Patent Application
20100129400
The invention relates generally to the fields of molecular biology, virology and immunology. More particularly, the invention relates to viral attenuation and anti-viral vaccines.
Many successful viral vaccines use live attenuated virus strains. The rate-limiting step for live-virus vaccine development is the identification of a suitable attenuated virus. Conventional methods of developing an attenuated virus involve propagation of the virus under novel conditions, such as passage of the virus in “foreign” or non-permissive cell lines, so that it becomes less pathogenic to its original host as it evolves under the new conditions. Although this methodology has shown remarkable success, little is known about the process by which the attenuating mutations arise and evolve, and isolating an attenuated virus is a random, slow process. In addition, the outcome of an attempted attenuation is largely unpredictable, and depending on the nature of the attenuation, an attenuated virus may revert to virulence.
Some currently used attenuation methods result in virus vaccine strains that are too virulent to produce. Highly virulent strains are deleterious to the host and developing vaccines requires inactivation of the virus. This has certain drawbacks. For example, inactivated viruses as orally or nasally applied vaccines must be given in high concentrations in order to bring about a significant increase of antibodies. As another example, the administration of inactivated influenza virus or antigen in convenient commercial doses, free of side effects, with nasal or oral administration, does not produce a satisfactory immune response without the use of an adjuvant. (Chen et al., 1989, Current Topics in Microbiology and Immunology146:101 106, Couch et al., 1997, J. Infect. Dis. 176:38 44). Thus, for example, for the optimum induction of the immune response with oral administration of an emulsion-inactivated vaccine, an antigen content between 66 μg antigen/dose and 384 μg antigen/dose is required (Avtushenko et al., 1996, J. Biotechnol. 44:21 28). Thus, this dose lies far above that of an inactivated vaccine for parenteral administration, which is at approximately 15 μg antigen/dose.
A cold-adapted, live attenuated influenza virus vaccine to be found in clinical studies for nasal administration is based on virus antigens from which reassortmants must be produced annually by means of genetic methods, in which the genes for the hemagglutinin and neuramidase antigens of the corresponding influenza A or B strain are transferred to an attenuated, cold-adapted master virus strain. This method is very time consuming and labor intensive. In addition, there is the danger that through reversion the attenuated virus back mutates into a virulent virus and thus can trigger viremia. When immunization is carried out with living viruses there is also a further spread in the body of the immunized individual. When cold-adapted viruses are used, there is also the constant necessity of storing the virus vaccine below the freezing point, as close to −20° C. as possible, which then requires the absolute maintenance of a chain of refrigeration to ensure sufficient storage life of the vaccine.
Eggs are used for the production of the live attenuated influenza virus vaccine virus reassortmants and the propagation of the vaccine viruses, which entails the risk that any contaminating infectious agents that may be present may be transferred into the eggs. The purification of live viruses is also not without problems because they represent infectious material and thus a higher standard of security must be maintained.
The availability of a technique for attenuating viruses in a rapid and non-random way would eliminate the disadvantages associated with current methods.
The invention relates to the development of modified viral polymerases that have an active site lysine residue substitution resulting in an altered rate of replication and altered fidelity compared to wild-type (WT) polymerases. In a typical embodiment, when incorporated into a virus, a modified polymerase as described herein exhibits a decreased rate of replication and a higher fidelity than a corresponding WT polymerase. In the experiments described herein, a poliovirus (PV) 3Dpol active site residue, Lys-359, was identified as a general acid catalyst during the phosphoryl transfer step of the nucleotide incorporation reaction. Surprisingly, subtle changes in polymerase speed and accuracy were found to have dramatic attenuation effects on the virus. Mutant PV viruses were made having a modified polymerase gene resulting in either a K359R or K359H substitution. These viruses were shown to be infectious and attenuated in cells relative to WT virus. The well-conserved residues in a polymerase active site as described herein modulate polymerase speed and/or accuracy, and are therefore targets for attenuating viruses. Active site lysine residues in polymerases from other classes were also identified and analyzed. Thus, the compositions and methods described herein can be applied to any virus, including viruses with regular intervals of antigenic shift (e.g., influenza), newly emerging and re-emerging viruses (e.g., SARS, West Nile, Dengue), and viruses used as agents of terror or biological weapons (e.g., Ebola, smallpox). Thus, the compositions and methods described herein provide a universal strategy for attenuating viruses for anti-viral vaccines. The present invention also solves the problem of generating vaccines of both highly virulent viruses and in amounts that induce either the humoral or cellular or both, immune responses.
Accordingly, the invention includes an attenuated virus including a polymerase gene that encodes a polymerase having a modification that results in a substitution of a lysine residue to a leucine, histidine, or arginine residue, the lysine residue capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction and located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RNA-dependent RNA polymerases and reverse transcriptases. The substitution causes the polymerase to have increased fidelity compared to a polymerase encoded by a WT polymerase gene not having the modification. The polymerase can be, for example, a PV polymerase and the lysine residue at position 359 of an amino acid sequence encoded by a nucleic acid sequence having accession number V01148 (SEQ ID N0:9); an influenza polymerase and the lysine residue at position 481 of a sequence having accession number AAY44773 (SEQ ID NO:10); or an HIV-1 reverse transcriptase and the lysine residue at position 220 of a sequence having accession number 4139739 (SEQ ID NO:11). The polymerase can be, for example, an A-family polymerase, a B-family polymerase, or a DNA-dependent DNA polymerase. In a typical embodiment, the virus is infectious.
Also within the invention is a vaccine that includes (a) an attenuated virus including a polymerase gene that encodes a polymerase, the polymerase gene having a modification that results in a substitution of a lysine residue to a leucine, histidine, or arginine residue, the lysine residue capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction and located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RNA-dependent RNA polymerases and reverse transcriptases, and a pharmaceutically acceptable carrier. The substitution causes the polymerase to have increased fidelity compared to a polymerase encoded by a WT polymerase gene not having the modification. The polymerase can be, for example, a PV polymerase and the lysine residue at position 359 of an amino acid sequence encoded by a nucleic acid sequence having accession number V01148; an influenza polymerase and the lysine residue at position 481 of a sequence having accession number AAY44773; or an HIV-1 reverse transcriptase and the lysine residue at position 220 of a sequence having accession number 4139739. The polymerase can be an A-family polymerase, a B-family polymerase, or a DNA-dependent DNA polymerase. The vaccine can further include an adjuvant.
In another embodiment, the invention includes a purified nucleic acid including a polymerase gene that encodes a polymerase, the polymerase gene having a modification that results in a substitution of a lysine residue to a leucine, histidine, or arginine residue, the lysine residue capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction and located on helix 0 of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RNA-dependent RNA polymerases and reverse transcriptases. The substitution causes the polymerase to have increased fidelity compared to a polymerase encoded by a WT polymerase gene not having the modification. The nucleic acid can be within a vector.
In still a further embodiment, the invention includes a method of attenuating a virus including modifying a viral polymerase gene that encodes a polymerase, such that the modification results in a substitution of a lysine residue to a leucine, histidine, or arginine residue, the lysine residue capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction and located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RNA-dependent RNA polymerases and reverse transcriptases. The substitution causes the polymerase to have increased fidelity compared to a polymerase encoded by a WT polymerase gene not having the modification.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the term “attenuated” means a virus that is modified to be less virulent (disease-causing) than WT virus.
As used herein, “vaccine” includes all prophylactic and therapeutic vaccines. According to one embodiment the vaccine contains an avirulent or attenuated infectious virus, the viral polymerase having amino acid changes in the conserved regions of the polymerase. These changes include for example, substituted amino acids, analogs, deletions and the like.
The vaccine compositions of the invention are suitable for administration to subjects in a biologically compatible form in vivo. The expression “biologically compatible form suitable for administration in vivo” as used herein means a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to any animal, preferably humans.
By the terms “RNA-dependent polymerase” and “RDP” is meant a viral polymerase that transcribes either RNA or DNA from an RNA template. RNA viruses described herein produce an RNA-dependent RNA polymerase (RDRP), and retroviruses described herein produce an RNA-dependent DNA polymerase, also referred to as a “reverse transcriptase.”
By the term “DNA-dependent DNA polymerase” is meant a viral polymerase that transcribes DNA from a DNA template.
As used herein, a “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid). A “purified” nucleic acid molecule is one that has been substantially separated or isolated away from other nucleic acid sequences in a cell or organism in which the nucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants). The term includes, e.g., a recombinant nucleic acid molecule incorporated into a vector, a plasmid, a virus, or a genome of a prokaryote or eukaryote.
As used herein, “protein” or “polypeptide” are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.
When referring to a nucleic acid molecule, polypeptide, or virus, the term “native” refers to a naturally-occurring (e.g., a WT) nucleic acid, polypeptide, or virus.
As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.”
A first sequence is “operably” linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
Although compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.
The invention encompasses compositions and methods relating to the development of modified viral polymerases that have an active site lysine residue substitution resulting in an altered rate of replication and altered fidelity compared to WT polymerases. The compositions and methods described herein provide a universal strategy for attenuating viruses for viral vaccines.
A two-metal ion mechanism has been proposed for polymerase-catalyzed nucleotidyl transfer reactions (Liu, J. & Tsai, M.-D. (2001) Biochemistry 40, 9014-9022; Steitz, T. A. (1993) Current Opinion in Structural Biology 3, 31-38). In this mechanism (
In the studies described herein, the mechanism of nucleotidyl transfer led to identification of a residue in the active site of all polymerases that facilitates nucleotide addition by protonating the pyrophosphate leaving group; this residue is Lys-359 in PV polymerase (
1The calculated average number of mutations per genome based upon sequencing 36,000 nucleotides of capsid coding sequence from 18 viral isolates.
2The calculated transition mutation frequency based upon the ratio of the kinetic parameters for correct and incorrect nucleotide incorporation by the PV RdRp allele.
3The maximal observed rate constant, kpol, for correct nucleotide incorporation.
4Not determined.
The below described preferred embodiments illustrate adaptations of these compositions and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.
Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Methods of propagating viruses for vaccine production and administering viral vaccines are also generally known in the art and are described in detail, for example, in Vaccine Protocols (Methods in Molecular Medicine) by Andrew Robinson, Martin P. Cranage, and Michael J. Hudson, 2nd ed., Humana Press, Totowa, N.J., 2003; Vaccine Adjuvants and Delivery Systems, by Manmohan Singh, 1st ed., Wiley-Interscience, Hoboken, N.J., 2007; Arvin A. M. and Greenberg H. B., Virology 344:240-249, 2006; and R. Morenweiser, Gene Therapy suppl. 1:S103-S110, 2005. Viral polymerase amino acid sequences and nucleic acid sequences encoding the viral polymerases are known in the art. For example, an HIV-1 RT amino acid sequence having accession number 4139739 is described in Huang et al. (Science 282:1669-1675, 1998). This HIV-1 RT is based upon a WT background, but has some modifications that allow for proper expression, crystallography and for trapping the enzyme in a covalent attachment with its RNA/DNA substrate. As another example, a human PV nucleic acid sequence having accession number V01148 has been described (Kitamura and Wimmer, Proc. Natl. Acad. Sci. 77:3196-3200, 1980). In yet another example, a human influenza A amino acid sequence having accession number AAY44773 has been described (Ghedin et al., Nature 437:1162-1166, 2005). These references are herein incorporated by reference.
The invention includes attenuated viruses having at least one mutation (e.g., substitution, deletion, insertion) at a conserved amino acid residue in the active site of the viral polymerase. Typically, such a modified polymerase includes a substitution of a lysine residue in the active site of the polymerase that is capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation to a leucine, histidine, or arginine residue. This lysine residue as well as neighboring conserved amino acid residues are located on helix-O of A-family polymerases, helix P of B-family polymerases, and on a loop of motif D of RNA dependent RNA polymerases (RdRps) and reverse transcriptases (RTs).
Viral RNA-dependent RNA polymerases have a conserved structure. Lys-359 of PV is present in the “palm” subdomain of the enzyme (
The comparable residue is located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RdRps and RTs (Table 6). Many DNA virus polymerases are B-family polymerases, for example herpes viruses and poxviruses, and have this conserved Lys residue. These viruses can also be attenuated by using the polymerase-mechanism-based strategies described herein. In the experiments described below, PV mutant viruses K359R and K359H were found to replicate.
In some embodiments described herein, an attenuated virus is replication-deficient or non-replicating and includes a foreign nucleic acid that is expressed in a host cell. Examples of such nucleic acids include therapeutic molecules such as for example, cytokines, enzymes and the like.
Accordingly, the present invention also includes a method of preventing or treating a viral infection comprising administering a vaccine of the present invention to an animal in need thereof. For example, viral organisms which cause human diseases according to the present invention include (but are not restricted to) Filoviruses, Herpes viruses, Hepatitisviruses, Retroviruses, Orthomyxoviruses, Paramyxoviruses, Togaviruses, Picornaviruses, Papovaviruses and Gastroenteritisviruses.
In some embodiments, an attenuated virus is replication deficient or replication incompetent and is used in the manufacture of a vaccine. For example, fast replicating virulent viruses used in the preparation of vaccines, such as for example, influenza (e.g. H5N1), HIV, are deleterious to the host cells. An attenuated vaccine strain is advantageous in that the slower replication rate, and if desired, the virus is rendered non-replicating, prevents the virus strain from being lethal to the host due to a fast replication rate. Therefore, in a preferred embodiment, the replication rate is controlled by incorporating a mutation that changes the general acid from lysine to another residue (e.g., leucine, arginine, histidine). Generally, viral replication is decreased at least 24-fold compared to WT virus using the modified polymerases and methods described herein. In another embodiment, the attenuated virus strain is grown in host cells which are normally lysed by the normal virus. Examples include an attenuated HIV strain wherein the HIV strain is grown in human lymphocytes without the immediate lysis or death of these cells.
In one embodiment, a vaccine for a pathogenic virus includes an effective amount of attenuated virus for providing protection from challenge with the pathogenic virus and a pharmaceutically acceptable carrier. In this embodiment, the attenuated virus includes a polymerase gene that encodes a polymerase, the polymerase gene having a modification that results in a substitution of a lysine residue to a histidine or arginine residue, the lysine residue capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction and located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RNA-dependent RNA polymerases and reverse transcriptases. This substitution causes the polymerase to have increased fidelity compared to a polymerase encoded by a wild-type polymerase gene not having the modification. For example, the pathogenic virus can be poliovirus and the polymerase a poliovirus polymerase, the lysine residue present at position 359 of an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:9. In another example, the pathogenic virus can be an influenza virus and the polymerase an influenza polymerase, the lysine residue present at position 481 of an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:10. In yet another example, the pathogenic virus can be HIV-1 and the polymerase an HIV-1 reverse transcriptase, the lysine residue present at position 220 of an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:11. In such a vaccine, the polymerase can be an A-family polymerase, a B-family polymerase, a DNA-dependent DNA polymerase, an RNA-dependent RNA polymerase, or a reverse transcriptase. A vaccine as described herein can further include an adjuvant. A pathogenic virus is any virus that causes pathology in a subject or patient (e.g., a mammal such as a human). An effective amount of attenuated virus for providing protection from challenge with a pathogenic virus is an amount effective for preventing pathology caused by the pathogenic virus (e.g., paralysis caused by poliovirus, flu symptoms caused by influenza virus, acquired immune deficiency syndrome (AIDS) caused by HIV, etc.) in the subject. The attenuated virus is generally an attenuated version of the pathogenic virus (e.g., an attenuated poliovirus for protecting a subject from challenge with a naturally occurring or non-naturally occurring pathogenic poliovirus).
Described herein is a method of protecting a subject against a pathogenic virus including administering to the subject an effective amount of a vaccine for the pathogenic virus. The vaccine includes a pharmaceutically acceptable carrier and an attenuated virus including a polymerase gene that encodes a polymerase, the polymerase gene having a modification that results in a substitution of a lysine residue to a histidine or arginine residue, the lysine residue capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction and located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RNA-dependent RNA polymerases and reverse transcriptases. The substitution causes the polymerase to have increased fidelity compared to a polymerase encoded by a wild-type polymerase gene not having the modification.
For example, the pathogenic virus can be poliovirus and the polymerase a poliovirus polymerase, the lysine residue present at position 359 of an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:9. In another example, the pathogenic virus can be an influenza virus and the polymerase an influenza polymerase, the lysine residue present at position 481 of an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:10. In yet another example, the pathogenic virus can be HIV-1 and the polymerase an HIV-1 reverse transcriptase, the lysine residue present at position 220 of an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:11. In such a vaccine, the polymerase can be an A-family polymerase, a B-family polymerase, a DNA-dependent DNA polymerase, an RNA-dependent RNA polymerase, or a reverse transcriptase.
Also described herein is a method of inducing an immune response to a pathogenic virus antigen in a subject including administering to the subject an effective amount of a vaccine for the pathogenic virus wherein administration of the vaccine to the subject results in prevention of disease from the pathogenic virus. The vaccine includes a pharmaceutically acceptable carrier and an attenuated virus including a polymerase gene that encodes a polymerase, the polymerase gene having a modification that results in a substitution of a lysine residue to a histidine or arginine residue, the lysine residue capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction and located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RNA-dependent RNA polymerases and reverse transcriptases. The substitution causes the polymerase to have increased fidelity compared to a polymerase encoded by a wild-type polymerase gene not having the modification. In one embodiment, the pathogenic virus is poliovirus and administration of the vaccine prevents paralysis in the subject. In other embodiments, the pathogenic virus is influenza or HIV. The vaccine can further include an adjuvant.
Modified viral polymerases as described herein are encoded by a viral polymerase gene that has a modification resulting in a mutation (e.g., substitution, deletion, insertion) at an amino acid residue in the active site of the polymerase that is capable of functioning as a general acid catalyst during a phosphoryl transfer step of a nucleotide incorporation reaction. One or more mutations in the conserved region of a viral polymerase modulate the activity of the polymerase (e.g., rate of replication, fidelity, etc.).
In the experiments described below, a PV polymerase was modified to include a substitution of leucine, arginine or histidine for the lysine corresponding to position 359 of the WT PV polymerase amino acid sequence encoded by a nucleic acid sequence having accession number V01148. Corresponding residues in HIV-1 and influenza were also identified. For example, the corresponding lysine in HIV-1 RT corresponds to position 220 of HIV-1 RT amino acid sequence having accession number 4139739. Similarly, the corresponding lysine in influenza corresponds to position 481 of the WT influenza gene segment PB1 amino acid sequence having accession number AA444773.
In some embodiments, a plurality of contiguous nucleic acids in the polymerase gene can be substituted with oligonucleotides which do not code for the conserved amino acid residues. Examples of some preferred oligonucleotides envisioned for this invention include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2—NH—O—CH2, CH, —N(CH3)—O—CH2 [known as a methylene(methylimino) or MMI backbone], CH2—O—N (CH3)—CH2, CH2—N (CH3)—N (CH3)—CH2 and O—N(CH3)—CH2—CH2 backbones, wherein the native phosphodiester backbone is represented as O—P—O—CH,). The amide backbones disclosed by De Mesmaeker et al. Acc. Chem. Res. 1995, 28:366-374) are also preferred. Also preferred are oligonucleotides having morpholino backbone structures (Summerton and Weller, U.S. Pat. No. 5,034,506). In other preferred embodiments, such as the peptide nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleobases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al. Science 1991, 254, 1497). Oligonucleotides may also comprise one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3, O(CH2)nCH3, O(CH2)n NH2 or O(CH2)n CH3 where n is from 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3 ; OCF3; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy [2′-O—CH2 CH2 OCH3, also known as 2′-O-(2-methoxyethyl)] (Martin et al., Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2′-methoxy (2′-O—CH3), 2′-propoxy (2′-OCH2 CH2CH3) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
Oligonucleotides may also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine and often referred to in the art as 5-Me—C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp75-77; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15:4513). A “universal” base known in the art, e.g., inosine, may be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions.
Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553), cholic acid (Manoharan et al. Bioorg. Med. Chem. Let. 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al. Ann. N.Y. Acad. Sci. 1992, 660, 306; Manoharan et al. Bioorg. Med. Chem. Let. 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al. EMBO J. 1991, 10, 111; Kabanov et al. FEBS Lett. 1990, 259, 327; Svinarchuk et al. Biochimie 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al. Tetrahedron Lett. 1995, 36, 3651; Shea et al. Nucl. Acids Res. 1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al. Nucleosides & Nucleotides 1995, 14, 969), or adamantane acetic acid (Manoharan et al. Tetrahedron Lett. 1995, 36, 3651). Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides are known in the art, for example, U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255.
It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide. The present invention also includes oligonucleotides which are chimeric oligonucleotides as hereinbefore defined.
In another embodiment, a nucleic acid molecule of the present invention is conjugated with another moiety including but not limited to abasic nucleotides, polyether, polyamine, polyamides, peptides, carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in the art will recognize that these molecules can be linked to one or more of any nucleotides comprising the nucleic acid molecule at several positions on the sugar, base or phosphate group.
RNA Interference (RNAi): RNAi is a remarkably efficient process whereby double-stranded RNA (dsRNA, also referred to herein as siRNAs, for small interfering RNAs, or ds siRNAs, for double-stranded small interfering RNAs) induces the sequence-specific degradation of homologous mRNA in animals and plant cells (Hutvagner and Zamore, Curr. Opin. Genet. Dev., 12:225-232 (2002); Sharp, Genes Dev., 15:485-490 (2001)). In mammalian cells, RNAi can be triggered by duplexes of small interfering RNA (siRNA) (Chiu et al., Mol. Cell., 10:549-561 (2002); Elbashir et al., Nature, 411:494-498 (2001)), or by micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which are expressed in vivo using DNA templates with RNA polymerase III promoters (Zeng et al., Mol. Cell, 9:1327-1333 (2002); Paddison et al., Genes Dev., 16:948-958 (2002); Lee et al., Nature Biotechnol., 20:500-505 (2002); Paul et al., Nature Biotechnol., 20:505-508 (2002); Tuschl, T., Nature Biotechnol., 20:440-448 (2002); Yu et al., Proc. Natl. Acad. Sci. USA, 99(9):6047-6052 (2002); McManus et al., RNA, 8:842-850 (2002); Sui et al., Proc. Natl. Acad. Sci. USA, 99(6):5515-5520 (2002)).
The agents described herein can include dsRNA molecules that are targeted to (i.e., bind to) regions of residues conserved between polymerases and/or structural homologous regions between different polymerases. For example, lysine at position 359 of the poliovirus polymerase. (See, also, for example, Tables 1 and 6). Structurally homologous regions are readily identifiable using various programs, such as BLAST etc.
The dsRNA molecules typically include 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the mRNA, and the other strand is identical or substantially identical to the first strand. Each strand can also have one or more overhanging (i.e., non-complementary) nucleotides, e.g., one, two, three, four or more overhanging nucleotides, e.g., dTdTdT.
The dsRNA molecules can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from, e.g., shRNA. The dsRNA molecules can be designed using any method known in the art; a number of algorithms are known in the art, see, e.g., Tuschl et al., Genes Dev 13(24):3191-7 (1999), and many are available on the internet, e.g., on the websites of Dharmacon (Lafayette, Colo.) or Ambion (Austin, Tex.).
Negative control siRNAs should have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.
micro RNA (miRNAs) of approximately 22 nucleotides can be used to regulate gene expression at the post transcriptional or translational level. miRNAs can be excised in the cell from an approximately 70 nucleotide precursor RNA stem-loop by Dicer, an RNase III-type enzyme, or a homolog thereof. By substituting the stem sequences of the miRNA precursor with miRNA sequence complementary to the target mRNA, a vector construct that expresses the novel miRNA can be used to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells (Zeng (2002), supra). When expressed by DNA vectors containing polymerase III promoters, micro-RNA designed hairpins can silence gene expression (McManus (2002), supra).
dsRNA can be delivered directly into cells in vivo or in vitro using methods known in the art, e.g., cationic liposome transfection, nanoparticles, and electroporation, or expressed in vivo or in vitro from recombinant DNA constructs that allow longer-term target gene suppression in cells, including mammalian Pol III promoter systems (e.g., H1 or U6/snRNA promoter systems (Tuschl (2002), supra) capable of expressing functional double-stranded siRNAs; (Bagella et al., J. Cell. Physiol. 177:206-213 (1998); Lee et al. (2002), supra; Miyagishi et al. (2002), supra; Paul et al. (2002), supra; Yu et al. (2002), supra; Sui et al. (2002), supra).
Viral-mediated delivery mechanisms can also be used to induce specific silencing of targeted genes through expression of siRNA, for example, by generating recombinant adenoviruses harboring siRNA under RNA Pol II promoter transcription control (Xia et al. (2002), supra). Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, providing a mechanism to end the siRNA transcript at a specific sequence. The dsRNA thus produced is complementary to the sequence of the target gene in 5′-3′ and 3′-5′ orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs. Hairpin siRNAs, driven by H1 or U6 snRNA promoter and expressed in cells, can inhibit target gene expression (Bagella et al. (1998), supra; Lee et al. (2002), supra; Miyagishi et al. (2002), supra; Paul et al. (2002), supra; Yu et al. (2002), supra; Sui et al. (2002) supra). Constructs containing siRNA sequence under the control of T7 promoter also make functional siRNAs when cotransfected into cells with a vector expression T7 RNA polymerase (Jacque (2002), supra).
In an animal, whole-embryo electroporation can efficiently deliver synthetic siRNA into post-implantation mouse embryos (Calegari et al., Proc. Natl. Acad. Sci. USA, 99(22):14236-40 (2002)). In adult mice, efficient delivery of siRNA can be accomplished by “high-pressure” delivery technique, a rapid injection (within 5 seconds) of a large volume of siRNA containing solution into animal via the tail vein (Liu (1999), supra; McCaffrey (2002), supra; Lewis, Nature Genetics 32:107-108 (2002)). Local delivery can also be used, e.g., with a carrier such as lipiodol (iodine in oil) to facilitate delivery into cells.
Engineered RNA precursors, introduced into cells or whole organisms as described herein, can be used for the production of a desired siRNA molecule. Such an siRNA molecule can then associate with endogenous protein components of the RNAi pathway to bind to and target a specific mRNA sequence for cleavage and destruction. In this fashion, the mRNA to be targeted by the siRNA generated from the engineered RNA precursor will be depleted from the cell or organism, leading to a decrease in the concentration of the protein encoded by that mRNA in the cell or organism. Additional information regarding the use of RNAi can be found in RNA Interference Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), Gott, Ed. (Humana Press, 2004);
Antisense Polynucleotides: An “antisense” nucleic acid can include a nucleotide sequence that is complementary to a “sense” nucleic acid encoding regions of residues conserved between polymerases and/or structural homologous regions between different polymerases. For example, lysine at position 359 of the poliovirus polymerase. (See, also, for example, Tables 1 and 6).
Based upon the sequences disclosed herein, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. For example, a “gene walk” comprising a series of oligonucleotides of 15-30 nucleotides spanning the length of regions of residues conserved between polymerases and/or structural homologous regions between different polymerases. For example, lysine at position 359 of the poliovirus polymerase. (See, also, for example, Tables 1 and 6). The nucleic acid can be prepared, followed by testing for inhibition of viral replication. Optionally, gaps of 5-10 nucleotides can be left between the oligonucleotides to reduce the number of oligonucleotides synthesized and tested. Other methods, including computational analysis, RNAse H mapping, and antisense-oligonucleotide scanning microarrays, can also be used (see, e.g., DNA Microarrays: A Practical Approach, Schena, Ed. (Oxford University Press 1999; Scherr and Rossi, Nucl. Acids Res., 26(22):5079-5085 (1998)).
An antisense nucleic acid can be designed such that it is complementary to the entire coding region of a target polymerase but can also be an oligonucleotide that is antisense to only a portion of the coding or noncoding region of the target mRNA. For example, the antisense oligonucleotide can be complementary to a region surrounding the translation start site of the target mRNA, e.g., between the −10 and +10 regions of the target gene nucleotide sequence of interest. Preferably, the antisense nucleic acid is targeted to regions of residues conserved between polymerases and/or structural homologous regions between different polymerases. For example, lysine at position 359 of the poliovirus polymerase. (See, also, for example, Tables 1 and 6). An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
Antisense nucleic acid molecules are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to mRNA and/or DNA encoding regions of residues conserved between polymerases and/or structural homologous regions between different polymerases. For example, lysine at position 359 of the poliovirus polymerase.
Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter can be used.
In yet another embodiment, an antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res. 15:6625-6641 (1987)). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. Nucleic Acids Res. 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al. FEBS Lett., 215:327-330 (1987)).
The potential sequences that can be targeted for triple helix formation can be increased by creating a so called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
Ribozymes: Ribozymes are a type of RNA that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. By cleaving the target RNA, ribozymes inhibit translation, thus preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme genes can be introduced into cells through gene-delivery mechanisms known in the art. A ribozyme having specificity for regions of residues conserved between polymerases and/or structural homologous regions between different polymerases can include one or more sequences complementary to these nucleotide sequences and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach, Nature, 334:585-591 (1988)).
The following examples are offered by way of illustration, not by way of limitation. While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon review of the specification.
All publications and patent documents cited in this application are incorporated by reference in pertinent part for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is “prior art” to their invention.
The vaccine compositions, attenuated viruses and compositions including attenuated viruses described herein can be introduced into a cell or administered to a subject (e.g., a human) in any suitable formulation by any suitable method. For example, the compositions and anti-viral vaccines described herein may be injected directly into a cell, such as by microinjection. As another example, the compositions and anti-viral vaccines described herein may be directly introduced into a subject (e.g., human), including by intravenous (IV) injection, intraperitoneal (IP) injection, subcutaneous injection, intramuscular injection, or in situ injection into target tissue. For example, a conventional syringe and needle can be used to inject a suspension containing an anti-viral vaccine as described herein into a subject. Depending on the desired route of administration, injection can be in situ (i.e., to a particular tissue or location on a tissue), IM, IV, IP, or by another parenteral route. Compositions and anti-viral vaccines as described herein can also be administered by oral administration, inhalation, transdermal administration, or suppository applications.
The vaccines of the present invention may additionally contain suitable diluents, adjuvants and/or carriers. Preferably, the vaccines contain an adjuvant which can enhance the immunogenicity of the vaccine in vivo. The adjuvant may be selected from many known adjuvants in the art including the lipid-A portion of gram negative bacteria endotoxin, trehalose dimycolate of mycobacteria, the phospholipid lysolecithin, dimethyldictadecyl ammonium bromide (DDA), certain linear polyoxypropylene-polyoxyethylene (POP-POE) block polymers, aluminum hydroxide, and liposomes. The vaccines may also include cytokines that are known to enhance the immune response including GM-CSF, IL-2, IL-12, TNF and IFNγ.
The dose of the vaccine may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances.
The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.
Materials: [γ-32P]ATP (>7000 Ci/mmol) was purchased from ICN; [α-32P] ATP (3000 Ci/mmol was from New England Nuclear; nucleoside 5′-triphosphates, (ultra pure solutions), were from Amersham Pharmacia Biotech, Inc; adenosine 5′-O-(1-thiotriphosphate) (ATPαS) was from Axxora Biochemicals (San Diego, Calif.); D2O was purchased from Isotec (Miamisburg, Ohio); deuterated glycerol was from Cambridge Isotope Laboratories (Andover, Mass.); all RNA oligonucleotides were from Dharmacon Research, Inc. (Boulder, Colo.); all DNA oligonucleotides were from Integrated DNA Technologies (Coralville, Iowa); T4 polynucleotide kinase was from New England Biolabs, Inc; all other reagents were of the highest grade available from Sigma, Fisher, or VWR.
Abbreviations: DdRp, DNA-dependent RNA polymerase DdDp, DNA-dependent DNA polymerase; RNAp, DNA-dependent RNA polymerase; RT, reverse transcriptase; PV, poliovirus; S/S, symmetrical primer/template substrate; CQF, chemical quench flow instrument; SF, stopped-flow instrument; 2AP, 2-aminopurine; SDIE, solvent deuterium isotope effect; DTT, dithiothreitol; BME, beta-mercaptoethanol; EDTA, ethylenediamino tetra acetic acid; NP-40, nonident P-40; PMSF, phenylmethylsulfonyl fluoride; PEI, Polyethyleneimine.
Stopped-flow experiments: Stopped-flow experiments were performed using a Model SF-2001 stopped-flow apparatus (Kintek Corp., Austin, Tex.) equipped with a water-bath. All reactions were performed at 30° C. Reactions were performed in two different sets of buffers. Reactions in HEPES refer to reactions performed by incubating 1 μM enzyme with 1 μM sym/sub (0.5 μM duplex) at room temperature for 3 minutes in 50 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol, 5 mM MgCl2, 60 μM ZnCl2, and then allowed to equilibrate to 30° C. in the sample compartment then rapidly mixed with the nucleotide prepared by using the same buffer. Reactions in MTCN or MHCN refer to reactions performed by incubating 1 μM enzyme with 1 μM sym/sub (0.5 μM duplex) in 1 mM HEPES pH 7.5 at room temperature for 3 minutes and then allowed to equilibrate to 30° C. in the sample compartment then rapidly mixed with the nucleoside triphosphate substrate in a solution containing 2×MTCN buffer at the indicated pH. 1×MTCN buffer is: 50 mM MES, 25 mM TRIS, 25 mM CAPS and 50 mM NaCl. The other buffer components for both enzyme-sym/sub and nucleotide solutions were the same. 1×MHCN buffer is: 50 mM MES, 25 mM HEPES, 25 mM CAPS and 50 mM NaCl. 3Dpol was diluted into enzyme buffer (50 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol, 60 μM ZnCl2, and 20% glycerol) immediately prior to use. The volume of enzyme added to any reaction was always equal to one-twentieth the total reaction volume. For reactions with a final ATP concentration higher than 1 mM, the amount of free Mg2+ was kept constant by increasing the amount of MgCl2 in the reaction to 5 mM plus the ATP concentration above 1 mM. For example, in a reaction containing 5 mM ATP, the final Mg2+ concentration was adjusted to 9 mM. The excitation wavelength used was 313 nm. Fluorescence emission was monitored by using a 370 nm cut-on filter (model E370LP, Chroma Technology Corp., Rockingham, Vt.). Reactions utilizing the nonhydrolyzable ATP analog α,β-methyleneadenosine 5′-triphosphate (AMPCPP) were done to investigate the possibility that observed changes in fluorescence resulted from alterations in the 2AP environment as a consequence of nucleotide binding. This proved not to be the case as changes in fluorescence were minimal over the time frame under investigation. The concentrations of nucleotide substrates and contents of buffers used are indicated in the appropriate figure and table legends. Rate constants for nucleotide incorporation were obtained by using the data analysis software of the instrument.
Rapid mixing/quenching experiments were performed by using a Model RQF-3 chemical-quench-flow apparatus (KinTek Corp., Austin, Tex.). 3Dpol-sym/sub complexes and all buffer solutions were prepared in the same way as for the stopped-flow experiments described above. Reactions were quenched by addition of HCl to a final concentration of 1 M. Immediately after the addition of HCl, the solution was neutralized by addition of 1 M KOH and 300 mM Tris base (final concentration). The concentrations of nucleotide substrates and contents of buffers used are indicated in the appropriate figure and table legends.
An equal volume of loading/quenching dye (85% formamide, 0.025% bromophenol blue, and 0.025% xylene cyanol, 90 mM EDTA) was added to 10 μl of the quenched reaction mixtures and heated to 70° C. for 2-5 min prior to loading 5 μl on a denaturing 23% polyacrylamide gel containing 1×TBE (2 mM EDTA, 89 mM boric acid, 87 mM Tris) and 7 M urea. Electrophoresis was performed in 1×TBE at 90 W. Gels were visualized by using a phosphorimager and quantified by using the ImageQuant software (Molecular Dynamics).
Steady-State Incorporation of AMP into Sym/Sub.
Steady-state experiments were performed by incubating 1 μM 3Dpol with 30 μM sym/sub (15 μM duplex) in 1 Mm HEPES buffer at pH 7.5 for 90 seconds at 30° C. then mixed with ATP in 2×MTCN buffer at pH 7.5. Reactions were quenched at various times by mixing with an equal volume of loading/quenching dye. Product analysis was performed by using denaturing PAGE as described above. All buffers and solutions were prepared in either H2O or D2O for each experiment. The pD was used instead of pH for the solutions in D2O and was adjusted according to pD=pH+0.4.
Enzymes, substrates and buffers for the proton-inventory experiments were prepared in 100% water or 100% D2O followed by mixing at the appropriate ratio to obtain 0, 25, 50, 75 or 100% D2O. Deuterated glycerol was used in all solutions in D2O. All data collection was performed in the stopped-flow instrument as indicated above. The pD was used instead of pH for the solutions in D2O as described above.
Time courses at fixed nucleotide concentrations were fit to the single exponential function:
[product]=A*exp(−kobs*t)+C (1)
where A is the amplitude of the burst, kobs is the observed first-order rate constant describing the burst, t is the time, and C is a constant. Time courses from the stopped-flow instrument were fit to the same equation using the instrument software. Data were fit by nonlinear regression using the program KaleidaGraph (Synergy Software, Reading, Pa.). The apparent binding constant (KD,app) and maximal rate constant for nucleotide incorporation (kpol) were determined using the equation:
k
obs
=k
pol
[NTP]/K
D,app
+[NTP] (2)
The pKa values for the pH dependence of kpol were obtained by fitting to a model describing two-ionizable groups (Bevilacqua, P. C. (2003) Biochemistry 42, 2259-65.):
k
pol
=k
ind/(1+10̂(pKa1−pH)+(pH−pKa2)) (3)
or one-ionizable group (Bevilacqua, P. C. supra.):
k
pol
=k
ind/(1+10̂(pKa−pH)) (4)
where kind is the pH-independent rate constant.
The proton-inventory data were fit to the modified Gross-Butler equation for a twoproton-transfer model (Schowen, R. L. & Venkatasubban, K. S. (1985) CRC Crytical reviews in Biochemistry 17, 1-44.):
k
n
/k
H2O=(1−n+n*φ1)(1−n+n*φ2) (5)
or for a one-proton-transfer model (Schowen et al):
k
n/kH2O=(1−n+n*φ) (6)
where kn is the observed rate constant at the different percentages of D2O, kH2O is the observed rate constant in water, n is the mole fraction of D2O and φ is the inverse of the isotope effect for each ionizable group.
Equilibrium and rate constants reported were determined independently at least twice. Error was propagated through products and quotients by using equation 6:
Δz=((Δx/x)2+(Δy/y)2)̂½*z (7)
where x and y are the primary data, z is the product or quotient of x and y, and Δx, Δy and Δz are the corresponding error values (Aikens, D. A., Bailey, R. A., Moore, J. A., Giachino, G. G. & Tomkins, R. P. T. (1984) Principles and techniques for an integrated chemistry laboratory (Waveland Press, Prospect Heights, Ill.)).
Expression and purification of RB69 DdDp.
RB69 DdDp was expressed in BL21 (DE3) cells. Frozen cells were thawed and suspended in lysis buffer (100 mM KPO4 pH 8.0, 20% glycerol, 20 mM DTT, 0.5 mM EDTA pH 8.0, 2.8 μg/mL pepstatin A, 2.0 μg/mL leupeptin) and lysed by passing through a French press at 1000 psi. Immediately after lysis, NP-40 was added to 0.1% (v/v) and PMSF was added to 2 mM. Polyethyleneimine (PEI) was added drop wise at 4° C. to a final concentration of 0.25% (v/v) and stirred for 30 min. This suspension was then centrifuged at 75,000×g for 30 min at 4° C. The supernatant was decanted and collected, and ground ammonium sulfate powder was slowly added to 60% saturation at 4° C. and stirred for 30 min. The ammonium sulfate precipitate was suspended in Buffer A (50 mM Tris pH 8.0, 20% glycerol, 10 mM DTT, 0.1% NP-40) and dialyzed overnight against 1 L Buffer A containing 75 mM NaCl using a 12-14,000 Da MWCO membrane. After dialysis, the sample was diluted in Buffer A to a final NaCl concentration of 50 mM. The sample was loaded onto a phosphocellulose column (approximately 1 mL bed volume/25 mg total protein) at a flow rate of 1 mL/min. The column was washed with ten column volumes of Buffer A containing 50 mM NaCl and the protein was eluted with Buffer A containing 150 mM NaCl. Fractions were pooled based upon their purity on 8% SDS-PAGE gels. The pooled fractions were diluted in Buffer A to a final salt concentration of 50 mM. The sample was loaded onto a Q-Sepharose column (1 mL bed volume/40 mg of protein) at 1 mL/min. The column was washed with ten column volumes of Buffer A containing 50 mM NaCl and the protein eluted with Buffer A containing 150 mM NaCl. Protein-containing fractions were pooled as before. The protein was concentrated using a small (0.5 mL) Q-Sepharose column. Loading and washing of the column was as described above. Elution was performed with Buffer A containing 500 mM NaCl.
Purification of T7 DdRp was done using the same protocol as for RB69 DdDp with the following modifications: 1. Ammonium sulfate was added to 40% saturation. 2. The phosphocellulose column was eluted using a linear gradient (6 column volumes) from 50 mM-700 mM NaCl in Buffer A. 3. The Q-Sepharose column was loaded and washed, and the protein eluted using a linear gradient (6 column volumes) from 50 mM-400 mM NaCl in Buffer A. Protein-containing fractions were pooled as before. Additional steps to concentrate the protein were not necessary.
Expression and purification of HIV RT.
HIV RT was expressed and purified as described previously (Le Grice, S. F. & Gruninger-Leitch, F. (1990) Eur J Biochem 187, 307-14).
Experiments were done in the stopped-flow instrument at 25° C. essentially as described (Yang, G., et al. (2002) Biochemistry 41, 2526-34.) with minor modifications. The assays were done in 1×MTCN pH 7.5, 10 mM MgCl2, 1 mM dATP with 1 μM RB69 WT enzyme and 150 nM primer-template substrate. The DNA primer used was: 5′-CCGACCAGCCTTG-3′ (SEQ ID NO: 1); the DNA template used was: 5′-AAAGC(2AP)TCAAGGCTGGTCGG-3′ (SEQ ID NO: 2). The solvent deuterium isotope effect and proton inventory were performed as described above for 3Dpol.
Experiments were done in the stopped-flow instrument at 30° C. essentially as described for 3Dpol with minor modifications. One μM RNA primer, 5′-UUUUGCCGCGCC-3′(SEQ ID NO: 3), was annealed to 1 μM DNA template, 5′-GGAATGC(2AP)TGGCGCGGC-3′ (SEQ ID NO: 4), and then incubated with 1.6 μM T7 WT for 10 minutes at room temperature in 1×MTCN pH 7.5, 10 mM BME, and 5 mM MgCl2. The reaction was started by mixing the T7-primer-template complex with 3 mM ATP in MTCN pH 7.5, 10 mM BME, 7 mM MgCl2, 100 mM NaCl (additional). The solvent deuterium isotope effect and proton inventory were performed as described above for 3Dpol.
Pre steady state experiments were done in the chemical-quench-flow instrument at 37° C. essentially as described (Gotte, M., et al (2000) Journal of Virology, 3579-3585) with some modifications: 2 μM 32P-labeled DNA primer, 5′-TTAAAAGAAAAGGGGGGACTGGA -3′ (SEQ ID NO: 5), was annealed with 2.2 μM DNA template, 5′-CGTTGGGAGTGAATTAGCCCTTCCAGTCCCCCCTTTTCTTTTAAAAAGTGGCTAAGA-3′ (SEQ ID NO: 6), and incubated with 4 μM RT in a solution containing no metals. The reaction was started by addition of 200 μM dATP in reaction buffer containing 10 mM MgCl2. After mixing, reactant concentrations were reduced by 50%. Reactions were performed by using a chemical quench flow instrument. Product analysis was performed essentially as described for PV 3Dpol with the following modifications. 1. Unlabeled DNA primer was added to the loading/quenching dye at a concentration 100-fold that of the labeled primer as described previously (Arnold, J. J., Ghosh, S. K., Bevilacqua, P. C. & Cameron, C. E. (1999) Biotechniques 27, 450-2, 454, 456). 2. A 15% polyacrylamide gel containing 40% formamide was used.
A rapid fluorescence assay for evaluation of 3Dpol catalyzed nucleotide incorporation was developed. This assay has been employed to evaluate the pH dependence of this reaction, providing evidence for two ionizable groups in the rate limiting step for nucleotide incorporation. Under physiological conditions, a solvent deuterium isotope effect was observed. A proton inventory experiment provided the first observation of a two-proton transfer reaction during polymerase-catalyzed nucleotide incorporation. Two proton-transfer reactions were also observed for nucleotidyl-transfer reactions catalyzed by the RB69 DNA-dependent DNA polymerase, T7 DNA-dependent RNA polymerase and HIV reverse transcriptase. Together, these data provide very compelling evidence for use of a general base and a general acid in polymerase-catalyzed nucleotidyl-transfer reactions.
Two ionizable groups are required for RdRp-catalyzed nucleotidyl transfer. Because chemistry is at least partially rate limiting for 3Dpol-catalyzed nucleotide incorporation, we reasoned that it should be possible to obtain insight into the proton transfers occurring during the rate-limiting transition state by evaluating the pH dependence of the reaction. In order to maximize the amount of kinetic data obtained, we developed and validated a stopped-flow fluorescence assay for 3Dpol that employed an RNA template containing 2-aminopurine (
aBuffers are described in materials and methods. “SF” indicates that experiments were performed in a stopped flow instrument. “CQF: indicates that experiments were performed in a chemical-quench-flow instrument. Kinetic constants are reported to one significant figure.
The KD,app and kpol values for nucleotide incorporation were measured at different pH values in Mg2+ or Mn2+ (Table 4). Experiments were limited to pH 10 in Mg2+ and pH 9 in Mn2+ due to nucleotide precipitation at higher pH values. Values for kpol wereplotted as a function of pH (
aExperiments were performed as described in materials and methods.
bUnits for KD, app and kpol are μM and s−1 respectively. Values are reported to one significant figure.
Rate limiting steps as a function of pH. In order to determine whether rate-limiting steps were changing as a function of pH, we evaluated the phosphorothioate (thio) effect over the pH range evaluated above (Table 5). For 3Dpol, chemistry is partially rate limiting in Mg2+ at pH 7.5. Under these conditions, the observed thio effect was 3±0.3 (Table 5). At pH values lower than 7.5, chemistry was clearly at least partially rate limiting as the value for the thio effect either did not change (pH 7.0) or increased (pH 6.0) (Table 5). A thio effect was also observed at pH values of 8.0 and 9.0; however, none was observed at pH 10 (Table 5). Interestingly, the decrease in the value of the thio effect above pH 7.5 was due to a specific increase in the observed rate constant for AMPαS incorporation without any significant effect on that for AMP incorporation (Table 5). In Mn2+ at pH 7.5, chemistry is the sole rate limiting step. From pH 6.0 to 9.0, the thio effect ranged from 7 to 5 (Table 5), consistent with chemistry remaining kinetically significant over this pH range.
Solvent deuterium isotope effect as a probe for rate-limiting steps during nucleotide incorporation: Because it was difficult to interpret the loss of the thio effect in Mg2+ at pH 10.0, the solvent deuterium isotope effect was investigated. If proton transfers are occurring during the rate-limiting steps measured by the nucleotide incorporation assay, then an isotope effect should be apparent. In Mg2+ at pH 7.5, an isotope effect of 3±0.5 was observed for AMP incorporation in the pre-steady-state (
The rate-limiting step in the steady state is dissociation of enzyme from the primer template substrate. An isotope effect on this step would not be expected. As shown in
Two proton-transfer reactions in the rate-limiting transition state for RdRp catalyzed nucleotidyl transfer. The existence of a solvent deuterium isotope effect on the rate constant for nucleotide incorporation permits quantitation of the number of protons transferred in the rate-limiting transition state (proton inventory). In this experiment, the observed rate constant for nucleotide incorporation (kn) is measured in reactions containing different mole fractions of D2O (n). A plot of the quotient knkH2O (kH2O is the observed rate constant in H2O) as a function of n will fall on the line defined by kH2O/kH2O and k100%D2O/kH2O if a single proton is transferred. The data will fit to a second-order polynomial if two protons are transferred, to a third order polynomial if three protons are transferred and so on. A proton-inventory experiment was performed for AMP incorporation by 3Dpol in Mg2+ (
However, the data fit well to a second-order polynomial, the Gross-Butler equation (equation 5) for two-proton transfers (solid lines in
Two proton-transfer reactions in the rate-limiting transition state for nucleotidyl transfer of other classes of nucleic acid polymerases. In order to determine whether the conclusions reached here for the RdRp applied to other classes of nucleic acid polymerases, similar experiments were performed with RB69 DdDp, T7 DdRp and HIV RT. In all cases, a solvent deuterium isotope effect was observed (
3Dpol-catalyzed nucleotide incorporation can be monitored by using 2-aminopurine containing primer/template substrates. Previous studies of 3Dpol mechanism revealed that the rate of nucleotide incorporation catalyzed by 3Dpol is partially limited by both a conformational change and chemistry in the presence of Mg2+. However, in the presence of Mn2+, the reaction is limited solely by chemistry. The experiments leading to these conclusions employed radioactively labeled substrates and a chemical quench flow instrument (CQF). This approach is slow and provides no direct information on conformational changes that may occur during the reaction. 2-Aminopurine (2AP) has been employed to study DNA-dependent DNA and RNA polymerase-catalyzed reactions. 2AP is similar in structure to adenine, which has its amino group on the 6-position (
Validation of the SF assay. We previously determined the complete kinetic mechanism of 3Dpol-catalyzed nucleotide incorporation using radiolabeled S/S in the CQF. The values obtained in those experiments for KD,app and kpol were 130±20 μM and 90±10 s−1, respectively. Use of S/S-+1 in CQF experiments produced values for KD,app and kpol of 70±10 μM and 80±10S−1, respectively (Table 3). We interpret the decrease in KD,app as a nearest neighbor effect caused by the 2AP. Note that the kpol values are the same—that is, within the error of the measurement.
Experiments with S/S-+1 in the SF were performed with an excitation wavelength of 313 nm, ensuring excitation of only 2AP and guaranteeing that the observed changes in fluorescence were attributable only to changes in the environment surrounding the 2AP probe in the active site of 3Dpol. The observed change in fluorescence was measured as a function of ATP concentration (
Interrogation of the chemical mechanism for nucleotidyl transfer catalyzed by DNA polymerases has been thwarted by the general belief that a conformational change is rate limiting for nucleotide incorporation, a belief that has relied on interpreting the magnitude of the thio effect, a highly controversial parameter (Joyce, C. M. et al. (2004) Biochemistry. 43, 14317-24; Showalter, A. K. et al. (2002) Biochemistry 41, 10571-6). The studies described herein of the RdRp from poliovirus, however, have shown that chemistry is at least partially rate limiting for nucleotide incorporation in Mg2+ and completely rate limiting in Mn2+. This system permits interrogation of the chemical mechanism by employing a nucleotide incorporation assay.
Evaluation of the pH dependence of PV polymerase-catalyzed nucleotide incorporation in Mg2+ revealed a dependence of the reaction on two ionizable groups with pKa values of 7.0 and 10.5 (
The observed pKa value for the 3′-OH is sensitive to the disposition of residues in the active site as distortions caused by increasing the size of the divalent cation or substituting ATPαS for ATP cause an increase in the observed pKa value (
The solvent deuterium isotope effect is a useful probe for chemistry in the rate limiting step for nucleotide incorporation by the RdRp (
The observation of a solvent deuterium isotope effect permitted the quantification of the number of proton-transfer reactions occurring during the rate-limiting transition state for nucleotide incorporation catalyzed by the RdRp (
A solvent deuterium isotope effect of seven was observed for the RdRp under conditions in which chemistry is the sole rate-limiting step for nucleotide incorporation (
It is generally assumed that the pKa value for the Mg2+-bound PPi is low enough to preclude protonation during catalysis. However, in solution the highest pKa value for PPi is on the order of 9.3. Therefore, protonation of PPi by a general acid should increase the rate of catalysis but may not be absolutely essential. Structural studies have revealed a basic amino acid, in most cases a lysine, in a position to serve as a general acid (Table 6). This residue is located on helix O of A-family polymerases, helix P of B-family polymerases and on the loop of structural motif D of RdRps and RTs. Motif D has been in search of a function other than structural scaffolding since solution of the first RT structures. In all cases in which this putative general acid has been changed, the observed rate constant for nucleotide incorporation has been diminished substantially. Particularly noteworthy is the observation that chemistry appears to become the rate-limiting step by changing the putative general acid of RB69 DNA polymerase, Lys-560, to alanine.
The RdRp from PV was used as a model in the studies described herein to produce the first comprehensive analysis of the chemical mechanism for a nucleic acid polymerase. This system has proven to be particularly attractive because of the extremely slow (0.0001 s−1) and pH-independent nature of the rate constant for dissociation of the primer-template substrate from the enzyme. These studies have provided very compelling evidence for a highly symmetrical transition state with proton transfers from the 3′-OH and to PPi occurring coordinately. Pyrophosphate protonation was not expected. All classes of polymerase appear to have a similar transition-state structure. Coordinated proton transfer reactions necessitate a well-positioned acceptor and donor. The observed pKa values required for nucleotidyl transfer and the absence of ordered water molecules in the appropriate position in all cases force one to re-propose general-acid-base-facilitated catalysis by all polymerases.
D654, E655
E. coli DNA Pol
D882, E883
Bacillus stear.
D830, E831
Therm. aquat.
D785, E786
D621, D623
D256
D185, D186
D812
D328, D329
aPutative general base indicated in bold-faced type.
The experiments described herein show that poliovirus RNA-dependent RNA polymerase (3Dpol) active site Lys-359 enhances the rate of phosphoryl transfer by acting as a catalytic general acid. This finding was extended to conserved active site lysine residues in polymerases from other classes by the demonstration of a role as catalytic general acid for the conserved active site lysine of HIV-1 RT RdDp and RB69 DdDp. General acid catalysis by a conserved active site residue capable of proton donation may therefore be a universal feature of the nucleic acid polymerase phosphoryl transfer reaction, adding an additional 2-3 orders of magnitude of rate enhancement to that already supplied by the two metal cations, and bringing rates of nucleotide incorporation to a level of biological sufficiency.
Nucleic acid polymerases are the primary agents of genome replication, maintenance and expression in all organisms and viruses. To fulfill this role, these enzymes must not only assemble nucleic acid chains at biologically sufficient rates but must also achieve a level of accuracy of nucleotide selection (fidelity) that is optimal for fitness.
Nucleic acid polymerases vary widely in their nucleic acid tasks. They require DNA or RNA as template. In most situations, they add nucleotides to a DNA, RNA or peptide primer-terminus 3′-OH. However, in some cases a primer-terminus requirement is lacking and nucleotide addition occurs de novo. Polymerase fidelity likewise varies widely. Misincorporation frequencies range from as low as 10−8 errors per incorporation in some genome-replicating DNA polymerases, to 10−5 for certain viral RNA polymerases to 10−2 or higher for some repair enzymes. In all cases, misincorporation rates are thought to reflect biological need. Enzyme size and architectural complexity also vary widely, ranging from small, single-subunit enzymes having only polymerase activity to large, multi-subunit macromolecule assemblages possessing additional activities.
The broad range in biological roles, incorporation accuracies and overall architecture thus impose multiple functional constraints on the polymerase nucleotide incorporation mechanism. Yet in spite of this complexity, the most basic underlying polymerase active site structural features are highly conserved. In addition, all nucleic acid polymerases accomplish the same fundamental chemical reaction of phosphoryl transfer and use essentially the same five-step mechanism in the single nucleotide incorporation cycle. In step one an incoming nucleotide binds to the enzyme-primer/template binary complex. In step two this ternary complex undergoes a conformational change, aligning reactive functional groups for phosphoryl transfer, which occurs in step three. A post-chemistry conformational change occurs in step four. Pyrophosphate release occurs in step five, resetting the cycle to start the next incorporation.
Polymerase amino acid sequence alignments in combination with a growing list of high-resolution x-ray crystal structures of polymerase-primer/template-nucleotide ternary complexes reveal the universal presence of an active site positively charged amino acid residue, usually Lys but sometimes Arg or His, in the precise location of covalent bond cleavage and leaving group formation during step three, phosphoryl transfer, of the nucleotide incorporation cycle. Conservation of a residue capable of proton donation at this specific location suggests a direct role as general acid catalyst during phosphoryl transfer for this active site amino acid. However, a long-standing experimental limitation of some well-studied nucleic acid polymerase model systems has been the inability to interrogate the phosphoryl transfer step directly and conveniently because of a rate-limiting conformational change immediately prior to chemistry. This limitation was removed with findings described herein that for PV polymerase (3Dpol), an RNA-dependent RNA polymerase (RdRp), phosphoryl transfer is partially or completely rate-limiting in Mg2+ or Mn2+, respectively, and therefore accessible to mechanistic examination.
One proton transfer during the chemistry step of nucleotide incorporation is the removal of the proton from the primer-terminus 3′-OH (and acceptance by an undetermined recipient) during creation of the attacking nucleophile. As described above, universal conservation of a positively charged amino acid residue capable of proton donation at the precise site of bond cleavage and pyrophosphate leaving group formation during phosphoryl transfer suggests that the second catalytically-important proton transfer may involve this active site amino acid residue in a role as general acid catalyst.
Materials: Materials were purchased and prepared as described in Example 1.
Expression and purification of polymerases: 3Dpol RdRp, HIV-1 RT, RB69 DdDp and T7 DdRp were expressed in E. coli and purified as described previously (Castro et al, Proc. Natl. Acad. Sci., 104:4267-4272, 2007) except for the following. Construction of the RB69 DdDp K560L mutant- The K560L mutant was constructed using the pSP72-RB69-pol template and introducing the K560L-encoding mutation into the polymerase gene (gp43) by Quick Change PCR using the forward oligonucleotide RB69-DdDp-K560L-fwd 5′-GCACAAATTAA TCGTCTGTTGCTTATCAACTCAC-3′ (SEQ ID NO:7) and reverse oligonucleotide RB69-DdDp-K560L-rev 5′-GTGAGTTGATAAGCAACAGACGATTAATTTGTGC-3′ (SEQ ID NO:8). The expression vector for RB69 K560A was kindly provided by Dr. William Konigsberg, Yale University.
PV 3Dpol-catalyzed nucleotide incorporation experiments: PV 3Dpol stopped-flow and chemical-quench-flow experiments were accomplished as described previously (Castro et al, Proc. Natl. Acad. Sci., 104:4267-4272, 2007). Enzymes, substrates and buffers for proton-inventory experiments were prepared in 100% water or 100% D2O followed by mixing at the appropriate ratio to obtain 0, 25, 50, 75 or 100% D2O. Deuterated glycerol was used in all solutions in D2O. All data collection was performed in the stopped-flow instrument. The pD was used instead of pH for the solutions in D2O as described above.
Solvent deuterium isotope effect and proton inventory for WT and a K220A HIV-RT were performed essentially as described in Example 1.
RB69 WT DdDp stopped-flow and chemical-quench-flow experiments: RB69 DdDp WT solvent deuterium isotope effect and proton inventory chemical-quench-flow experiments were done essentially as described in Example 1, with reactions quenched in 1 M HCl, neutralized and analyzed as described in Example 1.
Stopped-flow Kd,app and kpol, solvent deuterium isotope effect and proton inventories for RB69 K560A: Experiments were done using reaction conditions and DNA primer and template essentially as described for WT (Castro et al, Proc. Natl. Acad. Sci., 104:4267-4272, 2007). For Kd,app and kpol determinations, kobs was obtained using [dATP] of 0.5, 1, 2.5, 5 and 10 mM. For 2.5 and 10 mM dATP reactions, MgSO4 was supplemented to maintain free Mg2+ at 10 mM. Plots of kobs vs. [dATP] were fit with a hyperbola (eq. 2). Solvent deuterium isotope effect and proton inventory experiments were conducted as for WT except [dATP] was 10 mM and MgSO4 was increased to provide 10 mM free Mg2+ over that bound to dATP. Chemical-quench-flow solvent deuterium isotope effect for RB69 K560A-Experiments were done as described above in the stopped-flow except [dATP] was 5 mM. Reactions were quenched with 0.25 M EDTA as described in Example 1.
Chemical-quench-flow Kd,app, kpol, solvent deuterium isotope effect and proton inventories for RB69 K560L: Experiments were done using reaction conditions and DNA primer and template essentially as described for WT (Castro et al, Proc. Natl. Acad. Sci., 104:4267-4272, 2007). For Ka,app and kpol determinations, kobs was obtained using [dATP] of 0.3, 1, 3, 6 and 12 mM. For 3, 6 and 12 mM dATP reactions, MgSO4 was supplemented to maintain free Mg2+ at 10 mM. Plots of kobs vs. [dATP] were fit with a hyperbola (eq. 2). Solvent deuterium isotope effect and proton inventory experiments were conducted as for WT except [dATP] was 5 mM and MgSO4 was increased to provide 10 mM free Mg2+ over that bound to dATP. Chemical-quench-flow RB69 K560L reactions were quenched with 0.25 mM EDTA as described in Example 1.
Data analysis: Data analysis was performed as described in Example 1.
The conserved active site positively charged amino acid residue in PV 3Dpol is Lys-359: Nucleic acid polymerase architecture resembles a cupped right hand consisting of palm, thumb and fingers subdomains. Conserved structural motifs comprise the palm subdomain, the location of the polymerase active site. Alignment of RNA polymerase palm structural motif D amino acid residues shows absolute conservation of a lysine residue (
Kinetic parameters for 3Dpol position-359 mutants suggest a catalytic role for Lys-359: To evaluate the influence of PV 3Dpol Lys-359 on the phosphoryl transfer step, this residue was changed to Leu, His or Arg (Table 7). K359L 3Dpol with Mg2+ as metal-ion co-factor showed a 50-fold reduction in single nucleotide incorporation catalytic rate (kpol) relative to WT K359 and a 3-fold weakening in nucleotide binding, as reported by Kd,app. These data suggest a profound role for Lys-359 in catalysis and a lesser role in nucleotide binding. The reduced catalytic performance of K359L 3Dpol is consistent with lack of ability to donate a proton by the chemically inert Leu side chain. The reduction, albeit small, in nucleotide binding is consistent with absence of a positive charge on the Leu side chain, presumably important for binding interactions with the negatively-charged nucleotide triphosphate moiety.
aKd,app is for ATP.
bSDKIE is solvent deuterium kinetic isotope effect, calculated as kobs in H2O/kobs in D2O at saturating [ATP].
cnd means not determined.
The solvent deuterium kinetic isotope effect (SDKIE) for WT K359 3Dpol was 2-fold larger in the presence of Mn2+ than in Mg2+, consistent with chemistry being completely rate-limiting in Mn2+ and partially rate-limiting in Mg2+. In contrast, identical and low kpol values and indistinguishable SDKIE values in Mg2+ and Mn2+ for K359L 3Dpol point to the importance of Lys-359 in catalyis and indicate that chemistry is completely rate-limiting with either metal ion co-factor for this mutant enzyme (Table 7).
Catalytic rates for the K359H and K359L 3Dpol variants were 5-fold faster than for K359L 3Dpol, but 10-fold slower than the WT K359 polymerase (Table 7). Nucleotide binding interactions for the K359H and K359L enzymes were similar to that of WT, consistent with retention of ability of the positively charged His and Arg side chains to bind a negatively-charged incoming nucleotide. However, while both His and Arg have exchangeable protons theoretically available to assist catalysis, their 10-fold diminished catalytic rates relative to the WT K359 enzyme suggests that the active site environment during phosphoryl transfer is not chemically (i.e. matching of pKas, etc.) and/or spatially optimized to make efficient catalytic use of these non-biological residues when at position 359.
As shown in
The kinetic behavior of the K359R 3Dpol was similar to that of K359H at lower pH values (
Proton inventories reveal general-acid catalysis by PV 3Dpol Lys-359: Proton inventories for PV WT Lys-359 3Dpol were bowl-shaped, indicating transfer of more than one proton in the transition state of the phosphoryl transfer step of nucleotide incorporation (
A conserved active site Lys is a general acid catalyst in other polymerase classes: Sequence alignments and high resolution crystal structures permit identification of a conserved Lys residue occupying the same active site functional position as PV 3Dpol Lys-359 in nucleic acid polymerases from other classes. HIV-1 RT RdDp Lys-220 (
aKd,app is for ATP.
bSDKIE is solvent deuterium kinetic isotope effect, calculated as kobs in H2O/kobs in D2O at saturating [ATP].
cValues listed for T7 K631L are not actual Kd,app and kpol but rather represent lower limits for these values because it was impossible to achieve saturation of reactions with ATP. Likewise, the SKDIE was obtained under sub-saturating ATP concentrations.
dnd means not determined
As described above for PV 3Dpol RdRp (
A proton inventory for the T7 DdRp K631L polymerase could not be accomplished because, as described above, mutation of Lys to Leu in this enzyme abolished the ability to saturate the enzyme-primer/template binary complex with nucleotide experimentally.
In spite of wide diversity in the biological roles fulfilled by nucleic acid polymerases as agents of DNA and RNA replication and maintenance, the underlying active site architecture and mechanistic steps used to accomplish single nucleotide incorporation are highly conserved. In particular, the catalytic role during phosphoryl transfer has been assigned nearly exclusively to the two active site Mg2+ ions. These metal ions position reacting functional groups, facilitate deprotonation of the primer-terminus 3′-OH to create the attacking nucleophile and alleviate negative charge build-up during the transition state.
However, the finding that two transition-state proton transfers influence the rate chemistry reveals the importance of protonic catalysis in the phosphoryl transfer reaction of nucleic acid polymerases. Abstraction of the primer-terminus 3′-OH proton likely comprises one critical proton transaction. Numerous lines of experimental evidence described in this paper converge upon the conclusion that the other critical proton exchange is proton donation by a conserved active site positively charged amino acid residue, usually lysine, acting as a general-acid catalyst. Recent high-resolution x-ray crystal structures reveal that presence of this positively charged active site residue is absolutely conserved and, even more significantly, also conserved is its spatial position near the bridging oxygen between the α- and β-phosphorus atoms of the bound nucleotide triphosphate moiety, the ideal location to facilitate bond cleavage and pyrophosphate leaving group departure. Such specific conservation demands consideration of an extremely important function. Direct participation of a conserved active site basic residue in catalysis has been largely overlooked because 1) metal ion catalysis is clearly profoundly important, 2) a role in nucleotide binding was thought to explain presence of the conserved basic residue and 3) the ability to clearly interrogate the phosphoryl transfer step has been lacking in many model polymerase systems.
In PV 3Dpol RdRp, sequence alignments and molecular modeling identified Lys-359 as the putative active site general acid (
Studies with K359H and K359R 3Dpols are also supportive of a role as catalytic general acid for PV 3Dpol Lys-359. The pH-rate profile of K359H 3Dpol revealed this enzyme to be kinetically superior to the K359L enzyme up to approximately one pH unit beyond the pKa of the His imidazole moiety but to then converge toward the catalytic inefficiency of K359L 3Dpol at higher pHs (
In further support of a role as catalytic general acid for the conserved polymerase active site positively charged amino acid residue is extension of this conclusion described above for PV 3Dpol RdRp to other classes of polymerases. The combination of sequence alignments and high-resolution x-ray crystal structures permitted identification of a conserved active site lysine putative general acid in HIV-1 RT RdDp (K220) (
Proton inventory plots for WT HIV-1 RT RdDp, RB69 DdDp and T7 DdRp were clearly bowl-shaped, revealing transfer of more than one rate-influencing proton in the transition state of phosphoryl transfer (FIGS. 16Ai, 16Bi and 16C). In contrast, proton inventory plots for the HIV-1 RT K220L and RB69 K560L polymerases were linear, indicating that only one proton was being transferred in the transition state (FIGS. 16Aii and 16Bii). These proton inventory findings were similar to those described above for PV 3Dpol RdRp (
How nucleic acid polymerases enforce fidelity is currently highly controversial. Lack of ability to saturate T7 DdRp K631L reactions with nucleotide substrate experimentally appears consistent with the observation that in yeast, RNAP II nucleotide binding and catalysis may be coupled by an active site basic residue, His-1085. It has been further suggested that coupling of substrate selection and catalysis may enforce fidelity in RNA polymerases. The T7 RNAP O-helix, which bears Lys-631, may be functionally analogous to the yeast RNAP II trigger loop, which bears His-1085; both are mobile structural elements involved in nucleotide entry into the polymerase active site. The findings reported here that mutation of RB69 Lys-560 to Leu resulted in a 25-fold reduction in Kd,app (to go along with a 2500-fold reduction in kpol) with dATP as substrate (Table 8) suggests a significant role for this residue in nucleotide binding and a possible coupling with catalysis. However, lack of substantial changes in nucleotide binding upon mutation of the catalytic Lys to Leu in PV 3Dpol RdRp and HIV-1 RdDp, in spite of decreases of approximately two orders of magnitude in catalytic rate (Tables 7 and 8) suggest that coupling of substrate binding and catalysis may not be a feature of these polymerases.
The mechanistic model favored here involves donation of a proton from the active site general acid to the pyrophosphate leaving group to facilitate bond cleavage and leaving group departure. However, it should be noted that complete physical donation of the proton may not be a requirement for successful catalysis and that proton sharing may be sufficient. In summary, multiple lines of evidence described herein collectively argue that a conserved active site positively charged amino acid residue serves an important function as general acid catalyst in nucleic acid polymerases. Proton donation/sharing is the simplest, most fundamental means of dissipating energetically unfavorable negative charge development in enzyme active-site reactions. The findings reported here are important because they reveal a more complete picture of how the catalysis of phosphoryl transfer is accomplished by this universally important group of enzymes. While two-metal-ion catalysis is clearly profoundly important and provides the primary basis for acceleration of nucleotide incorporation, rates imparted by metal-ion-catalysis alone may be inadequate. The additional two-three orders of magnitude of catalytic-rate enhancement supplied by the active site general acid may be necessary to bring nucleotide incorporation rates to a level of biological sufficiency. Indeed, cell culture studies of 3Dpol K359H and K359R mutant polioviruses show substantially-slowed plaque formation by these variants, suggesting that infectivity and pathogenesis may prove to be attenuated due to inadequate rates of viral genome replication.
PV mutant viruses K359R and K359H were made and shown to replicate (
Analysis of the structural model for the RdRp from Norwalk virus (NV) in complex with primed template RNA and nucleotide showed that Lys374 of conserved structural motif D was located in the vicinity of the triphosphate moiety of incoming nucleotide (
Derivatives of each of the polymerases described above were produced that contained a leucine residue instead of the candidate lysine proton donor (methods described in Example 5). Leucine was selected instead of alanine to prevent water from binding to the site and serving as a proton donor. For all polymerases tested, the maximal rate constant for nucleotide incorporation (kpol) was reduced by 50-2000 fold for the Leu derivative relative to the wild-type, lysine-containing (wt) enzyme (Table 9). This observation is consistent with a role for the lysine in the rate-limiting step for nucleotide incorporation. For all of the wt polymerases employed here, chemistry is at least partially rate limiting (see Example 1). With the exception of the leucine derivative of T7 DdRp, the kpol values derive from experiments that included a saturating nucleotide concentration (5×Kd,app) (methods described in Example 5). In the case of the leucine derivative of T7 DdRp, the highest concentration of nucleotide attainable was two times the Kd,app (methods described in Example 5). This circumstance increases the error on the kpol value measured; however, our ability to reach conclusions for the leucine derivative is not affected because the observed 100-fold reduction in kpol value for this derivative is much greater than the error of the measurement.
Kinetic analysis of K560A RB69 DdDp is consistent with the existence of two forms of the enzyme. Referring to Table 10, K560A DdDp exhibits a reduced affinity for nucleotide substrate and slower rate of catalysis. The kinetic parameters for WT, K560L and K560A RB69 DdDp are shown in Table 10. The maximal rate constant for nucleotide incorporation (Kpol) was obtained for dAMP incorporation. Biphasic kinetics observed for K560A DdDP indicated two populations of polymerase-primer/template-deoxynucleotide complex. The results showed that one fraction (the faster fraction) of the K560A DdDp contains a water molecule that can serve as a proton donor; the slower fraction lacks the water molecule. Kinetic analysis results also showed that an ambiguous isotope effect for K560A DdDp was caused by multiple forms of the reactive complex.
A substantial difference was not observed in the apparent dissociation constant (Kd,app) for the nucleotide substrate measured for the leucine derivatives of PV RdRp or HIV RT (Table 9). This observation is consistent with residues in conserved, structural motif (F) of these enzymes functioning independently in triphosphate binding. In contrast, the Kd,app value for nucleotide substrate measured for the leucine derivatives of RB69 DdDp and T7 DdRp increased 25-fold and 167-fold, respectively (Table 9). This observation is consistent with structural studies which show that these lysines interact with the nucleotide substrate.
Unlike many other polymerases, the stability of PV RdRp on its nucleic acid primer-template is not affected by pH, permitting the pH dependence of the kinetics of nucleotide incorporation to be interpreted (methods described in Examples 1 and 5). Consistent with Lys359 of PV RdRp functioning as a proton donor during nucleotidyl transfer, the descending limb of the pH rate profile observed for wt PV RdRp was lost for the K359L derivative. The ionization observed for the K359L derivative likely reflects deprotonation of the 3′-hydroxyl. Theoretical studies of nucleotidyl transfer by rat DNA polymerase beta have suggested that the primer 3′-hydroxyl has a pKa in the 8-9.5 regime. Because the pKa values measured are kinetic pKa values, the lack of equivalence in the pKa value for the K359L derivative to any measured for wt enzyme likely reflects a change in the rate-determining step for nucleotidyl transfer (see Example 1). Chemistry should be solely rate limiting for the K359L derivative whereas a conformational-change step and chemistry are equally rate limiting for wt enzyme.
Theoretical studies of the free energy landscape for phosphoryl transfer almost always show proton transfer in the transition state that would manifest experimentally as a solvent deuterium kinetic isotope effect. The solvent deuterium kinetic isotope effect is defined as the ratio of kpol values obtained when the reaction is performed in H2O relative to values obtained when the reaction is performed in D2O (methods described in Example 5). The observation of a solvent deuterium kinetic isotope effect supports a proton-transfer reaction representing one of the microscopic steps reflected in the macroscopic rate constant, kpol. Each wt polymerase exhibited a solvent deuterium kinetic isotope effect of 2-5 (Table 9). This observation is consistent with chemistry contributing to the rate-limiting step(s) reported by the nucleotide-incorporation assay as observed for the wt enzymes (see Example 1). The solvent deuterium kinetic isotope effect measured for each leucine derivative was smaller than observed for the corresponding wt enzyme (Table 9). This observation could be interpreted in one of two ways. First, it is possible that the extent to which chemistry limits nucleotide addition by the leucine derivatives is less than occurs for the wt enzymes. Second, it is possible that fewer protons are being transferred. This latter possibility would be consistent with the candidate lysine residues contributing one proton transfer during nucleotide incorporation.
In order to count the number of protons being transferred during the nucleotidyl-transfer reaction, a proton-inventory experiment (methods described in Examples 1 and 5) was performed. This experiment obtains kpol values in the presence of different mole fractions of D2O (kn). The ratio of kn/k0 (k0 is kpol in H2O) is plotted as a function of mole fraction of D2O (n). If a single proton is transferred during the reaction, then the data should fit to a line. This experiment was performed for all leucine derivatives except for T7 DdRp derivative, which could not be saturated with nucleotide. The data obtained for all of the leucine derivatives evaluated fit well to a straight line, consistent with a single proton transfer reaction occurring during nucleotidyl transfer for these enzymes. In contrast, the data for the corresponding wt enzymes failed to fall on a line defined by kn/k0 values at n=0 and n=1, consistent with a model for more than one proton transfer reaction occurring during nucleotidyl transfer. More extensive analysis of the proton-inventory experiment for the wt enzymes, including statistical analysis, is described in Example 1, convincingly demonstrating two-proton transfer reactions in the transition state for nucleotidyl transfer by the wt enzymes employed here. These data provide compelling evidence for the use of an active-site amino acid residue as a general acid to protonate the pyrophosphate leaving group to facilitate nucleotidyl transfer.
All of the experiments described to this point have been performed in the presence of Mg2+ because this divalent cation is considered to be the biologically relevant cofactor. In the presence of Mg2+, chemistry is only partially rate limiting for PV RdRp at pH 7.510. In addition, the extent to which chemistry contributes to the observed rate constant for nucleotide addition changes as a function of pH (see Example 1). In the presence of Mn2+, however, chemistry is the primary determinant of the rate constant for nucleotide addition by PV RdRp from pH 6.0 to pH 9.0 (see Example 1). Experiments cannot be performed above pH 9.0 in the presence of Mn2+ due to precipitation of the metal hydroxide.
The activity of the K359L derivative of PV RdRp in the presence of Mn2+ was evaluated. The observed rate constant for nucleotide incorporation was reduced by 10-fold relative to wt enzyme to 1 s−1 without a substantial change in the apparent dissociation constant for nucleotide. The phosphorothioate effect observed for the K359L derivative (6±1) was essentially the same as measured for wt enzyme (7±1), consistent with chemistry remaining as the rate-limiting step. The solvent deuterium kinetic isotope effect observed for the K359L derivative of PV RdRp was 3±1, a value that is more than two-fold lower than the value of 7±1 observed for wt enzyme. These data are consistent with the loss of a proton-transfer reaction as observed in the presence of Mg2+. As expected, the dependence of the rate constant for nucleotide incorporation on pH was now essentially identical within the experimentally accessible pH range. These data are consistent with chemistry serving as the rate-limiting step for both the K359L derivative and the wt enzyme in the presence of Mn2+. The reduced rate constant for nucleotide incorporation and solvent deuterium kinetic isotope effect observed for the K359L derivative relative to wt enzyme provide additional support for Lys359 being employed as a general acid. It was thus concluded that PV RdRp, and likely the other polymerases, employ general acid catalysis regardless of the divalent cation cofactor employed.
In order to test further the use of a general acid for nucleotidyl transfer, a PV RdRp derivative with Lys359 changed to arginine was produced. In addition to testing the model for general acid catalysis, the ability of an arginine to function at this position as a general acid would be consistent with the use of arginine as a general acid in X-family polymerases like DNA polymerase beta (polβ). An arginine at this position should produce a derivative that functions better than the K359L derivative but still worse than wt enzyme, exhibits unique pH dependence relative to wt enzyme and shows two proton-transfer reactions during nucleotidyl transfer. At pH 7.5, the K359R derivative bound nucleotide well and turned over ˜3-fold faster than the K359L derivative and 20-fold slower than wt enzyme. At pH 10, the kpol value for the K359R derivative had risen to within 2-fold of the maximum value observed for wt enzyme at pH 8.5 and continued to rise. In contrast, the value for the wt enzyme was falling at pH 10. This difference can be explained by the ΔpKa value of 2 between lysine and arginine. The descending limb of the pH rate profile for the K359R derivative should appear at pH values greater than 11. Unfortunately, experiments cannot be performed at pH values higher than 10 because of the insolubility of metal hydroxide. Finally, the solvent deuterium kinetic isotope effect was 3.4±0.1, essentially the same as wt enzyme (Table 9) and proton inventory data fit well to a two proton model. These data strongly support the use of a general acid for nucleotidyl transfer and suggest that both lysine and arginine can serve this function.
Multi-subunit RNA polymerases contain a histidine in the trigger loop that may serve as the general acid for these enzymes. Histidine can function at this position of the PV RdRp. A 10-fold reduction in the kpol value was observed relative to wt enzyme with only a modest (50%) increase in the Kd,app value for nucleotide at pH 7.5. The pKa value for the ascending limb of the pH rate profile for the K359H derivative was identical to wt enzyme; the descending limb was absent, consistent with a pKa value of 6-7 for histidine.
The general acid of all polymerases appears to be associated with exceptionally dynamic structural elements, and the movement of these elements have been implicated in translocation. A comparison of enzyme-primer/template complexes to enzyme-primer/template-nucleotide complexes revealed movement of the general acid in response to nucleotide binding. It is possible that the protonation-deprotonation cycle of the general acid during catalysis toggles helix O (and its structural equivalents) between the closed (protonated) and open (deprotonated) states, thus driving the translocation reaction. Consistent with this possibility is the observation that changing the general acid to Leu leads to substantial reductions in processivity for all nucleic acid polymerases employed in this study. Severe defects to processive incorporation of nucleotides was observed for leucine derivatives and are consistent with defects to translocation arising from loss of the general acid.
Derivatives of all polymerases: PV RdRp, RB69 DdDp, T7 DdRp and HIV RT were prepared by using standard recombinant DNA protocols as described in Example 5. All polymerases were expressed in E. coli and purified as described in Example 1 above. Briefly, cells were transformed with the appropriate plasmid and these cells were used to produce an inoculum for large-scale growth. Gene expression was induced during exponential growth by addition of isopropyl-β-D-thiogalactopyranoside. Induced cells were lysed in appropriate buffers and the enzymes were purified to apparent homogeneity by using standard column chromatography resins and protocols as described in Example 5.
Nucleotide incorporation experiments were performed as described in Examples 1 and 5. SDKIE and PI experiments were performed by monitoring pre-steady state nucleotide incorporation either using the chemical quench flow or stopped-flow instruments. Enzymes, substrates and buffers were prepared in 100% water or 100% D2O followed by mixing at the appropriate ratio to obtain 0, 25, 50, 75 or 100% D2O. Deuterated glycerol was used in all solutions in D2O. The pD was used instead of pH for the solutions in D2O and was adjusted according to pD=pH+0.4. SDKIE values were calculated as the ratio of kpol values obtained in H2O divided by that obtained in D2O. PI plots consisted of kn/kH2O as a function of n where kn is the observed rate constant for nucleotide incorporation at a particular mole fraction of D2O, kH2O is the observed rate constant for nucleotide incorporation in H2O and n is the solvent mole fraction of D2O. Proton-inventory data were fit to the modified Gross-Butler equation for a two-proton-transfer model (Venkatasubban, K. S. & Schowen, R. L. CRC Crit Rev Biochem vol. 17, 1-44:1984) or for a one-proton-transfer model (Venkatasubban, K. S. & Schowen, R. L. CRC Crit Rev Biochem vol. 17, 1-44:1984).
Observed rate constants (kobs) for nucleotidyl transfer at various concentrations of nucleotide were obtained by fitting product-versus-time data to an equation defining a single exponential. Values for Kd,app and kpol were obtained by fitting kobs-versus-[NTP] data to an equation defining a hyperbola. Data were fit by nonlinear regression using the program KaleidaGraph (Synergy Software, Reading, Pa.). Specific equations employed are described in Example 5.
[γ-32P]ATP (>7000 Ci/mmol) was purchased from MP Biomedical; [α-32P] ATP (3000 Ci/mmol was from Perkin Elmer-New England Nuclear; nucleoside 5′-triphosphates, (ultra pure solutions), were from GE-Amersham Pharmacia Biotech, Inc; D2O was purchased from Isotec (Miamisburg, Ohio); deuterated glycerol was from Cambridge Isotope Laboratories (Andover, Mass.); all RNA oligonucleotides were from Dharmacon Research, Inc. (Boulder, Col.); all DNA oligonucleotides were from Integrated DNA Technologies (Coralville, Iowa); T4 polynucleotide kinase was from New England Biolabs, Inc; all other reagents were of the highest grade available from Sigma, Fisher or VWR.
Introduction of the K359L mutation into the poliovirus (PV) 3D gene required overlap extension PCR by using oligos 1-4 (Table 11) and the expression vector, pET26Ub-PV 3D1, as the template. First, two PCR fragments were amplified using oligonucleotides 1, 3 and 2, 4 (Table 11) encoding the mutation K359L. These two fragments were then used as templates for the amplification of a new fragment using oligonucleotides 1 and 4 (Table 11). The PCR product, 3D K359L, was then cloned into pET26Ub-PV-3D using KpnI and BamHI sites. Introductions of the K359R and K359H mutations were performed essentially as described above for K359L, except oligonucleotides 5-8 were used encoding the mutations K359R and K359H. The QuikChange® site-directed mutagenesis kit (Stratagene) was employed to introduce the mutation, K220L, into HIV RT-coding sequence with oligonucleotides 9 and 10 (Table 11). The QuikChange® site-directed mutagenesis kit (Stratagene) was employed to introduce the mutation, K560L, into RB69 DdDp-coding sequence with oligonucleotides 11 and 12 (Table 11). The QuikChange® site-directed mutagenesis kit (Stratagene) was employed to introduce the mutation, K631 L, into T7 DdRp-coding sequence with oligonucleotides 13 and 14 (Table 11). Wild-type (wt) and K359H, K359L and K359R 3Dpol derivatives were expressed and purified as described above for wild-type. HIV RT wt and K220L polymerases were expressed and purified as described previously (Le Grice, S. F. & Gruninger-Leitch, Eur J Biochem vol. 187:307-314, 1990).
RB69 wt and K560L polymerases were expressed in BL21 (DE3) cells. Frozen cells were thawed and suspended in lysis buffer (100 mM KPO4 pH 8.0, 20% glycerol, 20 mM DTT, 0.5 mM EDTA pH 8.0, 2.8 μg/mL pepstatin A, 2.0 μg/mL leupeptin) and lysed by passing through a French press at 1000 psi. Immediately after lysis, NP-40 was added to 0.1% (v/v) and PMSF was added to 2 mM. Polyethyleneimine (PEI) was added drop wise at 4° C. to a final concentration of 0.25% (v/v) and stirred for 30 mM. This suspension was then centrifuged at 75,000×g for 30 min at 4° C. The supernatant was decanted and collected, and ground ammonium sulfate powder was slowly added to 60% saturation at 4° C. and stirred for 30 min. The ammonium sulfate precipitate was suspended in Buffer A (50 mM Tris pH 8.0, 20% glycerol, 10 mM DTT, 0.1% NP-40) and dialyzed overnight against 1 L Buffer A containing 75 mM NaCl using a 12-14,000 Da MWCO membrane. After dialysis, the sample was diluted in Buffer A to a final NaCl concentration of 50 mM. The sample was loaded onto a phosphocellulose column (approximately 1 mL bed volume/25 mg total protein) at a flow rate of 1 mL/min. The column was washed with ten column volumes of Buffer A containing 50 mM NaCl and the protein was eluted with Buffer A containing 150 mM NaCl. Fractions were pooled based upon their purity on 8% SDS-PAGE gels. The pooled fractions were diluted in Buffer A to a final salt concentration of 50 mM. The sample was loaded onto a Q-Sepharose column (1 mL bed volume/40 mg of protein) at 1 mL/min. The column was washed with ten column volumes of Buffer A containing 50 mM NaCl and the protein eluted with Buffer A containing 150 mM NaCl. Protein-containing fractions were pooled as before. The protein was concentrated using a small (0.5 mL) Q-Sepharose column. Loading and washing of the column was as described above. Elution was performed with Buffer A containing 500 mM NaCl.
Purification of T7 wt and K631L polymerases was done using the same protocol as for the RB69 enzymes with the following modifications: 1. Ammonium sulfate was added to 40% saturation. 2. The phosphocellulose column was eluted using a linear gradient (6 column volumes) from 50 mM-700 mM NaCl in Buffer A. 3. The Q-Sepharose column was loaded and washed, and the protein eluted using a linear gradient (6 column volumes) from 50 mM-400 mM NaCl in Buffer A. Protein-containing fractions were pooled as before. Additional steps to concentrate the protein were not necessary.
Pre-steady state nucleotide incorporation stopped-flow experiments were performed using a Model SF-2001 stopped-flow apparatus (Kintek Corp., Austin, Tex.) equipped with a water-bath. All reactions were performed at 30° C. 3Dpol was diluted into enzyme buffer (50 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol, 60 μM ZnCl2, and 20% glycerol) immediately prior to use. The volume of enzyme added to any reaction was always equal to one-twentieth the total reaction volume. Reactions were performed in MTCN buffer by incubating 1 μM enzyme with 1 μM sym/sub RNA primer-template3 (0.5 μM duplex) in 1 mM HEPES pH 7.5, 10 mM 2-mercaptoethanol, 60 μM ZnCl2 and 5 mM MgCl2 at room temperature for 3 minutes, allowing equilibration to 30° C. and then rapidly mixing with ATP in a solution containing 2×MTCN buffer, 10 mM 2-mercaptoethanol, 60 μM ZnCl2 and 5 mM MgCl2 at the pH or pD indicated for individual experiments. 1×MTCN buffer is: 50 mM MES, 25 mM TRIS, 25 mM CAPS and 50 mM NaCl. For reactions with a final ATP concentration higher than 1 mM, the amount of free Mg2+ was kept constant by increasing the amount of MgCl2 in the reaction to 5 mM plus the ATP concentration above 1 mM. For example, in a reaction containing 5 mM ATP, the final Mg2+ concentration was adjusted to 9 mM. Values for kpol in pH-rate profiles were obtained by first determining kobs, the observed first order rate constant for single nucleotide incorporation, across a range of [ATP] and then fitting plots of kobs dependence on [ATP] to eq 2 (see below) to obtain estimates of Kd,app and kpol. The excitation wavelength used was 313 nm. Fluorescence emission was monitored by using a 370 nm cut-on filter (model E370LP, Chroma technology corp., Rockingham, Vt. Rate constants for nucleotide incorporation were obtained by using the data analysis software of the instrument.
Pre-steady state nucleotide incorporation rapid mixing/quenching experiments were performed by using a Model RQF-3 chemical-quench-flow apparatus (KinTek Corp., Austin, Tex.). 3Dpol-sym/sub complexes and all buffer solutions were prepared in the same way as for the stopped-flow experiments described above. Reactions were quenched by addition of HCl to a final concentration of 1 M. Immediately after the addition of HCl, the solution was neutralized by addition of 1 M KOH and 300 mM Tris base (final concentration). Rapid-quench product analysis was done after fractionation on denaturing PAGE gels. An equal volume of loading/quenching dye (85% formamide, 0.025% bromophenol blue, and 0.025% xylene cyanol, 90 mM EDTA) was added to 10 μl of the quenched reaction mixtures and heated to 70° C. for 2-5 min prior to loading 5 μl on a denaturing 23% polyacrylamide gel containing 1×TBE (2 mM EDTA, 89 mM boric acid, 87 mM Tris) and 7 M urea. Electrophoresis was performed in 1×TBE at 90 W. Gels were visualized by using a phosphorimager and quantified by using the ImageQuant software (Molecular Dynamics).
PV 3Dpol SDKIE and PI experiments were accomplished by monitoring pre-steady state nucleotide incorporation in the stopped-flow instrument. Enzymes, substrates and buffers were prepared in 100% water or 100% D2O followed by mixing at the appropriate ratio to obtain 0, 25, 50, 75 or 100% D2O. Deuterated glycerol was used in all solutions in D2O. The pD was used instead of pH for the solutions in D2O and was adjusted according to pD=pH+0.44. SDKIE values were calculated as the ratio of kpol values obtained in H2O divided by that obtained in D2O. PI plots consisted of kn/kH2O as a function of n where kn is the observed rate constant for nucleotide incorporation at a particular mole fraction of D2O, kH2O is the observed rate constant for nucleotide incorporation in H2O and n is the solvent mole fraction of D2O. Proton inventory experimental data were fit to equations 5 (two-proton transfer model) or 6 (one- proton transfer model).
HIV-RT pre-steady state nucleotide incorporation experiments were done in the chemical quench flow instrument at 37° C. essentially as described in Gotte et al., (J Virol. Vol 74:3579-3585, 2000) with some modifications: 2 μM 32P-labeled DNA primer was annealed with 2.2 μM DNA template, and incubated with 4 μM RT in a solution containing no metals. The reaction was started by addition of 200 μM dATP in reaction buffer containing 10 mM MgCl2. After mixing, reactant concentrations were reduced by 50%. Product analysis was performed essentially as described for PV 3Dpol with the following modifications. 1. Unlabeled DNA primer was added to the loading/quenching dye at a concentration 100-fold that of the labeled primer. A 15% polyacrylamide gel containing 40% formamide was used. SDKIE and PI data were evaluated as described above for PV 3Dpol.
RB69 wt K560 pre-steady state experiments were done in the stopped-flow instrument at 25° C. essentially as described in Example 1. The assays were done in 1×MTCN pH 7.5, 10 mM MgSO4, 1 mM dATP with 1 μM RB69 enzyme and 150 nM primer-template substrate. RB69 K560L experiments were done in the chemical quench flow instrument with reaction components as described above for WT except using 5 mM dATP and 14 mM MgSO4. The solvent deuterium isotope effect and proton inventory experiments were performed and evaluated essentially as described above for PV 3Dpol.
T7 wt K631 pre-steady state nucleotide incorporation experiments were done in the stopped-flow instrument at 30° C. essentially as described above for poliovirus 3Dpol with minor modifications as described in Example 1. One μM RNA primer was annealed to 1 μM DNA template, and then incubated with 1.6 μM T7 for 10 minutes at room temperature in 1 mM Hepes pH 7.5, 10 mM BME, and 5 mM MgCl2.The reaction was started by mixing the T7-primer-template complex with 3 mM ATP in 2×MTCN pH 7.5, 10 mM BME, 9 mM MgCl2 and 200 mM NaCl. The solvent deuterium isotope effect and proton inventory experiments were performed and evaluated essentially as described above for poliovirus 3Dpol. T7 K631 L pre-steady state nucleotide incorporation experiments were done in the stopped-flow instrument with the goal of obtaining estimates of kpol, the maximal rate constant for nucleotide incorporation and Kd,app, the apparent binding constant for dATP, as described above for PV 3Dpol. However complete saturation with ATP was not achievable because of precipitation within the reaction mixture above [ATP] of 75 mM. Proton inventory experiments were therefore not performed with T7 K631L due to inability to saturate the reactions with ATP.
Time courses at fixed nucleotide concentrations were fit to the single exponential function
[product]=A×exp(−kobs×t)+C (1)
where A is the amplitude of the burst, kobs is the observed first-order rate constant describing the burst, t is the time, and C is a constant. The apparent binding constant (Kd,app) and maximal rate constant for nucleotide incorporation (kpol) were determined using the equation:
The pKa values for the pH dependence of kpol were obtained by fitting to a model describing two ionizable groups9:
or one-ionizable group9
where kind is the pH-independent rate constant.
Proton-inventory data were fit to the modified Gross-Butler equation for a two-proton-transfer model:
or for a one-proton-transfer model:
where kn is the observed rate constant at the different percentages of D2O, kH2O is the observed rate constant in water, n is the mole fraction of D2O and Φ is the inverse of the isotope effect for each ionizable group. Data were fit by nonlinear regression using the program KaleidaGraph (Synergy Software, Reading, Pa.). Equilibrium and rate constants reported were determined independently at least twice. Error was propagated through products and quotients by using equation 7:
where x and y are the primary data, z is the product or quotient of x and y, and Δx, Δy and Δz are the corresponding error values (Aikens et al., Principles and techniques for an integrated chemistry laboratory, (Waveland Press, Prospect Heights, Ill. 1984).
Challenge experiments were performed in mice. In the first experiment, it was found that PV encoding the K359H mutation confers partial protection to lethal doses of wild-type PV. In this experiment, 15 cPVR mice were immunized, intramuscularly (IM), with 1×105 plaque forming units (PFU) of 3D-P3565-K359H PV. Four weeks after immunization, all animals were challenged IM with 5LD50, 5×106 PFU, of wild-type PV and monitored for signs of paralysis. The percent protection is shown in the table below.
Another challenge experiment was performed which conclusively showed that in addition to PV encoding the K359H mutation, a PV encoding the K359R mutation is an effective vaccine. An illustration of the vaccination protocol used in this challenge experiment is shown in
Any improvement may be made in part or all of the compositions and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Although modified polymerases are described herein having a substitution at a lysine residue in the active site of the polymerase, a modified polymerase as described herein includes mutations, deletions, and substitutions of proton-accepting and proton-donating amino acid residues. Any molecule that changes the activity (e.g., rate of replication, fidelity, etc.) of the polymerase can be utilized. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.
The present application is a continuation-in-part application of U.S. patent application Ser. No. 11/963,930 filed Dec. 24, 2007 that claims the priority of U.S. provisional patent application No. 60/871,570 filed Dec. 22, 2006, and U.S. provisional patent application No. 60/892,086 filed Feb. 28, 2007. The present application also claims priority from U.S. provisional patent application No. 61/144,334 filed Jan. 13, 2009. Applicants herein claim the benefit of all these prior patent applications, the entire contents of which are incorporated herein by reference.
This invention was made with government support under grant number AI45818, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60871570 | Dec 2006 | US | |
| 60892086 | Feb 2007 | US | |
| 61144334 | Jan 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11963930 | Dec 2007 | US |
| Child | 12686200 | US |
