Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-05-25
2003-02-04
McGarry, Sean (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S091100, C536S023100, C536S024200, C536S024300, C536S024310, C536S024320
Reexamination Certificate
active
06515120
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention provides aptamers that recognize and bind to guanosine (GMP), deoxyguanosine (dGMP), adenosine (AMP), deoxyadenosine (dAMP), cytosine (CMP) and deoxycytosine (dCMP). The present invention also relates to a method for sequencing a polymeric biomolecule and a method for structurally characterizing the same comprising the use of aptamers. In a preferred embodiment of this invention, these methods relate to the sequencing or characterization of a single polymeric biomolecule. The invention also relates to a method for selecting aptamers useful for sequencing nucleic acids.
BACKGROUND OF THE INVENTION
Knowing the primary structure and composition of polymeric biomolecules, such as DNA, RNA, polysaccharides, lipids and polypeptides, is important for scientific and medical research and the development of medical treatments. For example, information regarding the primary structure of certain polymeric biomolecules is important for studying the genetic basis of certain diseases, understanding role that polysaccharides play in cellular recognition processes, determining the DNA sequence of a purified protein and producing recombinant proteins for assays for screening drugs. Thus, fast, accurate and efficient methods for determinating the primary structure and composition of a polymeric biomolecule, especially a biomolecule that is long and/or is in short supply, are important for progress in research.
1.1 DNA Sequencing
Approaches to sequencing DNA have varied widely. The Maxam-Gilbert technique for sequencing (Maxam and Gilbert, 1977, PNAS USA 74:560) involves four separate chemical cleavage reactions using the same DNA molecules. The partial or total cleavage of the DNAs, which are end-labeled, produce varying sized DNAs which are run on a gel electrophoresis apparatus. The sequence of the DNA molecule is determined from the migratory position of the bands in the gel. The dideoxy method of sequencing (Sanger et al., 1977, PNAS USA 74:5463) involves four enzymatic reactions using DNA polymerase to synthesize fragments of varying lengths due to the incorporation of a chain terminating dideoxy nucleotide into each fragment. Typically, radioactively-labeled nucleotide(s) are incorporated into the growing chains. Variations on the Sanger method comprise the use of fluorescent dye-labeled primers or nucleotide chain terminators. The reactions are then run on a gel electrophoresis apparatus. The sequence of the DNA molecule is determined from the migratory position of the cleaved bands in the gel. Fluorescence emissions from the dyes are monitored. These gel-based, ladder-like output methods are disadvantageous, in part, because they (1) require substantial amounts of template DNA for the reactions to occur, (2) produce a relatively small number of resolvable, visual fragments per reaction, (3) require time for the separation of the fragments and generation of the ladder, (4) require resequencing and overlapping sequencing reactions to determine the primary sequence of a long piece of DNA. A typical DNA sequencing as described above may yield the sequence of 300-500 nucleotides of a desired nucleic acid.
Alternatively, sequencing methods involving the use of an exonuclease to cleave off a terminal nucleotide of a single DNA molecule have been described. Jett et al. (U.S. Pat. No. 4,962,037) describes a method wherein a complementary strand of the DNA to be sequenced is synthesized with nucleotides covalently bonded to a fluorescent dye. Then, the labeled complementary strand of the desired DNA is sequenced using exonuclease cleavage. In practice, the exonuclease cleavage is hindered by the presence of dye on each nucleotide. Ishikawa (U.S. Pat. No. 5,528,046) describes the use of monoclonal antibodies against nucleotides A, G, T or C for detecting nucleotides freed from the DNA being sequenced. The monoclonal antibody in Ishikawa may be conjugated to a light emitting reagent, particularly a luminescent enzyme, to facilitate detection of the freed nucleotide. However, the use of monoclonal antibodies is disadvantageous, inter alia, because the production of monoclonal antibodies is labor intensive and requires considerable animal and cell culture resources for production and screening.
Thus, there is a need for alternative, sensitive methods for rapidly and accurately obtaining the nucleic acid sequence information. This is especially true for nucleic acid sequences that are long (greater than 1000 bp) and/or in short supply (less than nanomolar range).
1.2 Protein Sequencing
Chemical protein sequencing has been and continues to be one of the most popular methods for determining the primary structure of proteins. See Stolowitz, “Chemical Protein Sequencing and Amino Acid Analysis,”
Curr. Opin. Biotech.
4:9-13 (1993) and Hunkapiller, M. W., “Contemporary Methodology for the Determination of the Primary Structure of Proteins,”
Macromol. Seq. and Synthesis,
Ed. D. H. Schlesinger, pp.45-58, Alan R. Liss: New York, N.Y. (1988).
Traditional chemical amino-terminal sequencing includes a degradation step such as Edman degradation and a detection step. Edman degradation typically includes a coupling step, a cleavage step, and a conversion step. For example, in an Edman degradation, the amino terminus of a target polypeptide is coupled to an isothiocyanate reagent and then the derivatized N-terminal amino acid is cleaved from the polypeptide with a strong organic acid. The reagents of the Edman process may be delivered to the target polypeptide in a vapor (gas-phase method) or in a liquid pulse (pulsed-liquid method). The target polypeptide may be covalently (e.g., with carbonyldiimidazole) or non-covalently (e.g., with polybrene) attached to a solid support. Solid supports used in protein sequencing include polyvinylidene difluoride (PVDF), glass beads or polystyrene beads. The cleaved amino acid is typically converted to a more stable phenylthiohydantoin (PTH) form by treatment with an aqueous solution of strong organic acid. The PTH amino acid may be detected, for example, by high pressure liquid chromatography (HPLC) with UV absorbance detectors or by mass spectrometry (Aebersold, R., et al., “Design, Synthesis, and Characterization of a Protein Sequencing Reagent Yielding Amino Acid Derivatives with Enhanced Detectability by Mass Spectrometry,”
Protein Science
1:494-503 (1992)).
In an alternative chemical sequencing method, the degradation step involves the thioacetylation of the amino-terminal amino acid, which is detected by gas chromatography/mass spectrometry (Stolowitz, M L et al., “Thioacetylation Method of Protein Sequencing: Gas Chromatography/Ion Trap Mass Spectrometric Detection of 5-acetoxy-2-Methylthiazoles,”
J. Protein Chem.
11:360-361 (1992)). In another chemical sequencing process, a peptide ladder generated by Edman degradation is analyzed using matrix-assisted, laser desorption, time-of-flight mass spectrometry (Chait, et al., “Protein Ladder Sequencing,”
Science
262:89-92 (1993)).
Chemical cleavage of carboxy-terminal amino acids has been achieved through a variety of methods (Inglis, A. S., “Chemical Procedures for C-Terminal Sequencing of Peptides and Proteins,”
Analytical Biochemistry
195:183-196 (1991)). For example, the carboxy-terminus of a polypeptide has been coupled to a thiocyanate salt or thiocyanic acid (HSCN) to form a thiohydantoin or a peptidyl isothiocyanate which may be cleaved to form a thiohydantoin. The thiohydantoin-carboxy terminal amino acid can be detected by its UV absorption. Other carboxy-terminal cleavage reactions which do not involve the formation of a thiohydantoin can be characterized by the formation of (1) an acyl urea; (2) an O-peptidyl amino alcohol; (3) an N-peptidyl-2-oxazolidone; (4) an oxazole; and (5) an azide which is converted into an isocyanate. See, supra, Table 1 in Inglis.
Enzymatic digestion of terminal amino acids have been used to sequence polypeptides. Some amino-terminal and carboxy-terminal specific exopeptidases known in the art are carboxypeptidases (i.e. Y,
Kwagh Jae-Gyu
Macklin John J.
Mitsis Paul G.
Ulmer Kevin M.
Fish & Neave
Haley Jr. James F.
McGarry Sean
Praelux Incorporated
Zara Jane
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