English:
[0003] The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art in the present invention.
[0004] PCR is likely the most widely used method in modern molecular biology and biotechnology, and is rapidly being applied to genetic testing, diagnostics, forensics and biodefense. Kolmodin, L.A., et al., Nucleic Acid Protocols, 569-580 (2000); Budowle, B., et al., 301 Science, 1852-53 (2003); Y. Sato, et al., 5 (Suppl. 1) Legal Medicine, S191-S193 (2003); Saldanha, J., et al., 43 J. Medical Virol., 72-76 (1994); Dahiya, R., et al., 44 Biochemistry and Molecular Biology International, 407-15 (1998); and Elnifro, E.M., et al., 13 Clin. Microbiol. Rev., 559-70 (2000). PCR is described in U.S. Pat. No. 4,683,195 and 4,683,202. In each cycle of the PCR amplification process there are typically several steps. The double-stranded DNA target sequence is first thermally denatured at elevated temperatures (~95°C). The first occurrence of denaturation is referred to herein as the "initial denaturation step." This is followed by annealing of a synthetic oligonucleotide primer to each strand at lower temperatures (~60°C). These forward and reverse oriented oligonucleotide primers are then each extended from their 3' termini at an elevated temperature (~70°C) by a thermally stable, magnesium ion-dependent, DNA polymerase which incorporates 5'-deoxynucleotide triphosphates (dNTPs) and generates pyrophosphate (PPi), as depicted in the top portion of Figure 1 for the forward oligonucleotide primer.
[0005] The utility of PCR is driven by its ability to rapidly provide target amplifications of ~106- fold as well as high specificity, which depends in part on the specificity of oligonucleotide primer hybridization. Oligonucleotide primer sequences and length are therefore designed to hybridize to only the intended target sequence, at the temperatures used for annealing. However, PCR amplification reactions are typically prepared over a period of minutes or hours at ambient room temperatures which are well below the temperature range needed to ensure the specificity of oligonucleotide primer hybridization. Under such less stringent sample preparation conditions, the oligonucleotide primers may bind non- specifically to other sequences having substantial non-complementarity and potentially initiate the synthesis of undesired extension products, which can be amplified along with the target sequence. As has been discussed by Chou, Q., et al., amplification of non-specific sequences by this "mis-priming" can compete with amplification of the desired target sequences, and can therefore significantly decrease the efficiency of the amplification of the desired sequence, especially for low-copy number targets. Chou, Q., et al., 20 Nucleic Acids Res., 1717-23 (1992).
[0007] While the aforementioned problems due to mis-priming and primer dimer formation can be encountered in all applications of PCR, these issues can be particularly challenging for high-sensitivity analytical PCR schemes, such as those used for detection of blood-borne infectious agents (Saldanha, J., et al. and Elnifro, E.M., et al.), biohazardous microbes (Budowle, B., et al.), defective or cancerous genes (Dahiya, R., et al), and forensics (Budowle, B., et al. and Y. Sato, et al.). In addition, there is a much greater chance for formation of spurious amplification products in multiplex PCR. Markoulatos, P., et al., 16 J. of Clin. Laboratory Analysis, 47-51 (2002). In reverse transcriptase PCR (RT-PCR), the most sensitive means for detection of a target RNA sequence is to use a gene-specific oligonucleotide primer in the RT step. Zhang, J., et al., 337 Biochem. J., 231-41 (1999); Lekanne Deprez, R.H., et al., 307 Analytical Biochem., 63-69 (2002); and Bustin, S.A., et al., 15 J. of Biomolecular Techniques, 155-66 (2004). In view of the importance of these high- sensitivity applications requiring high specificity to avoid serious, adverse consequences of "false negatives" and "false positives", it is critical to have reagents and protocols which provide assays that are functionally free of artifacts due to mis-prirning and primer dimer formation.
[0008] A number of general strategies have been investigated for reducing non-specific PCR amplification based on the so-called "hot start" process which aims at impairing undesired amplification due to mis-priming and oligonucleotide primer dimer formation under low-stringency conditions at room temperature during sample preparation. PCR amplification subsequently begins when the amplification reaction mixture reaches high- stringency, "hot" temperatures to "start" polymerase-mediated extension of oligonucleotide primers hybridized only to target sequences. Thus temperature is used to trigger enzymatic extension of the oligonucleotide primers only at elevated temperatures when the stringency of primer/target hybridization conditions are optimal for specificity.
[0009] These general strategies for "hot start" include the use of (1 ) temperature-sensitive materials, such as waxes as barriers or sequestrants to control mixing of the reagents (Q. Chou, et al, and Tanzer, L.R., et al., 273 Anal. Biochem., 307-310 (1999)); (2) oligonucleotide aptamers (Dang, C, et al., 264 J. MoI. Biol., 268-78 (1996)) or antibodies (Eastlund, E., et al., 2 LifeScience Quarterly, 2-5 (2001) and Mizuguchi, H., et al., 126 J. Biochem (Tokyo), 762-68 (1999)) that inhibit the function of DNA polymerases; (3) use of a second thermostable enzyme, such as pyrophosphatase (Clark, D.R., et al., International Patent Application No. WO 2002088387) to remove suppression by added pyrophosphate (PPi); (4) chemically modified polymerases with hydrolytically reversible reagents, such as citraconic acid-modified lysine (Birch, D.E., et al., U.S. Patent No. 5,773,258) in AmpliTaq Gold (Moretti, T., et al., 25 BioTechniques, 716-722 (1998) and Saldanha, J., et al.) and (5) oligonucleotide primer sequence constructs that disfavor low-temperature mis-priming, such as competitor sequences (Puskas, L.G., et al, 5 Genome Research, 309-31 1 (1995) or "touch- up and loop-incorporated oligonucleotide primers " (TULIPS-PCR) (Ailenberg, M., et al., 29(5) BioTechniques, 1018-23 (2000)).
[0012] In particular embodiments, the modification group is attached creating a phosphotriester (PTE) internucleotide linkage. The modification impairs DNA polymerase mediated oligonucleotide primer extension prior to the initial incubation period at an elevated temperature of amplification such as in PCR. Oligonucleotide primers made of nucleotides and nucleosides of the present invention have two states. First, the oligonucleotide primer is in an inactive state due to the presence of a modification group until the initial denaturation temperature is reached, often 95°C. Upon reaching the initial denaturation temperature, the oligonucleotide primer becomes active by thermally induced intramolecular fragmentation which converts the oligonucleotide to the second state. This second state of the oligonucleotide primer is the corresponding unmodified oligonucleotide primer which has an active state phosphodiester bond and is extendable by polymerase. Partial or complete dissociation of the modification group preferably occurs after incubation at approximately 95°C for approximately 2-10 minutes. In certain embodiments, dissociation of the modification group from the oligonucleotide primer occurs in respect to temperature and does not require enzymes, chemicals, or amplification reaction conditions such as pH. Phosphotriester linkages are described in Miller, et al., 93(24) J. Am. Chemical Society, 6657-65 (1971); Zon, et al., 6(2) Protein J., 131-45 (1987); and Koziolkiewicz, M. and WiIk, A., Protocols for Oligonucleotides and Analogs (1993).
[0018] In a preferred embodiment, the modification group of Formula Ib is 4-methylthio- 1 -butyl, shown below:
[0019] In one embodiment, modification groups in accordance with the invention provide compounds of Formula Ic:
[0020] In a preferred embodiment, the modification group of Formula Ic is 3-(N-tert- butylcarboxamido)-! -propyl, shown below:
[0023] In another embodiment, modification groups in accordance with the invention provide compounds of Formula II:
[0025] In another embodiment, modification groups in accordance with the invention provide compounds of Formula III:
[0026] In another embodiment, modification groups in accordance with the invention provide compounds of Formula IV:
[0027] In one embodiment of a compound of Formula IV wherein R5 and R6 cooperate to form a ring the modification group is methoxyrnethyl-cyclohex-l,3-yl-ethyl, shown below:
[0035] In certain embodiments of the methods and compositions provided herein, amplification is by reverse transcriptase (RT). One of ordinary skill in the art is familiar with the conditions needed to perform RT. The denaturation and extension temperatures may vary and depend on the reverse transcriptase enzyme being used. For example, denaturation and extension by reverse transcriptase occurs at about 37-700C. Modification groups provided herein can be selected for use according to a desired dissociation condition, such as a particular temperature, or particular temperature in conjunction with a particular time frame. For example, modification groups can be used for RT such that the group dissociates at 37- 700C; preferably 37-60 0C; preferably at about 500C; preferably at about 420C, and preferably at about 37°C. In other embodiments, the modification group dissociate within 0.1-60 minutes; preferably 1-30 minutes; preferably 1-15 minutes; preferably 1-10 minutes; preferably 0.1-5 minutes at the desired temperature.
[0036] In other embodiments of the methods and compositions provided herein, amplification can be one or more than one amplification reaction in a single reaction mixture, such as reverse transcriptase polymerase chain reaction (RT-PCR) or other examples, such as those described in U.S. Patent No. 6,399,304. In certain embodiments, reagents needed for more than one enzymatic reaction may be added and carried out in a single reaction vessel. In such reactions, various combinations of modified and/or unmodified oligonucleotides may be used. For example, modified oligonucleotides could be used for one, one or more, two or more, three or more, five or more, ten or more of the reactions. In one instance, an oligonucleotide with a modification group could be used for reverse transcriptase that dissociates at temperatures ideal for RT, such as 42°C and another oligonucleotide with a different modification group could be used for PCR that dissociates at temperatures ideal for PCR, such as 95°C. By providing such a combination, the PCR primers do not interfere or substantially interfere with the RT reaction. Alternatively, the RT primers may be unmodified primers and the PCR primers may be modified with a group that dissociates at temperatures ideal for PCR, such as 95°C. In yet further embodiments, the subsequent PCR includes multiplex PCR, real-time PCR, or quantitative PCR.
[0039] As used herein, "nucleic acid," "nucleotide sequence," or "nucleic acid sequence" refer to nucleotides, oligonucleotide, polynucleotide, or any fragment thereof and to naturally occurring or synthetic molecules. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, or to any DNA-like or RNA-like material. An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose. Additional alternative nucleic acid backbones suitable for the invention include but are not limited to phosphorothioate, phosphoroselenoate, alkyl phosphonate, aryl phosphonate, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA) and boronate. RNA may be used in the methods described herein and/or may be converted to cDNA by reverse-transcription for use in the methods described herein.
[0040] As used herein, the term "modified oligonucleotide" refers to an oligonucleotide primer having at least one modification group. Modified oligonucleotides include, for example, an oligonucleotide containing a modified nucleoside, an oligonucleotide containing a modified internucleotide linkage, or an oligonucleotide having any combination of modified nucleosides and internucleotide linkages (even if a natural nucleoside is present in the oligonucleotide chain). Oligonucleotides whose nucleosides are connected via modified internucleotide linkages can be found, for example, in Waldner et al., 6 Bioorg. Med. Chem. Letters, 2363-66 (1996), which describes the synthesis of oligonucleotides containing various amide internucleotide linkages.
[0043] As used herein, the term "terminus" with respect to oligonucleotide refers to the nucleotides at or near the 3' or 5' end of an oligonucleotide. Preferably the terminus of a nucleotide includes the terminal 6 nucleotides, more preferably the terminal 5 nucleotides, more preferably the terminal 4 nucleotides, more preferably the terminal 3 nucleotides, more preferably the teπninal 2 nucleotides, or more preferably the terminal nucleotide.
[0045] As used herein, the term "internucleotide linkage" refers to the bond between two nucleotides of an oligonucleotide primer.
[0046] As used herein, the term "target nucleic acid sequence" or "nucleic acid target" refers to a sequence of nucleotides to be identified.
[0048] As used herein, the term "heat induction" refers to a process by which the oligonucleotide primer modification is removed from the oligonucleotide primer generating an active state oligonucleotide primer by applying heat thus making it extendable by polymerases.
[0049] As used herein, the term "hot start" refers to a nucleic acid amplification reaction wherein polymerase induced nucleic acid amplification is impaired until the reaction reaches an initial temperature above the extension temperature of the enzyme. In hot start PCR applications, initial temperatures reach between about 80-1050C; or until the amplification reaction reaches an initial temperature of at least about 800C, or until the amplification reaction reaches an initial temperature of at least about 85°C, or until the amplification reaction an initial temperature of at least about 900C, or until the amplification reaction reaches an initial temperature of at least about 95°C. The term "hot start" is well known in the art and there are a number of methods known to impair amplification such as modified polymerases, oligonucleotides with secondary structures impairing hybridization and reagents contained in temperature sensitive barriers such as wax. In a preferred embodiment, hot start amplification is caused by heat induced removal of an oligonucleotide modification group.
[0056] As used herein, the term "stringent hybridization condition" refers to hybridization conditions which do not allow for hybridization of two nucleic acids which are not completely complementary.
[0057] As used herein, the term "sample" or "test sample" refers to any liquid or solid material believed to comprise nucleic acids of interest. A test sample may be obtained from any biological source (i.e., a biological sample), such as cells in culture or a tissue sample or synthetically produced including a chemically synthesized template.
[0058] As used herein, the terms "complement" "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) according to standard Watson/Crick pairing rules. A complement sequence can also be a sequence of RNA complementary to the DNA sequence or its complement sequence, and can also be a cDNA. For example, for the sequence "5'-A- G-T-3"' is complementary to the sequence "3'-T-C-A-5\" Certain nucleotides not commonly found in natural nucleic acids may be included in the nucleic acids described herein; these include, modified nucleosides, nucleotides, and nucleic acids, such as inosine, 7- deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA). Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.
[0059] Complementarity may be "partial" in which only some of the nucleotide bases of two nucleic acid strands are matched according to the base pairing rules. Complementarity may be "complete" or "total" where all of the nucleotide bases of two nucleic acid strands are matched according to the base pairing rules. Complementarity may be absent where none of the nucleotide bases of two nucleic acid strands are matched according to the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids. Either term may also be used in reference to individual nucleotides, especially within the context of polynucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity, or lack thereof, to a nucleotide within another nucleic acid strand, in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid strand.
[0064] As used herein, the terms "nucleoside analogs," "modified nucleosides," or "nucleoside derivatives" include synthetic nucleosides as described herein. Nucleoside derivatives also include nucleosides having modified base moieties, with or without protecting groups. Such analogs include, for example, deoxyinosine, 2,6-diaminopurine-2'- deoxyriboside, 5-methyl-2'-deoxycytidine, and the like. The base rings most commonly found in naturally occurring nucleosides are purine and pyrimidine rings. Naturally occurring purine rings include, for example, adenine, guanine, and N6 -methyladenine. Naturally occurring purine rings include, for example, cytosine, thymine, and 5- methylcytosine. The compounds and methods of the present invention include such base rings and synthetic analogs thereof, as well as unnatural heterocycle-substituted base sugars, and even acyclic substituted base sugars. Moreover, nucleoside derivatives include other purine and pyrimidine derivatives, for example, halogen-substituted purines (e.g., 6- fluoropurine), halogen-substituted pyrimidines, N6-ethyl adenine, N6-(alkyl)-cytosines3 5- ethylcytosine, and the like. Nucleoside derivatives and analogs encompass a wide variety of modifications, such as those described in U.S. Pat. No. 6,762,298.
[0069] As used herein, the term "alkenylaryl" refers to alkenyl -substituted aryl groups and "substituted alkenylaryl" refers to alkenylaryl groups further bearing one or more substituents as set forth herein.
[0075] As used herein, the term "alkynylaryl" refers to alkynyl-substituted aryl groups and "substituted alkynylaryl" refers to alkynylaryl groups further bearing one or more substituents as set forth herein.
[0076] As used herein, the term "alkoxy" denotes the group -OR0, where R° is lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heteroalkyl, heteroarylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, or substituted cycloheteroalkyl as defined.
[0077] As used herein, the term "lower alkoxy" denotes the group -ORd, where Rd is lower alkyl.
[0079] As used herein, the term "alkylcarbonylamino" denotes the group -NReC(O)Rf, wherein Re is optionally substituted alkyl, and Rf is hydrogen or lower alkyl.
[0080] As used herein, the term "alkylsulfinyl" denotes the group -S(O)R8, wherein Rg is optionally substituted alkyl.
[0085] As used herein, the term "alkynylene" refers to divalent straight or branched chain hydrocarbyl groups having at least one carbon — carbon triple bond, and typically having in the range of about 2-12 carbon atoms, preferably 2-8 carbon atoms, and "substituted alkynylene" refers to alkynylene groups further bearing one or more substituents as set forth herein.
[0087] As used herein, the term "substituted amido" denotes the group -C(O)NRkRk', wherein Rk and Rk are independently hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, provided, however, that at least one of Rk and Rk is not hydrogen. RkRk in combination with the nitrogen may form an optionally substituted heterocyclic or heteroaryl ring.
[0089] As used herein, the term "amino" or substituted amine denotes the group -NRnRn , where Rn and Rn may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl as defined herein, acyl or sulfonyl. A "divalent amine" denotes the group -NH-. A "substituted divalent amine" denotes the group -NR- wherein R is lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonyl or substituted sulfonyl.
[0090] As used herein, the term "substituted amino" or "substituted amine" denotes the group -NRPRP , wherein Rp and Rp> are independently hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonyl or substituted sulfonyl, provided, however, that at least one of Rp and Rp is not hydrogen. RPRP in combination with the nitrogen may form an optionally substituted heterocyclic or heteroaryl ring.
[0094] As used herein, the term "arylalkyl" refers to aryl-substituted alkyl groups and "substituted arylalkyl" refers to arylalkyl groups further bearing one or more substituents as set forth herein.
[0096] As used herein, the term "arylcarbonylamino" denotes the group -NRqC(O)Rr, wherein Rq is hydrogen or lower alkyl and Rr is optionally substituted aryl.
[0099] As used herein, the term "arylsulfonylamino" denotes the group -NRqS(O)2Rr, wherein Rq is hydrogen or lower alkyl and Rr is optionally substituted aryl.
[00101] As used herein, reference to "a dithiocarbamate group" embraces substituents of the structure -S-C(S)-NR2, wherein each R is independently H, alkyl, substituted alkyl, aryl or substituted aryl as set forth herein.
[00103] As used herein, the term "cycloalkenyl" refers to cyclic ring-containing groups containing in the range of 3 up to 20 carbon atoms and having at least one carbon-carbon double bond, and "substituted cycloalkenyl" refers to cycloalkenyl groups further bearing one or more substituents as set forth herein.
[00104] As used herein, the term "cycloalkyl" refers to a monocyclic or polycyclic alkyl group containing 3 to 15 carbon atoms, and "substituted cycloalkyl" refers to cycloalkyl groups further bearing one or more substituents as set forth herein.
[00105] As used herein, the term "cycloalkyl ene" refers to divalent ring-containing groups containing in the range of about 3 up to about 12 carbon atoms, and "substituted cycloalkyl ene" refers to cycloalkylene groups further bearing one or more substituents as set forth herein.
[00109] As used herein, the term "substituted heteroaryl" refers to a heterocycle optionally mono or poly substituted with one or more functional groups, e.g., halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.
[00110] As used herein, the term "heteroarylcarbonylamino" denotes the group - NRqC(O)Rr, wherein Rq is hydrogen or lower alkyl and Rr is optionally substituted aryl.
[00116] As used herein, the term "substituted hydrocarbyl" comprises any of the above- referenced hydrocarbyl groups further bearing one or more substituents selected from hydroxy, hydrocarbyloxy, substituted hydrocarbyloxy, alkylthio, substituted alkylthio, arylthio, substituted arylthio, amino, alkylamino, substituted alkylamino, carboxy, -C(S)SR, - C(O)SR, -C(S)NR2, wherein each R is independently hydrogen, alkyl or substituted alkyl, nitro, cyano, halo, -SO3M or , -OSO3M, wherein M is H, Na, K, Zn Ca, or meglumine, guanidinyl, substituted guanidinyl, hydrocarbyl, substituted hydrocarbyl, hydrocarbylcarbonyl, substituted hydrocarbylcarbonyl, hydrocarbyloxycarbonyl, substituted hydrocarbyloxycarbonyl, hydrocarbylcarbonyloxy, substituted hydrocarbylcarbonyloxy, acyl, acyloxy, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, heteroarylcarbonyl, substituted heteroarylcarbonyl, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, aryl carbamoyl, a carbamate group, a dithiocarbamate group, aroyl, substituted aroyl, organosulfonyl, substituted organosulfonyl, organosulfinyl, substituted alkylsulfinyl, alkylsulfonylamino, substituted alkylsulfonylamino, arylsulfonylamino, substituted arylsulfonylamino, a sulfonamide group, sulfuryl, and the like, including two or more of the above-described groups attached to said hydrocarbyl moiety by such linker/spacer moieties as -O-, -S-, -NR-, wherein R is hydrogen, alkyl or substituted alkyl, - C(O)-, -C(S)-, -C(=NR'K -C(=CR'2)-, wherein R' is alkyl or substituted alkyl, -O-C(O)-, -0-C(O)-O-, -0-C(O)-NR- (or -NR-C(O)-O-), -NR-C(O)-, -NR-C(O)-NR-, -S-C(O)-, -S-C(O)-O-, -S-C(O)-NR-, -0-S(O)2-, -0-S(O)2-O-, -0-S(O)2-NR-, -O-S(O)-, -0-S(O)-O-, - 0-S(O)-NR-, -O-NR-C(O)-, -0-NR-C(O)-O-, -0-NR-C(O)-NR-, -NR-O-C(O)-, -NR-O-C(O)-O-, -NR-O-C(O)-NR-, -O-NR-C(S)-, -0-NR-C(S)-O-, -0-NR-C(S)-NR-, -NR-O-C(S)-, -NR-O-C(S)-O-, -NR-O-C(S)-NR-, -O-C(S)-, -0-C(S)-O-, -0-C(S)-NR- (or -NR-C(S)-O-), -NR-C(S)-, -NR-C(S)-NR-, -S-S(O)2-, -S-S(O)2-O-, -S-S(O)2-NR-, -NR-O- S(O)-, -NR-O-S(O)-O-, -NR-O-S(O)-NR-, -NR-O-S(O)2-, -NR-O-S(O)2-O-, -NR-O-S(O)2- NR-, -O-NR-S(O)-, -0-NR-S(O)-O-, -0-NR-S(O)-NR-, -0-NR-S(O)2-O-, -0-NR-S(O)2-NR- , -0-NR-S(O)2-, -0-P(O)R2-, -S-P(O)R2-, -NR-P(O)R2-, wherein each R is independently hydrogen, alkyl or substituted alkyl, and the like.
[00124] As used herein, the term "organosulfonyl" refers to substituents having the structure -S(0)2-organo, wherein organo embraces alkyl-, alkoxy- and alkylamino- moieties, as well as substituted alkyl-, alkoxy- or alkylamino- moieties.
[00128] As used herein, the term "sulfonyl" denotes the group -S(O)2-.
[00130] As used herein, the term "sulfonyl amino" denotes the group — NRqS(O)2- where Rq is hydrogen or lower alkyl.
[00131] As used herein, the term "substituted sulfonylamino" denotes the group -NR11S(O)2R", where Rq is hydrogen or lower alkyl and Ru is lower alkyl, substituted lower alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroaralkyl, substituted heteroaralkyl, aralkyl or substituted aralkyl.
[00132] As used herein, the term "sulfuryl" refers to substituents of the structure =S(O)2.
[00133] As used herein in connection with numerical values, the terms "approximately" and "about " mean 10% of the indicated value.
[00134] Figure 1 is a schematic representation of the chemical modification (Q) impairing DNA polymerase mediated oligonucleotide primer extension prior to "hot start" activation.
[00135] Figure 2 is a schematic representation of the likely mechanism for thermally induced intramolecular fragmentation by which a phosphotriester linkage converts to an unmodified, active state phosphodiester linkage at the 3 * terminus which is extendable by polymerase.
[00136] Figure 3 is a general synthetic scheme for modifying oligonucleotide primers with phosphoramidite derivatives.
[00147] The modification group can be integrated into an oligonucleotide by using existing automated synthesis and purification methods. Oligonucleotide primers of the present invention may be synthesized by any methods well-known in the art, including automated solid-phase chemical synthesis using cyanoethylphosphoramidite precursors (Barone et al., 12 Nucleic Acids Research, 4051 (1984)), and as described in J. Sambrook, E. F. Fritsch and Maniatis, T., Molecular Cloning, ch. 11 (2d ed. 1989). Other approaches include phosphotri ester and phosphodiester synthesis methods can be used (Narang, et al., Meth. Enzymol. 68-90 (1979)). A comprehensive overview of a variety of methods for the synthesis of modified and unmodified oligonucleotides can be found in Beaucage, S.L. et al., Current Protocols in Nucleic Acid Chemistry (2006). Following synthesis and purification of a modified oligonucleotide primer, several different procedures may be utilized to determine the acceptability of the oligonucleotide primer in terms of size and purity. One such procedure is polyacrylamide gel electrophoresis. Another such procedure is High Performance Liquid Chromatography ("HPLC"). These procedures are well known to those skilled in the art. Current methods employed for purification and separation in the art are applicable to the modified oligonucleotide primers of the present invention as well.
[00160] The invention will now be described in greater detail by reference to the following non-limiting examples.
[00164] The tetranucleotide 3'-phosphotriester derivatives were deprotected and cleaved from CPG support by treatment with 50 raM potassium carbonate in methanol for several hours. Reverse phase HPLC purification (with the gradient of acetonitrile in triethylammonium acetate buffer (pH 7.2)) was used for isolation of the 3'-phosphotriester tetranucleotides containing 4-methylthio-l-butanol (MTB), 4-oxo-l-pentanol (OXP), and 3- (N-tert-butylcarboxamido)-l-propanol (TBCA) groups. In most cases, the conditions used during the synthesis, deprotection and cleavage from the support, resulted in a partial loss of PTE function (for the N-(2-hydroxyethyl) phthalimido PTE group, EPH, complete deprotection occurred during these steps). With further optimization, the degree of degradation of the PTE functionality maybe significantly diminished. Each of the isolated 3 '-phosphotriester tetranucleotides were shown to be stable for several months when kept in frozen solution at -700C. In addition to the tetranucleotide sequences, longer PTE-protected oligonucleotide sequences suitable for PCR were also prepared. Example are provided in Table 1 below.
[00170] The abilities of Taq DNA polymerase and Klenow fragment of DNA polymerase I to perform template-dependant elongation of 3'-PTE modified oligonucleotide primer (and non-modified PDE control oligonucleotide primers) were investigated. Under the amplification reaction conditions, it was found that a standard PDE primer can be elongated to a full-length extension product while PTE oligonucleotide primers are very poorly extended by Klenow fragment DNA polymerase I. During the timeframe of the primer extension reactions, there was a slight accumulation of truncated extension products for reactions containing the PTE oligonucleotide primer, an occurrence which may be attributed to either partial deprotection of the PTE oligonucleotide primer during the extension reaction or to preferential extension of one PTE stereoisomer. When the PDE and PTE oligonucleotide primers were preheated at 95°C, the extension of the oligonucleotide primer is no longer hindered and is consistent with a PDE oligonucleotide primer. Although the most detailed extension experiments were performed with the mesophilic Klenow fragment of DNA polymerase I, similar results were obtained with the thermophilic Taq DNA polymerase, a DNA polymerase suitable for PCR. Overall, the extendibility of PTE containing oligonucleotide primers was significantly reduced relative to the corresponding active state PDE oligonucleotide primer, an essential criterion for a "hot start" PCR oligonucleotide primer of the present invention. EXAMPLE 5 Performance of PTE Oligonucleotide Primers in Template-Independent PCR
[00171] Due to poor extendibility of the PTE oligonucleotide primer in the oligonucleotide primer extension experiments (Example 4), it was concluded that PTE oligonucleotide primers should greatly diminish the amount of primer-dimer formed during sample preparation and the initial heating step of PCR by restricting DNA polymerase extendibility. To further explore the effect of the PTE-modified group on primer-dimer formation, PCR conditions that generate primer dimers in high yields in the absence of template were evaluated. Therefore, PTE-modified oligonucleotide primers targeted to the tat HIV-I gene were preheated at 95°C in PCR buffer (pH 8.4 at 25°C) for increasing amounts of time prior to PCR amplification by Taq DNA polymerase. By preheating the PTE primers for incremental amounts of time, the effect of PTE modification on primer dimer formation can be evaluated. Primer dimers, which were detected by agarose gel electrophoresis, ran as a 50-80 base pair fragment.
[00175] To ensure that improved results of PTE oligonucleotide primers in PCR were not due to any differences in the preparation and handling of the PDE and the PTE oligonucleotide primers, the PTE oligonucleotide primers were preheated in PCR buffer for conversion to the corresponding PDE oligonucleotide primer. Subsequently, the heated PTE oligonucleotide primers (PTE 95°C) and heated PDE oligonucleotide primers (PDE 950C) were employed in PCR, and it was found that primer dimers formed to a similar if not greater extent to the unheated PDE oligonucleotide primers. Integration of the amplicon and primer dimers bands for. the Lambda DNA and HIV system (for cycle 35) revealed a much higher ratio of amplicon to primer dimer for the unheated PTE oligonucleotide primers, in comparison to PCR amplifications that were performed with either PDE oligonucleotide primers or with heated PTE oligonucleotide primers. When the ratio of amplicon to primer dimer for all amplification reactions were normalized to the ratio for the unheated PTE oligonucleotide primer, a marked improvement in PCR specificity for amplicon formation was seen for both the Lambda DNA and for the HIV-I DNA systems. The PTE oligonucleotide primer caused between a 2.6 and a 15-fold improvement in PCR performance. Overall, the utility of the hot start PTE oligonucleotide primers of the present invention in PCR has been demonstrated as, in comparison to the PDE oligonucleotide primers. The amount of primer dimer decreased significantly, with a concurrent increase in amplicon production.
[00177] The two-step protocol included reverse transcription, followed by PCR amplification. The reverse transcription conditions utilized 0.4 μM oligo(dT) primer, 0 or 1 μg of human liver total RNA, and 15 U of Cloned AMV reverse transcriptase in a 20 μL reaction. The reaction was subjected to the following reverse transcriptase thermal cycling protocol: 25°C for 10 min; 42°C for 60 min; and 95°C for 5 min. After completion, the reverse transcription product was diluted 1 in 10 and was used as the template for a series of PCR reactions. Two sets of PCR reactions were prepared. The first set contained unmodified PDE primers at 0.5 μM, and the second set contained PTE modified primers at 0.5 μM. Each set of reactions contained three reactions, each of which utilized different templates: reaction a) water, reaction b) 0 μg Human liver total RNA reverse transcription product, and reaction c) 1 μg human liver total RNA reverse transcription product. All PCR reactions contained 1.25 U of Taq DNA polymerase and were 50 μL in volume. The mixtures were subjected to thermal cycling protocol: 940C for 10 min; [94°C for 30 sec, 600C for 30 sec, 72°C for 30 sec] 40 times. The reaction products were then analyzed by agarose gel analysis. Reactions a) and b) from both sets functioned as negative controls. For the negative controls, unmodified, PDE primers formed a significant amount of primer dimer, while the modified, PTE primers displayed no detectable primer dimer formation. For both the PDE and the PTE primers, reaction c) formed the desired 264 bp product. However, as was evident for the negative controls, the PDE primer reactions contained a significant amount of primer dimer formation, while the PTE primer reactions did not. Overall, the benefit of PTE-modified primers in two-step RT-PCR is evident, the modified primers suppressed formation of undesired primer dimers.
[00178] The same primer pairs were also utilized in a one-step RT-PCR protocol. As in the two-step protocol, two sets of reactions were prepared. The first set contained unmodified PDE primers at 0.5 μM, and the second set contained PTE modified primers at 0.5 μM. Each set. consisted of three reactions, with reactions b) and c) as negative controls. Reaction a) consisted of 0.25 μg of human liver total RNA and 25 U of MMLV reverse transcriptase, reaction b) consisted of 0.25 μg of human liver total RNA and 0 U of MMLV reverse transcriptase, and reaction c) consisted of 0 μg of human liver total RNA and 25 U of MMLV reverse transcriptase. The reactions all contained oligo(dT) primers at 5 μM and 0.3 U of Taq DNA polymerase and were at a volume of 25 μL. The reactions were incubated as follows: 420C for 60 min; 940C for 5 min; [94°C for 30 sec, 6O0C for 30 sec, 72°C for 5 min] 30 times; and 72°C for 5 min. The reaction products were analyzed by agarose gel electrophoresis. For both primer pairs, the no RT controls, b), appeared the same, with a slight smudge appearing on the gel, which is likely due to the presence of total RNA in the samples. For the no RNA controls c), the PDE primers displayed a low molecular weight primer dimer band, while the PTE primers did not display any detectable amplification product. The low molecular weight primer dimer band is likely due to reverse-transcriptase- mediated extension of the PDE PCR primers, as has been discussed by Peters, I.R., et al., 286 Journal of Immunological Methods 203-217 (2004). For reaction a), both primer pairs formed the desired 264 bp product. However, the reaction product for the PDE primers contained several amplification products, including the primer dimer band seen in reaction b), while the PTE primers formed the desired reaction product without any additional amplification products. The use of PTE-modified primers in sequential enzymatic reactions, such as one-step RT-PCR allows for the gene specific primers to be partially or completely blocked from extension during the lower-temperature reverse transcription reaction. [00179] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
[00180] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.
[00181] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.
[00183] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

中文:
[0003]提供以下描述以有助于读者的理解。在本发明中,没有任何提供的信息或引用的文献被承认是现有技术。
[0004]在现代分子生物学和生物技术中,PCR(聚合酶链反应)可能是被最广泛使用的方法,并且被迅速应用于遗传检测、诊断学、法医学和生物防御。Kolmodin,L.A.等人的Nucleic Acid Protocols第569-580页(2000年);Budowle,B.等人的301Science第1852-53页(2003年);Y.Sato等人的5(Suppl.1)Legal Medicine第S191-S193页(2003页);Saldanha,J.等人的43J.Medical Virol.第72-76页(1994年);Dahiya,R.等人的44Biochemistry and Molecular Biology International第407-15页(1998年);和Elnifro,E.M.等人的13Clin.Microbiol.Rev.第559-70页(2000年)。PCR在美国专利第4,683,195号和第4,683,202号中被描述。在每一个PCR扩增过程的循环中有几个典型的步骤。双链DNA靶序列在高温(~95°C)下进行第一热变性。该第一变性的发生在本文称为“初始变性步骤(initial denaturation step)”。其后是在较低温度(~60°C)下合成的寡核苷酸引物与每条链的退火。然后这些正向或反向定向的寡核苷酸引物的每一个被延伸,该延伸是在高温(~70°C)下,从它们的3’末端,通过热稳定的、镁离子依赖性的DNA聚合酶进行的,所述DNA聚合酶与5’-脱氧核苷酸三磷酸(dNTPs)混合并产生焦磷酸(PPi),如图1顶部对正向寡核苷酸引物的描绘。
[0005]PCR的实用性是由其迅速提供约106倍的靶扩增的能力以及高特异性所达到的,这部分依赖于寡核苷酸引物杂交的特异性。寡核苷酸引物的序列和长度因此被设计为,在退火所使用的温度下,仅与希望的靶序列杂交。然而,PCR扩增反应通常在若干分钟或若干小时的期间在环境室温下准备,该温度远低于确保寡核苷酸引物杂交特异性所需的温度范围。在这样的不严格的样品制备条件下,寡核苷酸引物可能非特异性地与其它具有基本上非互补性的序列结合,并有可能开始合成不希望的延伸产物,该延伸产物可与靶序列一起被放大。如已经被Chou,Q.等人讨论的,通过此“误引导(mis-priming)”的非特异性序列的扩增可以与所需靶序列的扩增竞争,并能因此显著减少所需序列的扩增效率,特别是对于低拷贝数量的靶。Chou,Q.等人的20Nucleic Acids Res.第1717-23页(1992年)。
[0007]尽管由于误引导和引物二聚体形成引起的前述问题,可能在所有的PCR应用中遇到,但是这些问题在高敏分析PCR方案中是特别有挑战性的,如用于检测血源性传染物(blood-borne infectious agents)(Saldanha,J.等人和Elnifro,E.M.等人),生物有害微生物(biohazardous microbes)(Budowle,B.等人)缺陷或癌基因(defective or cancerous genes)(Dahiya,R.等人)以及法医学(forensics)(Budowle,B.等人和Y.Sato等人的)的那些。另外,对于在多重PCR中的假扩增产品的形成有大得多的机会。Markoulatos,P.等人的16 J.of Clin.LaboratoryAnalysis第47-51页(2002年)。在逆转录PCR(RT-PCR)中,对于靶RNA序列检测的最敏感方法是在RT步骤中使用基因特异性的寡核苷酸引物。Zhang,J.等人的337Biochem.J.第231-41页(1999年);Lekanne Deprez,R.H.等人的307AnalyticalBiochem.第63-69页(2002年);和Bustin,S.A.等人的15J.of Biomolecular Techniques第155-66页(2004年)。从需要高特异性以避免严重不利的“假阴性(false negatives)”和“假阳性(false positives)”后果的这些高敏应用的重要性的角度考虑,具有如此试剂和方案是关键,该试剂和方案提供功能上不与由于误引导和引物二聚体形成引起的人为产物相关的分析。
[0008]基于所谓“热启动”的方法,许多一般策略已经被研究用于减少非特异性PCR扩增,所述“热启动”的方法瞄准减弱在样品制备期间,在低严紧度条件、室温下,由于误引导和寡核苷酸引物二聚体形成引起的不期望的扩增。当扩增反应混合物达到高严紧度,“热”温度“启动”聚合酶介导的、仅与靶序列杂交的寡核苷酸引物延伸时,PCR扩增随即开始。因此,仅在引物严紧度/靶杂交条件对特异性为最佳时的高温下,温度被用于触发寡核苷酸引物酶促延伸。
[0009]这些对于“热启动”的一般策略包括使用(1)温度敏感材料,如蜡作为屏障或多价螯合剂以控制反应物的混合(Q.Chou等人和Tanzer,L.R.等人的273Anal.Biochem.第307-310页(1999年));(2)寡核苷酸适配体(Dang,C.等人的264J.Mol.Biol.第268-78页(1996年))或抗体(Eastlund,E.等人的2 LifeScienceQuarterly第2-5页(2001年)和Mizuguchi,H.等人的126J.Biochem(Tokyo)第762-68页(1999年))其抑制DNA聚合酶的功能;(3)使用第二热稳定酶,如焦磷酸酶(Clark,D.R.等人的国际专利申请号WO 2002088387)以除去通过加入焦磷酸(PPi)引起的抑制;(4)用水解可逆的反应物化学修饰的聚合酶,如柠康酸修饰的赖氨酸(Birch,D.E.等人的美国专利第5,773,258号)在AmpliTaq Gold(Moretti,T.等人的25BioTechniques第716-722页(1998年)和Saldanha,J.等人)中;和(5)不喜低温误引导的寡核苷酸引物序列构建物,如竞争序列(competitor sequence)(Puskas,L.G.等人的5Genome Research第309-311页(1995年)或“touch-up andloop-incorporated oligonucleotide primers“(TULIPS-PCR)(Ailenberg,M.等人的29(5)BioTechniques第1018-23页(2000年))。
[0012]在特定实施方式中,连接所述修饰基团产生磷酸三酯(PTE)核苷酸间键合。所述修饰削弱DNA聚合酶介导的寡核苷酸引物延伸,其在如PCR的扩增高温下的最初温育时期之前。本发明的核苷酸和核苷形成的寡核苷酸引物有两种状态。第一种,该寡核苷酸引物由于修饰基团的存在处于无活性状态,直至达到初始变性温度,通常95°C。一旦达到初始变性温度,通过将寡核苷酸转化为第二种状态的热诱导分子内断裂,该寡核苷酸引物变为有活性的。此寡核苷酸引物的第二状态是相应的未修饰的寡核苷酸引物,其具有活性状态的磷酸二酯键并且可以通过聚合酶延伸。修饰基团部分或完全解离优选发生在接近95°C下温育大约2-10分钟之后。在某些实施方式中,所述修饰基团从寡核苷酸引物发生解离,有赖于温度,并且不需要酶、化学制剂或扩增反应条件如pH值。磷酸三酯键合在Miller等人的93(24)J.Am.Chemical Society,6657-65(1971)中;Zon等人的6(2)Protein J.,131-45(1987)中;以及Koziolkiewicz,M.和Wilk,A.的(Protocols forOligonucleotides andAnalogs(1993))中描述。
[0018]在一个优选实施方式中,式Ib的修饰基团是4-甲硫基-1-丁基,如下所示:
[0019]在一个实施方式中,根据本发明的修饰基团提供式Ic的化合物:
[0020]在一个优选实施方式中,式Ic的修饰基团是3-(N-叔丁基酰胺基)-1-丙基,如下所示:
[0023]在另一个实施方式中,根据本发明的修饰基团提供式II的化合物:
[0025]在另一个实施方式中,根据本发明的修饰基团提供式III的化合物:
[0026]在另一个实施方式中,根据本发明的修饰基团提供式IV的化合物:
[0027]在式IV化合物的一个实施方式中——其中R5和R6联合形成环,修饰基团是甲氧基甲基-环己-1,3-基-乙基,如下所示:
[0035]在本文提供的方法和组合物的某些实施方式中,扩增是通过逆转录酶(RT)。本领域中的一个普通技术人员熟悉进行RT所需的条件。变性和延伸温度可以变化并有赖于所使用的逆转录酶。例如,通过逆转录酶的变性和延伸在约37-70°C下发生。本文提供的修饰基团可以根据所需的解离条件选择使用,如特定温度或与特定时间范围的结合。例如,修饰基团可以被用于RT,这样该基团在37-70°C下解离;优选为37-60°C下;优选为在约50°C下;优选为在约42°C下以及优选为在约37°C下。在其它实施方式中,在所需温度下,修饰基团在期望温度下在0.1-60分钟内解离;优选为1-30分钟;优选为1-15分钟;优选为1-10分钟;优选为0.1-5分钟。
[0036]在本文提供的方法和组合物的其它实施方式中,在单一反应混合物中,扩增可以是一个或多于一个扩增反应,如逆转录聚合酶链反应(RT-PCR)或其它例子,如那些在美国专利第6,399,304号中所述的。在某些实施方式中,可以加入多于一个酶促反应所需的试剂,并在单一反应容器中进行。在该反应中,可以使用各种修饰的和/或未修饰的寡核苷酸的组合。例如,修饰的寡核苷酸可以被用于其中的一个、一个或更多、两个或更多、三个或更多、五个或更多、十个或更多反应。在一个实例中,具有修饰基团的寡核苷酸可以被用于逆转录酶,其在RT的理想温度如42°C下解离,并且另一个具有不同修饰基团的寡核苷酸可被用于PCR,其在PCR的理想温度如95°C下解离。通过提供此组合物,PCR引物不干扰或基本不干扰RT反应。可选地,RT引物可以是未修饰的引物,并且所述PCR引物可以用基团修饰,所述基团在对PCR理想的温度如95°C下解离。在更进一步实施方式中,后续PCR包括多重PCR、实时PCR或定量PCR。
[0039]如在此使用的“核酸”、“核苷酸序列”或“核酸序列”是指核苷酸、寡核苷酸、多核苷酸或其任何片段并且指天然产生或合成分子。这些用语也是指基因组的或合成来源的DNA或RNA——其可以是单链的或双链的,并可以代表有义链或反义链——或指任何DNA-类似或RNA-类似材料。“RNA等价物”相对于DNA序列,由核苷酸的线性序列组成,该核苷酸的线性序列与参照DNA序列是相同的,除了所有存在的含氮碱基胸腺嘧啶被尿嘧啶取代,并且糖骨架由核糖,而不是脱氧核糖组成。适合本发明的另外可选的核酸骨架包括,但不限于,硫代磷酸酯、硒代磷酸酯(phosphoroselenoate)、烷基磷酸酯、芳基磷酸酯、锁定核酸(LNA)和肽核酸(PNA)和硼酸盐。RNA可以被用于在此描述的方法和/或可以通过逆转录被转变为cDNA用于在此描述的方法。
[0040]如在此使用的术语“修饰的寡核苷酸”是指具有至少一个修饰基团的寡核苷酸引物。修饰的寡核苷酸包括,例如,包含修饰的核苷的寡核苷酸、包含修饰的核苷酸间键合的寡核苷酸或具有修饰的核苷和核苷酸间键合的组合的寡核苷酸(即,使天然核苷存在于寡核苷酸链中)。可以发现其核苷经由修饰的核苷酸间键合连结的寡核苷酸,例如,在Waldner等人的6Bioorg.Med.Chem.Letters,2363-66(1996)中,其描述了包含各种酰胺核苷酸间键合的寡核苷酸的合成。
[0043]如在此使用的关于寡核苷酸的术语“末端”,指在寡核苷酸的3′或5′末端上或附近的核苷酸。优选地,核苷酸的末端包括末端的6个核苷酸,更优选为末端的5个核苷酸,更优选为末端的4个核苷酸,更优选为末端的3个核苷酸,更优选为末端的2个核苷酸或更优选为末端的1个核苷酸。
[0045]如在此使用的术语“核苷酸间键合”是指介于两个寡核苷酸引物的核苷酸之间的键。
[0046]如在此使用的术语“靶核酸序列”或“核酸靶”是指待被识别的核苷酸序列。
[0048]如在此使用的术语“热诱导”是指通过使用热,将寡核苷酸引物的修饰从寡核苷酸引物除去,产生活性状态的寡核苷酸引物,因此使其经聚合酶可延伸的方法。
[0049]如在此使用的术语“热启动”是指核酸扩增反应,其中聚合酶诱导的核酸扩增被减弱,直至反应达到高于酶的延伸温度的初始温度。在热启动PCR应用中,初始温度达到约80-105°C之间;或直至扩增反达到至少约80°C的初始温度或直至扩增反应达到至少约85°C的初始温度或直至扩增反应达到至少约90°C初始温度或直至扩增反应达到至少约95°C的初始温度。术语“热启动”在本领域中是熟知,并且有很多已知的减弱扩增的方法,如修饰的聚合酶、具有减弱杂交的二级结构的寡核苷酸以及包含在温度敏感屏障如蜡中的反应物。在一个优选实施方式中,热启动扩增是由热诱导去除寡核苷酸修饰基团引起的。
[0056]如在此使用的术语“严紧杂交(stringent hybridization)条件”是指不使两个非完全互补的核酸杂交的杂交条件。
[0057]如在此使用的术语“样品”或“测试样品”是指任何被认为包含目的核酸的液体或固体材料。测试样品可以来自任何生物源(即,生物样品),如培养基中的细胞或组织样品或合成产生的包括化学合成的模板。
[0058]如在此使用的,使用的术语“互补(complement)”“互补的(complementary)”或“互补性(complementarity)”用于指根据标准Watson/Crick配对原则的多核苷酸(即核苷酸序列,如寡核苷酸或靶核酸)。互补序列也可以是与DNA序列互补的RNA序列或其补体序列,并且也可以是cDNA。例如,对于序列“5′-A-G-T-3”的互补序列为“3′-T-C-A-5’”。某些在天然核酸中一般不被发现的核苷酸可以包括在此处所述的核酸中;这些包括修饰的核苷、核苷酸、和核酸,如肌苷,7-脱氮鸟嘌呤、锁定核酸(LNA)和肽核酸(PNA)。互补性不需要完全互补;稳定的双链体可以包含错配的碱基对、变性的或未配对的碱基。核酸技术领域的技术人员可以经验地决定双链体稳定性,这考虑到许多变量,包括,例如,寡核苷酸的长度、寡核苷酸的碱基组成和序列、离子强度和错配碱基对的发生率。
[0059]互补性可以是“部分的”,其中两条核酸链的仅仅一些核苷酸碱基根据碱基配对原则配对。互补性可以是“完全的”或“全部的”,其中两条核酸链的所有核苷酸碱基都根据碱基配对原则配对。互补性可以是不存在的,其中两条核酸链中没有核苷酸碱基根据碱基配对原则配对。核酸链之间的互补性的程度显著影响核酸链之间杂交的效率和强度。这在扩增反应以及依赖核酸之间结合的检测方法是特别重要的。术语也可以用于指单独的核苷酸,尤其是在多核苷酸的内容中。例如,在比较或对比在寡核苷酸的剩余部分和核酸链之间的互补性中,在寡核苷酸中的特定核苷酸被记下其与另一条核酸链中的核苷酸的互补性或互补性缺乏。
[0064]如在此使用的术语“核苷类似物”、“修饰的核苷”或“核苷衍生物”包括如此处所述的合成的核苷。核苷衍生物也包括具有修饰的碱基部分的核苷,其具有或不具有保护基。这种类似物包括,例如,脱氧肌苷、2,6-二氨基嘌呤-2′-脱氧核苷、5-甲基-2′-脱氧胞苷和类似物。在自然发生的核苷中最常见的碱基环是嘌呤和嘧啶环。自然发生的嘌呤环包括,例如,腺嘌呤、鸟嘌呤和N6-甲基腺嘌呤。自然发生的嘌呤环包括,例如,胞嘧啶、胸腺嘧啶和5-甲基胞嘧啶。本发明的化合物和方法包括这些碱基环和其合成的类似物,以及非自然的杂环-取代碱基糖和甚至非环取代碱基糖。而且,核苷衍生物包括其它嘌呤和嘧啶类似物,例如,卤素-取代嘌呤(例如,6-氟嘌呤)、卤素-取代嘧啶、N6-乙基腺嘌呤、N6-(烷基)-胞嘧啶、5-乙基胞嘧啶、以及类似物。核苷衍生物和类似物包含广泛的多种修饰,如那些在美国专利第6762298号中所描述的。
[0069]如在此使用的术语“烯基芳基”是指烯基-取代的芳基,“取代的烯基芳基”是指烯基芳基,其进一步包含一个或多个如前所述的取代基。
[0075]如在此使用的术语“炔芳基”是指炔基-取代的芳基,并且“取代炔芳基”是指炔芳基进一步包含一个或多个如前所述的取代基。
[0076]如在此使用的术语“烷氧基”表示基团-ORc,其中Rc是所限定的低碳烷基、取代的低碳烷基、酰基、芳基、取代的芳基、芳烷基、取代的芳烷基、杂烷基、杂芳烷基、环烷基、取代的环烷基、环杂烷基或取代的环杂烷基。
[0077]如在此使用的术语“低碳烷氧基”表示基团-ORd,其中Rd是低碳烷基。
[0079]如在此使用的术语“烷基羰基氨基”表示基团-NReC(O)Rf,其中Re是任选取代的烷基,并且Rf是氢或低碳烷基。
[0080]如在此使用的术语“烷基亚硫酰基”表示基团-S(O)Rg,其中Rg是任选取代的烷基。
[0085]如在此使用的术语“亚炔基(alkynylene)”是指二价的直链或支链的烃基团,其具有至少一个碳碳三键,并且通常具有约2-12个碳原子、优选为2-8个碳原子,以及“取代的亚炔基”是指亚炔基基团进一步包含一个或多个如前所述的取代基。
[0087]如在此使用的术语“取代的酰胺基”表示基团-C(O)NRkRk’,其中Rk和Rk’独立为氢、低碳烷基、取代的低碳烷基、芳基、取代的芳基、杂芳基或取代的杂芳基,然而条件是,Rk和Rk’的至少一个不是氢。RkRk’与氮的组合可以形成任选取代的杂环或杂芳环。
[0089]如在此使用的术语“氨基”或取代胺表示基团-NRnRn’,其中Rn和Rn’可以独立为氢、低碳烷基、取代的低碳烷基、芳基、取代的芳基、杂芳基或取代的杂芳基——如此处所定义的——、酰基或磺酰。“二价胺”表示基团-NH-。“取代的二价胺”表示基团-NR-,其中R是低碳烷基、取代的低碳烷基、芳基、取代的芳基、杂芳基、取代的杂芳基、酰基、取代的酰基、磺酰或取代的磺酰。
[0090]如在此使用的术语“取代的氨基”或“取代的胺”表示基团-NRpRp’,其中Rp和Rp’独立为氢、低碳烷基、取代的低碳烷基、芳基、取代的芳基、杂芳基、取代的杂芳基、酰基、取代的酰基、磺酰或取代的磺酰,然而条件是,Rp和Rp’的至少一个不是氢。RpRp’与氮的组合可以形成任选取代的杂环或杂芳环。
[0094]如在此使用的术语“芳基烷基”是指芳基-取代的烷基,和“取代的芳基烷基”是指芳基烷基进一步包含一个或多个如前所述的取代基。
[0096]如在此使用的术语“芳基羰基氨基”表示基团-NRqC(O)Rr,其中Rq是氢或低碳烷基,并且Rr是任选取代的芳基。
[0099]如在此使用的术语“芳基磺酰氨基”表示基团-NRqS(O)2Rr,其中Rq是氢或低碳烷基,并且Rr是任选取代的芳基。
[00101]如在此使用的,关于“二硫代氨基甲酸盐基团”包含结构为-S-C(S)-NR2的取代基,如前所述,其中每一个R独立为H、烷基、取代的烷基、芳基或取代的芳基。
[00103]如在此使用的术语“环烯基”是指环状基团,其包含3至20个碳原子并具有至少一个碳碳双键,并且“取代的环烯基”是指进一步具有一个或多个如前所述的取代基的环烯基。
[00104]如在此使用的术语“环烷基”是指单环或多环烷基,其包含3至15个碳原子,并且“取代的环烷基”是指进一步具有一个或多个如前所述的取代基的环烷基。
[00105]如在此使用的术语“亚环烷基”是指二价环状基团,其范围包含约3至约12个碳原子,并且“取代的亚环烷基”是指进一步具有一个或多个如前所述的取代基的亚环烷基。
[00109]如在此使用的术语“取代的杂芳基”指用如下的一个或多个官能团任选地单取代或多取代的杂环:例如卤素、低碳烷基、低碳烷氧基、烷基硫代、乙炔基、氨基、酰氨基、羧基、羟基、芳基、芳氧基、杂环、取代的杂环、杂芳基(hetaryl)、取代的杂芳基(hetaryl)、硝基、氰基、硫羟,磺氨基以及类似基团。
[00110]如在此使用的术语“杂芳基羰基氨基”表示基团-NRqC(O)Rr,其中Rq是氢或低碳烷基和Rr是任选取代的芳基。
[00116]如在此使用的术语“取代的烃基”包含上述引用的任意烃基,进一步包含一个或多个取代基,所述取代基选自羟基、烃氧基、取代的烃氧基、烷基硫代、取代的烷基硫代、芳基硫代、取代的芳基硫代、氨基、烷基氨基、取代的烷基氨基、羧基、-C(S)SR、-C(O)SR、-C(S)NR2——其中每个R独立为氢、烷基或取代的烷基——、硝基、氰基、卤素、-SO3M或-OSO3M——其中M是H、Na、K、Zn、Ca或葡甲胺(meglumine)、胍基、取代的胍基、烃基、取代的烃基、烃基羰基、取代的烃基羰基、烃氧基羰基、取代的烃氧基羰基、烃基羰氧基、取代的烃基羰氧基、酰基、酰氧基、杂环、取代的杂环、杂芳基、取代的杂芳基、杂芳基羰基、取代的杂芳基羰基、氨基甲酰基、单烷基氨基甲酰基、二烷基氨基甲酰基、芳基氨基甲酰基、氨基甲酸酯基团、二硫代氨基甲酸酯基团、芳酰基、取代的芳酰基、有机磺酰、取代的有机磺酰、有机亚硫酰基、取代的烷基亚硫酰基、烷基磺酰氨基、取代的烷基磺酰氨基、芳基磺酰氨基、取代的芳基磺酰氨基、氨磺酰基团、磺酰(sulfuryl)和类似物,包括两个或更多的上述基团,通过如连接/间隔部分(linker/spacer moiety)连接到所述烃基部分,所述连接/间隔部分如-O-、-S-、NR-,其中R是氢、烷基或取代的烷基、-C(O)-、-C(S)-、-C(=NR’)-、-C(=CR’2)-——其中R’是烷基或取代的烷基、-O-C(O)-、-O-C(O)-O-、-O-C(O)-NR-(或-NR-C(O)-O-)、-NR-C(O)-、-NR-C(O)-NR-、-S-C(O)-、-S-C(O)-O-、-S-C(O)-NR-、-O-S(O)2-、-O-S(O)2-O-、-O-S(O)2-NR-、-O-S(O)-、-O-S(O)-O-、-O-S(O)-NR-、-O-NR-C(O)-、-O-NR-C(O)-O-、-O-NR-C(O)-NR-、-NR-O-C(O)-、-NR-O-C(O)-O-、-NR-O-C(O)-NR-、-O-NR-C(S)-、-O-NR-C(S)-O-、-O-NR-C(S)-NR-、-NR-O-C(S)-、-NR-O-C(S)-O-、-NR-O-C(S)-NR-、-O-C(S)-、-O-C(S)-O-、-O-C(S)-NR-(或-NR-C(S)-O-)、-NR-C(S)-、-NR-C(S)-NR-、-S-S(O)2-、-S-S(O)2-O-、-S-S(O)2-NR-、-NR-O-S(O)-、-NR-O-S(O)-O-、-NR-O-S(O)-NR-、-NR-O-S(O)2-、-NR-O-S(O)2-O-、-NR-O-S(O)2-NR-、-O-NR-S(O)-、-O-NR-S(O)-O-、-O-NR-S(O)-NR-、-O-NR-S(O)2-O-、-O-NR-S(O)2-NR-、-O-NR-S(O)2-、-O-P(O)R2-、-S-P(O)R2-、-NR-P(O)R2-,其中每个R独立为氢、烷基或取代的烷基,和类似物。
[00124]如在此使用的术语“有机磺酰基”是指具有结构为-S(O)2-有机基团的取代基,其中有机基团包含烷基-、烷氧基-和烷基氨基-部分,以及取代的烷基-、烷氧基-或烷基氨基-部分。
[00128]如在此使用的术语“磺酰基”表示基团-S(O)2-。
[00130]如在此使用的术语“磺酰氨基”表示基团-NRqS(O)2-,其中Rq是氢或低碳烷基。
[00131]如在此使用的术语“取代的磺酰氨基”表示基团-NRqS(O)2Ru,其中Rq是氢或低碳烷基,并且Ru是低碳烷基、取代的低碳烷基、环烷基、取代的环烷基、杂环基、取代的杂环基、芳基、取代的芳基、杂芳基、取代的杂芳基、杂芳烷基、取代的杂芳烷基、芳烷基或取代的芳烷基。
[00132]如在此使用的术语“磺酰(sulfuryl)”是指结构是=S(O)2的取代基。
[00133]如在此使用的与数值相关的术语“接近”和“约”表示所示数值的10%。
[00134]图1是在“热启动”激活之前削弱DNA聚合酶介导的寡核苷酸引物延伸的化学修饰(Q)的示意图。
[00135]图2是对热诱导的分子内断裂的可能的机理的示意图,通过该断裂磷酸三酯键转化为未修饰的活性状态的磷酸二酯键,在其3’末端可以通过聚合酶延伸。
[00136]图3是用亚磷酰胺衍生物修饰寡核苷酸引物的一般合成方案。
[00147]修饰基团可以整合进寡核苷酸,这通过使用目前的自动合成和纯化方法进行。本发明的寡核苷酸引物可以通过任何本领域熟知的方法合成,包括使用氰基乙基亚磷酰胺前体的自动固相化学合成(Barone等人的12 Nucleic AcidsResearch,4051(1984))以及在J.Sambrook,E.F.Fritsch和Maniatis,T.,MolecularCloning,ch.11(2d ed.1989)中描述的。其它方法包括磷酸三酯和磷酸二酯的合成方法可以被使用(Narang等人的Meth.Enzymol.68-90(1979))。综合概述用于合成修饰的和未修饰的寡核苷酸的各种方法可以在Beaucage,S.L.等人的Current Protocolsin Nucleic Acid Chemistry(2006)中发现。在修饰的寡核苷酸引物的合成和纯化后,几种不同的方法可以被利用以测定寡核苷酸引物在尺寸和纯度方面的可接受性。一个这样的方法是聚丙烯酰胺凝胶电泳法。另一个这样的方法是高效液相色谱法(′′HPLC′′)。这些方法是本领域技术人员所熟知的。目前在本领域中使用的纯化和分离方法也同样适合本发明的修饰的寡核苷酸引物。
[00160]本发明现在将通过参考以下非限定性实施例更详细地描述。
[00164]通过使用甲醇中的50mM碳酸钾处理数小时,四核苷酸3’-磷酸三酯衍生物被去保护并从CPG支撑物切割。反向HPLC纯化(用醋酸三乙铵缓冲液中的乙腈梯度(pH 7.2))被用于分离包含4-甲硫基-1-丁醇(MTB),4-氧-1-戊醇(OXP)和3-(N-叔丁基酰胺基)-1-丙醇(TBCA)基团的3’-磷酸三酯四核苷酸。在很多例子中,在合成、去保护和从支撑物切割期间使用的条件,导致部分PTE功能的损失(对N-(2-羟乙基)苯二甲酰亚氨基PTE基团,EPH,完全的去保护发生在这些步骤期间)。随着进一步优化,PTE功能性的降解程度显著减少。每一个分离的3’-磷酸三酯四核苷酸当保存在-70°C下的冷冻溶液时,显示出数月的稳定。除了四核苷酸序列,适合PCR的更长的PTE-保护寡核苷酸序列也被制备。实施例在以下的表1中提供。
[00170]Taq DNA聚合酶和DNA聚合酶I的Klenow片段进行模板-依赖性的3’-PTE修饰的寡核苷酸引物(和未修饰的PDE对照寡核苷酸引物)的延伸的能力被研究。在扩增反应条件下,发现标准PDE引物可以延伸至全长程度产物,而PTE寡核苷酸引物通过Klenow片段DNA聚合酶I被非常差地延伸。在引物延伸反应的期限期间,对包含PTE寡核苷酸引物的反应,截短的延伸产物有轻微积聚,该发现可以归因于在延伸反应期间PTE寡核苷酸引物的部分去保护或一个PTE立体异构体的优选延伸。当PDE和PTE寡核苷酸引物被在95°C预热时,寡核苷酸引物的延伸不再被阻碍并与PDE寡核苷酸引物相符。虽然最详细的延伸实验用嗜温的DNA聚合酶I的Klenow片段进行,但是类似的结果也用嗜热的Taq DNA聚合酶——适合PCR的DNA聚合酶——获得。总体上,所述包含PTE的寡核苷酸引物的延伸能力相对于相应的活性状态的PDE寡核苷酸引物显著减少,这是本发明的“热启动”PCR寡核苷酸引物的重要标准。
[00171]由于PTE寡核苷酸引物在寡核苷酸引物延伸实验(实施例4)中延伸性差,推断PTE寡核苷酸引物应该大量减少在样品制备和限制DNA聚合酶延伸性的PCR的初始加热步骤期间形成的引物-二聚体的量。为进一步探索PTE-修饰基团对引物-二聚体形成影响,在缺少模板情况下以高收率产生引物二聚体的PCR条件被评价。因此,靶向tat HIV-1基因的PTE-修饰的寡核苷酸引物在95°C下在PCR缓冲液(25°C下pH 8.4)中被预热,以增加通过Taq DNA聚合酶的PCR扩增之前的时间量。通过预热PTE引物增加量的时间,PTE修饰对引物二聚体形成的影响可以被评价。引物二聚体,其通过琼脂糖凝胶电泳进行检测,作为50-80碱基对片段电泳。
[00175]为确保在PCR中PTE寡核苷酸引物结果的改善,不是由于在PDE和PTE寡核苷酸引物制备和处理中的任何差异,PTE寡核苷酸引物在PCR缓冲液中被预加热以转化为相应的PDE寡核苷酸引物。随后,加热的PTE寡核苷酸引物(PTE 95°C)和加热的PDE寡核苷酸引物(PDE 95°C)被用于PCR中,并且发现引物二聚体以与未加热的PDE寡核苷酸引物相似——如果不大于的话——的程度形成。对λDNA和HIV系统(35循环)的扩增子和引物二聚体带的积分,揭示:与使用PDE寡核苷酸引物或加热的PTE寡核苷酸引物进行的PCR扩增相比,对于未加热的PTE寡核苷酸引物,扩增子与引物二聚体的比率高得多。对于所有扩增反应,当扩增子与引物二聚体的比率被正归一化为对未加热的PTE寡核苷酸引物的比率时,在PCR中对扩增子形成显著改善特异性,在λDNA和对HIV-1DNA系统中都被发现。在PCR实行中,PTE寡核苷酸引物引起介于2.6和15-倍之间的改善。总体而言,本发明的热启动PTE寡核苷酸引物在PCR中的效用,作为与PDE寡核苷酸引物的比较,已经被证明。引物二聚体的量明显减少,并伴随扩增子产生的增加。
[00177]两步方案包括逆转录,接着PCR扩增。逆转录条件应用20μL反应中的0.4μM低聚(dT)引物,0或1μg的人肝脏总RNA,和15U的克隆的AMV逆转录酶。该反应进行以下逆转录酶热循环方案:25°C下10min;42°C下60min;和95°C下5min。完成以后,逆转录产物以1比10进行稀释,并且用作一系列PCR反应的模板。两组PCR反应被准备。第一组包含0.5μM的未修饰的PDE引物,第二组包含0.5μM的PTE修饰的引物。每一组反应包含三个反应,其每一个使用不同的模板:反应a)水,反应b)0μg人肝脏总RNA逆转录产物,和反应c)1μg人肝脏总RNA逆转录产物。所有的PCR反应包含1.25U的Taq DNA聚合酶,体积50μL。该混合物经历下述热循环方案:94°C对10min;[94°C对30sec,60°C对30sec,72°C对30sec]40次。该反应产物然后通过琼脂糖凝胶分析。两组的反应a)和b)都作为阴性对照。对于阴性对照,未修饰的、PDE引物形成明显量的引物二聚体,而修饰的、PTE引物未显示可检测到的引物二聚体形式。对PDE和PTE引物二者,反应c)形成所需的264bp产物。不过,阴性对照所显示的,PDE引物反应包含显著量的引物二聚体形式,而PTE引物反应没有。总体而言,在两步RT-PCR中,PTE-修饰的引物的益处是显然的,所述修饰的引物抑制不需要的引物二聚体的形成。
[00178]相同的引物对也被用于一步RT-PCR方案中。如在两步方案中,两组反应被准备。第一组包含0.5μM的未修饰的PDE引物,第二组包含0.5μM的PTE修饰的引物。每组由三个反应组成,反应b)和c)作为阴性对照。反应a)包括0.25μg的人肝脏总RNA和25U的MMLV逆转录酶,反应b)包括0.25μg的人肝脏总RNA和0U的MMLV逆转录酶,和反应c)包括0μg的人肝脏总RNA和25U的MMLV逆转录酶。所述反应均包含5μM的低聚(dT)引物和0.3U的Taq DNA聚合酶及25μL的体积。反应被进行如下:42°C下60min;94°C下5min;[94°C下30sec,60°C下30sec,72°C下5min]30次;和72°C下5min。反应产物通过琼脂糖凝胶电泳分析。对两种引物对,非RT对照b)表现相同,在胶上显现轻微污点,这可能是由于总RNA存在于样品中。对非RNA对照c),PDE引物显示低分子量引物二聚体带,而PTE引物未显示任何可探测扩增产物。低分子量引物二聚体带可能是由于逆转录酶-介导的PDE PCR引物的延伸,如已经被Peters,I.R.等人在286Journal of Immunological Methods第203-217页(2004)中所讨论的。对反应a),两个引物对都形成所需的264bp产物。不过,用于PDE引物所述反应产物包含数种扩增产物,包括在反应b)可见的引物二聚体带,而PTE引物形成所需的反应产物而没有任何另外的扩增产物。在随后的反应如一步RT-PCR中,PTE-修饰的引物的使用在低温逆转录反应期间,使基因特异性的引物部分地或完全地被阻断不再延伸。
[00180]本文阐述性描述的发明可适于在未在本文具体公开的任何要素或多个要素、限定或多个限定不存在的情况下实施。因此,例如,术语“包括(comprising)”、“包含(including)”或“含有(containing)”等应当作扩张性和非限制性的理解。此外,在本文中使用的术语和表达被用作描述性而非限制性术语,且这些术语和表达的使用不意图排除所示出和描述的特征的等价物或其部分,而应当认识到,在所要求保护的发明范围内各种变化是可能的。
[00181]因此,应当理解,尽管本发明已通过优选的实施方式和任选的特征加以具体公开,但是本领域普通技术人员可采用本文所公开的内在体现的发明的改变、改善和变化,且这类改变、改善和变化被认为在本发明的范围内。在此提供的所述材料、方法和实施例是优选的实施方式中的代表,是可示例性的,并且不希望限制本发明的范围。
[00183]另外,当本发明的特征或者方面针对马库什组(Markush groups)进行描述时,本领域技术人员会意识到,本发明因此也针对该马库什组成员中的任何单独成员或亚组进行描述。