATP) or the remaining three natural ribonucleoside triphosphates. The aptamer also undergoes a significant conformational change upon ligand binding, as determined by chemical probing studies. These characteristics were exploited to create a conjoined aptamer-ribozyme molecule that could be subject to ATP-dependent allosteric control.
The initial integrated design, H3, incorporates several key features into an otherwise unaltered bimolecular hammerhead ribozyme that is embodied by H1 (
Superficially, sequences at the 5′ and 3′ termini were appended to make the constructs amenable to amplification by reverse transcription-polymerase chain reaction methods for future studies. Surveyed independently as H2 (
The RNA-cleavage activity of H3 is significantly reduced when incubated with 1 mM ATP (
To investigate the mechanism of inhibition of H3 by ATP, two additional integrated constructs (
The inhibitory effect of ATP with H3 has been confirmed and quantitated by kinetic analysis. Ribozyme activity assays were conducted with trace amounts of substrate and excess ribozyme concentrations that significantly exceed Km. Replicate kobs values obtained for H1 and H2 at 200, 400 and 800 nM ribozyme concentration under identical assay conditions differed by less that two fold, suggesting that for each construct, kobs values approach Vmax. Reactions also contained 50 mM Tris-HCl (pH 7.5 at 23° C.) and 20 mM MgCl2, and were incubated at 23° C. with concentrations of effector molecules and incubation times as noted for each experiments. Ribozyme and substrate were preincubated separately for 10 min in reaction buffer and also with effector molecules when present, and reactions were initiated by combining preincubated mixtures. Assays with H8 were conducted in 50 mM HEPES (pH 7.3 at 23° C.), 500 mM NaCl and 10 mM MgCl2. Catalytic rates (kobs) were obtained by plotting the fraction of substrate cleaved versus time and establishing the slope of the curve that represents the initial velocity of the reaction by a least-squares fit to the data. Kinetic assays were analyzed by PAGE and were visualized and analyzed on a Molecular Dynamics Phosphorimager. When shorter effector-molecule preincubations are used, the catalytic burst was more prominent and when encountered, a post-burst slope was used in the calculations. Replicate experiments routinely gave kobs values that differed by less than 50% and the values reported are averages of two or more experiments. Equivalent rates were also obtained for duplicate ribozyme and substrate preparations.
The H3 ribozyme displays different cleavage rates, after a brief burst phase, with different concentrations of ATP (
Whether ATP could also be made to function as a positive effector of ribozyme function was investigated by designing H6 and subsequently H7 (
As with allosteric effectors of proteins, there is no true similarity between the effector molecule and the substrate of the ribozyme. Substrate and effector occupy different binding sites, yet conformational changes upon effector binding result in functional changes in the neighboring catalytic domain. The specificity of allosteric control of ribozymes can be exquisite, and in this example the ribozyme activity is sensitive to the difference of a single oxygen atom in the effector molecule.
With similar model studies, a palate of design options and strategic approaches that can be used to create ribozymes with controlled catalytic activity can be built. The principles used here (secondary binding sites, conformational changes, steric effects and structural stabilization) as well as others may be generally applicable and can be used to design additional allosteric ribozymes, or even allosteric deoxyribozymes (37). For example, an allosteric hammerhead (H8,
The isolation by in vitro selection of two distinct classes of self-cleaving DNAs from a pool of random-sequence oligonucleotides are reported in this example. Individual catalysts from ‘class I’ require both Cu2+ and ascorbate to mediate oxidative self-cleavage. Individual catalysts from class II were found to operate with copper as the sole cofactor. Further optimization of a class II individual by in vitro selection yielded new catalytic DNAs that facilitate Cu2+-dependent self-cleavage with rate a enhancement that exceed 1 million fold relative to the uncatalyzed rate of DNA cleavage.
DNA is more susceptible to scission via depurination/β-elimination or via oxidative mechanisms than by hydrolysis (27). To begin a comprehensive search for artificial DNA-cleaving DNA enzymes, DNAs that facilitate self-cleavage by a redox-dependent mechanism were screened for. Cleavage of DNA by chelates of redox-active metals (e.g., Fe3+, Cu2+) in the presence of a reducing agent is expected to be a more facile alternative to DNA phosphoester hydrolysis due to the reactivity of hydroxyl radicals that are produced by reduction of H2O2 (i.e., Fenton reaction). Moreover, a variety of natural and artificial ‘chemical nucleases’ rely on similar cleavage mechanisms (38-39).
Beginning with a pool of ˜2×1013 random-sequence DNAs (
Sequence analysis of individual DNAs from G8 reveals a diverse set of catalysts that were divided into two groups (
The distribution of cleavage products between the two sites in CA3 is expected to result in a significant disadvantage during the selection process. About 35% of CA3-like molecules cleave within the center of the molecule (and hence are probably not amplified), while only about 65% cleave at the expected site and can be perpetuated in the next round of selection via amplification by PCR. In contrast, 100% of the catalysts that cleave exclusively in the primer-binding region can be amplified, giving individuals from class I an apparent selective advantage. However, CA3-like catalysts were found to persist in additional rounds of in vitro selection and actually come to dominate the population by generation 13. The success of these catalysts can be understood, in part, by examining the catalytic rates of CA1 and CA3. The cleavage rate (kobs) of 0.018 min−1 was obtained for CA1 under the final selection conditions, while cleavage at Clv 1 of CA3 occurs with a kobs of 0.14 min−1. Despite a high frequency of miscleavage, class II catalysts more rapidly cleave at the correct site, giving CA3-like catalysts a distinct selective advantage over catalysts from class I.
Cleavage sites for both classes have been further localized by gel-mobility analysis of the 5′ 32P-labeled self-cleavage products (
Similarly, Clv 1 of CA3 consists of a series products that range in mobility from 9 to 14 nucleotides, with the major product corresponding to a 12-nucleotide DNA (
To gain insight into the secondary structure of CA1, an artificial phylogeny (2) of functional CA1 sequence variants for comparative sequence analysis (47) were produced. The 50 nucleotides that corresponds to the original random-sequence domain were mutagenized by preparing a synthetic DNA pool such that each wild-type nucleotide occurs with a probability of 0.85 and each remaining nucleotide occurs with a probability of 0.05. The resulting pool was subjected to five additional rounds of selection for activity in the presence of 10 μM each of Cu2+ and ascorbate. Sequence alignment of 39 resulting clones (
Using sequence data and truncation analyses, a partial secondary-structure model for CA1 was constructed (
CA1 has no detectable activity in the absence of ascorbate, but surprisingly, both the G8 population DNA and CA3 display significant cleavage when only Cu2+ is added (
The catalytic activity of the reselected CA3 pool improved by nearly 100-fold, with variant DNAs 1, 2 and 3 (
The isolation of a variety of self-cleaving DNAs with Cu2+/ascorbate-dependence is consistent with an earlier report (23) of site-specific cleavage of a single-stranded DNA under similar conditions. These results confirm that DNA is indeed capable of forming a variety of structures that promote chemical transformations. In addition, the catalytic rates for both classes of self-cleaving DNAs compare favorably to those attained by other deoxyribozymes and by natural and artificial ribozymes. The finding that DNA is also able to perform self-cleavage with Cu2+ alone is unexpected, since the mechanism for the oxidative cleavage of DNA also requires a reducing agent such as ascorbate or a thiol compound (38,39).
A number of chemical nucleases have been prepared by others and examined for their potential as site-specific DNA-cleaving agents. For example, 1,10-phenanthroline and similar agents bind DNA, presumably via intercalation, and positions copper ions near the ribose-phosphate backbone where formation of a reactive oxygen derivative favors cleavage of the DNA chain (39). Alternatively, metal-binding ligands have been attached to oligonucleotide probes, in order to construct highly-specific DNA cleaving agents that recognize DNA by triple-helix formation (26). The catalytic DNAs described in this report likely replace the role of chemical nucleases by forming their own metal-binding pockets so as to promote region-specific self-cleavage. In fact, the addition of 1,10-phenanthroline to a catalytic assay of a synthetic class II DNA actually inhibits catalytic function. The optimal Cu2+ concentration for the 87-nucleotide DNA is ˜10 μM, with catalytic activity dropping significantly at both 1 and 100 μM Cu2+. The inhibitory effect of 1,10-phenanthroline might be due to the reduction in concentration of free Cu2+ upon formation of Cu2+-phenanthroline complexes.
While not wishing to be bound to any theory, several different mechanisms for the oxidative cleavage of class II DNAs seem possible. For example, the class II DNAs may simply scavenge for trace amounts of copper and reducing agents that are present in the reaction buffer. Alternatively, these DNA molecules might make use of an internal chemical moiety as the initial electron donor. In each example, the catalytic DNAs could still cleave by an oxidative mechanism, but would at least appear to gain independence from an external source of reducing agent. The importance of H2O2 in oxidative processes can be examined with catalase, an enzyme that efficiently promotes the dismutation of H2O2 molecules to yield water and molecular oxygen. The catalytic activity of a representative DNA from class II is completely inhibited upon the addition of catalase, consistent with the notion that H2O2 is a necessary intermediate in an oxidative pathway for DNA cleavage. The catalytic rate of CA3 variants is greatly increased when incubated in the presence of added H2O2. For example, the 87-nucleotide DNA can be made to cleave quantitatively at Clv 1 (kobs=1.5 min−1) in the presence of 10 μM Cu2+ and 35 mM H2O2.
It has not been determined whether trace amounts of H2O2 in water are used by the catalysts, or if the DNA can produce H2O2 in the absence of a reducing agent. It was found that preincubation of separate solutions of catalytic DNA in reaction buffer (minus Cu2+) and of aqueous Cu2+, followed by thermal denaturation of the catalase, results in full self-cleavage activity upon mixing of the two solutions. We also find that self-cleavage of the 87-nucleotide variant reaches a combined maximum (Clv 1+Clv 2) of 70%, regardless of the concentration of catalytic DNA present in the reaction. Similarly, preincubation of a reaction mixture with excess unlabeled catalyst (1 μM) followed by the addition of a trace amount of identical 5′ 32P-labeled catalysts produces normal yields of labeled-DNA cleavage products. Finally, addition of fresh reaction buffer to a previously-incubated reaction mixture does not promote further DNA cleavage, as might be expected if limiting amounts of reducing agent were responsible for activity.
Certain constructs of the self-splicing ribozyme of Tetrahymena have been shown to catalyze the cleavage of DNA via a transesterification mechanism (19,33), and the ribozyme from RNase P has been found to cleave DNA by hydrolysis (31). Such ribozymes might also be made to serve as therapeutic DNA-cleaving agents, analogous to the function of RNA-cleaving ‘catalytic antisense’ ribozymes (9). The secondary-structure model of CA1 (
In summary, two distinct classes of DNAs that promote their own cleavage have been isolated. One class requires copper and catalyzes the oxidative cleavage of DNA with a rate in excess of 1 million fold. Extensive regions of both classes of self-cleaving DNAs are important for the formation of catalytic structures, as implicated by sequence conservation found with selected individuals. These results support the view that DNA, despite the absence of ribose 2′-hydroxyl groups, has considerable potential to adopt higher-ordered structures with functions that are similar to ribozymes.
All synthetic DNAs were prepared by automated chemical synthesis (Keck Biotechnology Resource Laboratory, Yale University). The starting pool is composed of DNAs that carry a 5′-terminal biotin moiety and a central domain of 50 random-sequence nucleotides. Primer 3 is an analogue of primer 1 (
In vitro Selection
A total of 40 pmoles of pool DNA in 40 μl buffer A (50 mM HEPES, pH 7.0 at 23° C., 0.5 M NaCl, 0.5 M KCl) was loaded on two streptavidin-matrix columns (Affinitip Strep20, Genosys Biotechnologies) and incubated for ˜5 min. Unbound DNAs were subsequently removed from each column by pre-elution with 500 μl of buffer A, then by 500 μl 0.2 N NaOH, and the resulting matrix-bound DNAs were equilibrated with 500 μl buffer A. Catalytic DNAs were eluted with three successive 20-μl aliquots of buffer B (buffer A, 100 μM CuCl2, 100 μM ascorbate) for rounds 1-3, or buffer C (buffer A, 10 μM CuCl2, 10 μM ascorbate) for rounds 4-8. Eluate from each column was combined with 120 μl 4 mM EDTA and 40 pmoles each of primers 1 and 2. Selected DNAs and added primers were recovered by precipitation with ethanol and amplified by PCR a 200 μl reaction containing 0.05 U μl-1 Taq polymerase, 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl (pH 8.3 at 23° C.), 0.01% gelatin, and 0.2 mM each dNTP for 25 cycles of 10 sec at 92° C., 10 sec at 50 ° C. and 30 sec at 72° C. The 5′-terminal region of each cleaved DNA, including the biotin moiety, was reintroduced at this stage. Subsequent rounds were performed by immobilizing 20 pmoles of pool DNA on a single streptavidin column and selected DNAs were amplified in a 100 μl reaction for 10 to 20 temperature cycles. Steps II-IV (
5′-32P-labeled precursor DNA was prepared by PCR-amplifying double-stranded DNA populations or plasmid DNA using 5′-32P-labeled primer 4 and either primer 5 or primer 3. The antisense strand is removed either by binding the biotinylated strand to a streptavidin matrix (primer 5) or by alkaline cleavage of the RNA phosphodiester-containing strand, followed by PAGE purification (primer 3). DNA self-cleavage assays (˜5 nM 5′ 32P-labeled precursor DNA) were conducted at 23° C. in buffer A, with cofactors added as detailed for each experiment. For both in vitro selection and for assays, reaction buffers that contained ascorbate were prepared just prior to use. Self-cleavage assays conducted with catalase (bovine liver, Sigma) contained 50 mM HEPES (pH 7.0 at 23° C.), 50 mM NaCI, 10 μM CuCl2, and 0.5 U/μl catalase, and were incubated at room temperature for 20 min. Catalase activity was destroyed by heating at 90° C. for 5 min. Products were separated by denaturing (8 M urea) polyacrylamide gel electrophoresis (PAGE) using a 10% gel and visualized by autoradiography or visualized and quantitated by PhosphorImager (Molecular Dynamics).
Cleavage Product Analysis
Primary cleavage sites for CA1 and CA3 were identified by incubating 5′ 32P-labeled precursor DNA in buffer C and assessing the gel mobility of the 5′-terminal cleavage fragments by analysis using a denaturing 20% PAGE as compared to a series of 5′ 32P-labeled synthetic DNAs that correspond in sequence to the 5′ terminus of the precursor DNAs. Products resulting from sission at Clv 2 were analyzed by denaturing 6% PAGE.
Catalytic rates were obtained by plotting the fraction of precursor DNA cleaved versus time and establishing the slope of the curve that represents the initial velocity of the reaction as determined by a least-squares fit to the data. Kinetic assays were conducted in buffer C or in buffer A plus 10 μM CuCl2 as indicated for each experiment. Rates obtained from replicate experiments differed by less than two fold and the values reported are averages of at least two analyses.
This example describes a DNA structure that can cleave single-stranded DNA substrates in the presence of ionic copper. This deoxyribozyme can self-cleave, or it can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. DNA strand scission proceeds with a kobs of 0.2 min−1, a rate that is ˜1012-fold faster than the uncatalyzed rate of DNA phosphoester hydrolysis. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ‘restriction enzymes’ for the site-specific cleavage of single-stranded DNA.
A Minimal Cu2+-Dependent Self-cleaving DNA. In Example 2, a variety of self-cleaving DNAs were isolated by in vitro selection from a pool of random-sequence DNAs. Most individual DNAs that were isolated after eight rounds (G8) of selection conformed to two distinct classes, based on similarities of nucleotide sequence and DNA cleavage patterns. Although individual DNAs from both class I and class II require Cu2+ and ascorbate for full activity, the G8 DNA population displays weak self-cleavage activity in the presence of Cu2+ alone. A representative class II DNA termed CA3 was further optimized for ascorbate-independent activity by applying in vitro selection to a DNA pool that was composed of mutagenized CA3 individuals. The sequence data from this artificial phylogeny of DNAs indicates that as many as 27 nucleotides, most of them located near the 3′ terminus of the molecule, are important for self-cleavage activity.
Beginning with the original G7 DNA population, an additional six rounds of in vitro selection was carried out for DNAs that self-cleave in the presence of 10 μM Cu2+, without added reducing agent. Analysis of the G13 population of DNAs revealed robust self-cleavage activity, demonstrating that catalytic DNAs can promote efficient cleavage of DNA using only a divalent metal cofactor. The G13 population displays the same cleavage pattern that was observed with individual class II DNAs, indicating that class II-like DNAs dominate the final DNA pool.
A total of 27 individual DNAs from G13 were sequenced and, without exception, each carried a 21-nucleotide sequence domain that largely conformed to the consensus sequence that was used previously to define class II self-cleaving DNAs. Although individuals that have a strictly conserved core (spanning nucleotides 11 to 31,
Whether the two pairing regions of the 69-mer that lie within the variable-sequence region could be replaced by a smaller stem-loop structure was tested by synthesizing a 46-mer DNA, in which 26 nucleotides of this imperfect hairpin were replaced by the trinucleotide loop GAA (
Bimolecular Deoxyribozyme Complexes: Substrate Recognition by Duplex and Triplex Formation. Separate ‘substrate’ and ‘catalyst’ DNAs can be created from the 46 mer by eliminating the connecting loop of stem I (
Stem II was examined by a similar approach using mutant versions of the 46 mer self-cleaving DNA. A series of variant deoxyribozymes with one or two mutations included in the putative stem II structure were synthesized and assayed for catalytic activity (
Although the existence of stem II is supported by the data derived from mutational analysis, the fact that total restoration of deoxyribozyme activity was not achieved with restoration of base complementation indicates that the identities of the base pairs in this structural element are important for maximal catalytic function. Moreover, it was found that mutation or deletion of nucleotides 1-7 of the 46 mer result in a dramatic loss of DNA cleavage activity. It was recognized that nucleotides 4-7 within this essential region of the substrate form a polypyrimidine tract that is complementary to the paired sequence of stem II for the formation of a YR*Y DNA triple helix (14).
To examine the possibility of triplex formation in the active structure of the deoxyribozyme, we modified both the base pairing sequence of stem II (c4) and the sequence of the polypyrimidine tract of the substrate (s4) to alter the specificity, yet retain the potential for forming four contiguous base triples (
Targeted Cleavage of DNA ‘Restriction Sites’ with Deoxyribozymes. The results described above demonstrate that class II deoxyribozymes identify substrate DNAs by simultaneously utilizing two distinct recognition domains that are formed separately by stems I and II. These structures might be further exploited as recognition elements to engineer deoxyribozymes that selectively cleave DNAs at different target sites. To demonstrate this capability, a 101-nucleotide DNA that carries three identical leader sequences, each followed by different stem I recognition sequences was synthesized (
The triplex interaction that is defined by the base-pairing sequence of stem II can also be exploited to target specific DNA substrates. We designed three new catalyst DNAs (c9, c10 and c11) that carry identical stem I pairing subdomains, but that have expanded and unique stem II subdomains (
Although DNA cleavage catalyzed by the deoxyribozyme is focused within the substrate domain, substantial (˜30%) cleavage occurs within the conserved core of the catalyst strand. This collateral damage causes inactivation of the deoxyribozyme and, as a result, super-stoichiometric amounts of catalyst DNA are needed to assure quantitative cleavage of DNA substrate. Cleavage of the substrate subdomain proceeds more rapidly than does cleavage within the catalytic core. In the presence of excess c1, s1 is cleaved at a rate of approximately 0.2 min−1 (reaction buffer containing 30 μM CuCl2), reaching a plateau of 80% cleaved after 20 min. In contrast, cleavage of c1 in the presence of excess s1 proceeds more than 2-fold slower, consistent with our earlier report that the ratio of self-cleavage localized in the substrate domain to self-cleavage in the catalytic core gives a ratio of 2:1. It was established that, barring inactivation by miscleavage, the catalyst strand can undergo multiple turnover.
Cleaving Double-stranded DNA by Thermocycling. Class II catalyst DNAs are not able to cleave target DNAs when they reside within a duplex. The catalyst DNA, with its short recognition sequence, presumably cannot displace the longer and more tightly-bound complementary strand of the target in order to gain access to the cleavage site. It was found that an effective means for specific cleavage of one strand of an extended DNA duplex makes use of repetitive cycles of thermal denaturation and reannealing. For example, c3 remains inactive against a double-stranded DNA target in the absence of thermal cycling, but efficiently cleaves the same DNA substrate upon repeated heating and cooling cycles. Cleavage of the radiolabeled target is quantitative after 6 thermal cycles. DNA cleavage by class II DNAs occurs within the base-pairing region corresponding to stem I, presumably when this region is in double-helical form. This, coupled with the observation of substrate recognition by triplex formation, suggests that different DNA enzymes might be engineered to cleave duplex DNA substrates without the need for thermal denaturation. Such deoxyribozyme activity would be similar to that performed by a number of triplex-forming oligonucleotides that have been engineered to bind and cleave duplex DNA using a chemically-tethered metal complex such as Fe-EDTA (24-27).
Conclusions. In its unimolecular arrangement, the class II deoxyribozyme could be used to confer the capacity for self-destruction to an otherwise stable DNA construct. In its bimolecular form, the deoxyribozyme can act as an artificial restriction enzyme for single-stranded DNA, whereas protein-based nucleases that cleave non-duplex DNA do not demonstrate significant sequence specificity. It is likely that Ymaximal discrimination by class II catalysts between closely related target sequences can be achieved through careful design of the duplex and triplex recognition domains. This is expected to eliminate the cross reactivity that was observed here. Although the role of most nucleotides within the substrate domain are involved in substrate recognition, the importance of each nucleotide within the leader sequence has yet to be fully delineated. However, guided by the basic rules of duplex and triplex formation, one w3can now engineer highly-specific deoxyribozymes that can catalyze the cleavage of single-stranded DNA at defined locations along a polynucleotide chain.
Synthetic DNAs were prepared by automated chemical synthesis (Keck Biotechnology Resource Laboratory, Yale University), and were purified by denaturing (8M urea) polyacrylamide gel electrophoresis (PAGE) prior to use. Double-stranded 101 mer DNA was prepared by the polymerase chain reaction (PCR) as described in Example 2 using the primer DNAs 5′ 32P-GTCGACCTGCGAGCTCGA, (SEQ ID NO: 51) 5′GTAGATCGTAAAGCTTCG (SEQ ID NO: 52) and the 101 mer DNA oligomer (
In vitro Selection
Optimization of class II self-cleaving DNAs was achieved by in vitro selection essentially as described In Example 2 using a reaction mixture for DNA cleavage composed of 50 mM HEPES (pH 7.0 at 23° C.), 0.5 M NaCl, 0.5 M KCl (buffer A), and that included 10 μM CUCl2. The selection process was initiated with 20 pmoles G7 PCR DNA in which the 5′ terminus of each catalyst strand carried a biotin moiety, thereby allowing DNA from this and subsequent generations to be immobilized on a streptavidin-derivatized chromatographic matrix. Reaction time was 15 min. for immobilized DNA from G8-G10 and 12, 7 and 5 min. for the G11-G13 DNA populations, respectively. Individual self-cleaving DNAs from G13 were analyzed by cloning (Original TA Cloning Kit, Invitrogen) and sequencing (Sequenase 2.0 DNA Sequencing Kit, U.S. Biochemicals).
DNA Cleavage Assays
To assess the DNA cleavage activity of self-cleaving molecules, radiolabeled precursor DNA was prepared by enzymatically tagging the 5′ terminus of synthetic DNAs in a reaction containing 25 mM CHES (pH 9.0 at 23° C.), 5 mM MgCl2, 3 mM DTT, 1 μM DNA, 1.2 μM (γ-32P)-ATP (˜130 μCi), and 1 U/μL T4 polynucleotide kinase, which was incubated at 37° C. for 1 hr. The resulting 5′ 32P-labeled DNA was isolated by denaturing PAGE and recovered from the gel matrix by crush-soaking in 10 mM Tris-HCl (pH 7.5 at 23° C.), 0.2 M NaCl, and 1 mM EDTA. The recovered DNA was concentrated by precipitation with ethanol and resuspended in deionized water (Milli-Q, Millipore). Self-cleavage assays using trace amounts of radiolabeled precursor DNA (˜100 pM) were conducted at 23° C. in buffer A containing CuCl2 as indicated for each experiment. Examinations of the DNA cleavage activity of bimolecular complexes were conducted under similar conditions using trace amounts of of 5′ 32P-labeled ‘substrate’ DNA. Cleavage products were separated by denaturing PAGE, imaged by autoradiography or by PhosphorImager (Molecular Dynamics) and product yields were determined by quantitation (ImageQuant) of the corresponding precursor and product bands.
Catalytic rates were estimated by plotting the fraction of precursor or substrate DNA cleaved versus time and establishing the slope of the curve that represents the initial velocity of the reaction as determined by a least-squares fit to the data. Upon close examination, DNA cleavage in both the substrate and enzyme domains displayed a brief lag phase that complicates the determination of the initial cleavage rate. In order to avoid the lag phase, the initial slope was calculated only using data collected after the reaction had proceeded for 1 min. Rates obtained from replicate experiments differed by less than 50% and the values reported are averages of at least three analyses.
The in vitro selection of a catalytic DNA that uses histidine as the active component for an RNA cleavage reaction is described in this example. An optimized deoxyribozyme only binds to L-histidine or to several closely-related analogues and subsequently catalyzes RNA phosphoester cleavage with a rate enhancement of ˜10-million fold over the uncatalyzed rate. While not wishing to be bound to any theory, the DNA-histidine complex apparently performs a reaction that is analogous to the first step of the catalytic mechanism of RNase A, in which the imidazole group of histidine acts as a general base catalyst.
The class of deoxyribozymes that catalyze the cleavage of an RNA phosphoester bond using the amino acid histidine as a cofactor described herein is depicted in
The catalytic rate for the original class II deoxyribozyme was 1000-fold slower (kobs=1.5×10−3 min−1) than most natural self-cleaving ribozymes (44). As a result, further optimization of catalytic activity was sought in order to provide an artificial phylogeny of variant catalysts for comparative sequence analysis. A new DNA pool was prepared based on the sequence of class II deoxyribozymes, such that the 39 nucleotides corresponding to the original random-sequence domain were mutagenized with a degeneracy of 0.21 (6). Beginning with a mutagenized pool that sampled all possible variant DNAs with seven or fewer mutations relative to the original class II sequence, parallel reselection was conducted using reaction solutions buffered with either 50 mM histidine, or with 5 mM histidine and 50 mM HEPES. Individual DNAs isolated from the populations resulting from five rounds of reselection are more active than the original class II deoxyribozyme, and show specific patterns of conserved sequences and mutation acquisition (
It was speculated that engineered pairing element i included in the original DNA construct (
A larger panel of histidine analogues were examined (Fogire 16b) in order to more carefully examine the chemical groups of histidine that are important for catalytic activity and to rule out the possibility that catalysis might be due to a contamination of a metal ion cofactor. HD1 discriminates against a variety of histidine analogues, but shows full activity with the methyl ester of L-histidine (
The rate constant for HD2-promoted catalysis (kobs of 0.2 min−1, 50 mM histidine) is similar to that of natural self-cleaving ribozymes and corresponds to a rate enhancement of 10 million fold over the uncatalyzed reaction (kobs <10−8 min−1 under in vitro selection conditions). The dependence of the rate constant on histidine concentration is characteristic of the presence of a saturable binding site for histidine, although neither HD2 nor HD1 reach saturation even at 100 mM concentration of cofactor. The established specificity for particular cofactors, however, indicates that both catalysts do indeed form a histidine binding site. HD2 demonstrates greater activity with lower histidine concentrations, perhaps reflecting a greater binding affinity for histidine as would be expected due to its isolation from a low-histidine selection regiment.
The pH-dependent activity profile for HD2 also implicates histidine as an integral component of the catalytic process (
The loss of catalytic activity at higher pH values is not expected to be due to the protonation state of histidine, unless the pKa of the imidazole group of a putative second histidine cofactor is dramatically shifted from its normal value. The β-amino group of histidine, which has a pKa of greater than 9, conceivably could be involved in catalysis as well. However, it is expected to find a loss of activity with pH values in excess of 9 or less than 4.5 due to the significant level of deprotonation of T and G residues or protonation of C and A residues, respectively.
Histidine was chosen as a candidate cofactor because of the potential for the imidazole side chain to function in both general acid and general base catalysis near neutral pH. This property is neither inherent to the four standard nucleotides of RNA nor to the remaining natural amino acids. As a consequence, histidine is one of the most-frequently used amino acids in the active sites of protein enzymes. For example, two active-site histidines are essential for the function of ribonuclease A from bovine pancreas, where both of these capacities are used to accelerate RNA cleavage. Although RNase A has long served as a model for the study of enzyme action, the specific roles that each active-site reside play in the catalytic process are still vigorously debated (31). The classical view holds that the histidine at position 12 acts as a general base for the deprotonation of the 2′ hydroxyl, while the histidine at position 119 acts as a general acid and protonates the 5′ oxyanion leaving group. Breslow and others (25,47) have proposed that the role for histidine 119 instead may be to protonate the phosphorane intermediate, thereby implicating general acid catalysis by the imidazole group as a priority step during the catalytic process. The data described herein indicate that the histidine cofactor for class II deoxyribozymes is not involved in a protonation step, but is functioning exclusively as a general base catalyst.
In comparison to proteins, the more repetitive nature of monomeric units that make up nucleic acids limits both the formation of fine structure in folded polynucleotides and the chemical reactivity of RNA and DNA. The fact that a nucleic acid enzyme can co-opt one of the favorite chemical units of protein-based enzymes supports the notion that RNA could rally its limited structure-forming potential and, using the catalytic tools of modern protein enzymes, could produce and maintain a complex metabolic state.
In vitro Selection and Reselection
In vitro selection was carried out essentially as described previously (5,7,47). The initial DNA pool was prepared by PCR amplification of the template 5′-CTAATACGACTCACTATAGGAAGAGATGGCGACATCTC (N)4GTGAGGTTGGTGTGGTTG (SEQ ID NOs: 53 and 54) (50 pmoles; N an equal probability of occurrence of the four nucleotides) in a 500-μL PCR reaction containing 400 pmoles of primer B2, 5′-biotin-GAATTCTAATACGACTCACTATrA (SEQ ID NO: 55), and 400 pmoles of primer 1, 5′-CAACCACACCAACCTCAC (SEQ ID NO: 56), with 4 thermocycles of 94° C. (15 sec), 50° C. (30 sec), and 72° C. (30 sec). PCR reaction mixture was prepared as described previously (16). Amplified DNA was precipitated with ethanol, resuspended in binding buffer (50 mM HEPES (pH 7.5 at 23° C.), 0.5 M NaCl, 0.5 M KCl, and 0.5 mM EDTA), and the solution was passed through a streptavidin-derivatized affinity matrix to generate inunobilized single-stranded DNA15. The matrix displaying the pool DNA was repeatedly washed with binding buffer (1.5 mL over 30 min), and subsequently eluted over the course of 1 hr with three 20-μL aliquots of reaction buffer in which HEPES was replaced with 50 mM histidine (pH 7.5, 23° C.). In rounds 8-11, reaction time was reduced to 25-15 min to favor those molecules that cleave more efficiently. Selected DNAs were preciptitated with ethanol and amplified by PCR using primer 1 and primer 2, 5′-GAATTCTAATACGACTCACTATAGGAAGAGATGGCGAC (SEQ ID NO: 57), and the resulting PCR was reamplified as described above to reintroduce the biotin and embedded ribonucleotide moieties.
Reselection of the class II deoxyribozyme was initiated with a pool of 1013 DNAs, each carrying a 39-nucleotide core that had been mutagenized with a degeneracy of 0.21 per position. Similarly, HD2 reselection was conducted with an initial pool in which 26 nucleotides was mutagenized to a degeneracy of 0.33 per position. Individual from the final selected pools were analyzed by cloning and sequencing. The DNA pools were prepared for this process by PCR amplification using primer 2 in place of primer B2. DNA populations and individual precursor DNAs were prepared for assays as described previously (7).
Deoxyribozyme Catalysis Assays
All catalytic assays were conducted in the presence of 0.5 M NaCl, 0.5 M KCl, 0.5 mM EDTA. Single turn-over assays contained a trace amount (˜50 nM) substrate oligonucleotide and an excess (1-10 μM) DNA catalyst as described for each assay. The cofactor used was L-histidine unless otherwise stated. Reactions were terminated by addition to an equal volume of a solution containing 95% formamide, 0.05% xylene cyanol, and 0.05% bromophenyl blue and stored on ice prior to gel electrophoresis. Termination buffers containing both urea and EDTA were incapable of completely terminating deoxyribozyme activity.
Caged histidine experiments were conducted with intact dipeptides or with a concentration of hydrolyzed dipeptide products. Hydrolysis of dipeptides was achieved by incubating solutions containing 100 mM dipeptide and 6 N HCl in a sealed tube at 115° C. for 23 hr. Samples were evaporated in vacuo, coevaporated with deionized water, and the resuspended samples were adjusted to neutral pH prior to use.
Catalytic rate constants (kobs) either were determined by determining the initial velocity of the reaction (16) or by plotting the natural log of the fraction substrate remaining over time, where the negative slope of the line obtained over several half lives represents kobs. The uncatalyzed rate was determined by incubating a trace amount of 5′ 32P-labeled substrate under reaction conditions in the absence of deoxyribozyme at 23° C. or at −20° C. for 21 days. Comparative analysis of RNA phosphoester cleavage indicates that the rate constant for uncatalyzed RNA cleavage in the presence of histidine does not exceed the speed of substrate degradation due to radiolysis. It is expected that the maximum uncatalyzed rate for cleavage of the embedded RNA linkage does not exceed 10−8 min−1. This value is ˜10-fold lower than the value obtained in the presence of 1 mM Mg2+ (7).
The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.
Tuschl, C. Gohlke, T. M. Jovin, E. Westhof, F. Eckstein, Science 266,785 (1994); S. T. Sigurdsson and F. Eckstein, Trends Biotech. 13, 286 (1995); W. G. Scott, J. T. Finch, A. Klug, Cell 81, 991 (1995).
The papers cited herein are expressly incorporated in their entireties by reference.