Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-04
2001-10-30
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007500
Reexamination Certificate
active
06309825
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to agents for use in a method of detection of 8-oxoguanine, 8-oxodeoxyguanosine, 8-oxoadenine and 8-oxodeoxyadenosine, in particular for the detection of damaged nucleic acids, diagnostic tests for same and methods for the detection of same.
2. Description of the Related Art
Nucleic acid base damage, in particular oxidative base-damage of DNA, may arise as a result of a number of causes including toxins, carcinogenesis and neurodegenerative disorders. The detection of such damage is important in many fields of medicine including medical diagnostics, pathology and occupational health.
Present techniques for the detection of nucleic acid base damage rely on detection of 8-oxoguanine, a sensitive marker of DNA base-damage caused by oxygen free radicals (Kasai, H. and Nishimura, S., In: Sies, H., ed. Oxidative Stress: Oxidants and Antioxidants. London: Academic Press, 1991:99-116; Ames, B. N., 1989, Free Rad. Res. Comm., 7: 121-128), although 8-oxoguanine is only one of at least 20 products formed.
The use of 8-oxoguanine as a marker of oxidative damage to DNA followed the description of its analysis as the deoxynucleoside (8-oxodeoxyguanosine) in DNA digests by HPLC (high pressure liquid chromatography) with electrochemical detection (Floyd, R. A. et al., 1986, Free Rad. Res. Commun. 1: 163-172). Alternatively, gas chromatography-mass spectrometry (GC-MS) methods have been used to quantitate 8-oxoguanine as the free base (Dizdaroglu, M., 1994, Methods Enzymol., 234: 3-16), but the technique is expensive and technically demanding to use. Nevertheless the two techniques have been compared (as reviewed in Halliwell, B. and Dizdaroglu, M., 1992. Free Rad. Res. Commun. 16: 75-87) and results obtained do not concur. In general. levels of 8-oxoguanine are higher when determined by the GC-MS technique and even in freshly isolated cells, 2-11 fold higher values have been reported by GC-MS.
A number of other approaches to the determination of 8-oxoguanine in DNA have been described.
32
P-postlabelling procedures are well established in the literature (Lu, L. J. W. et al., 1991, Chem. Pharmaceut. Bull. 39: 1880-1882; Povey, A. C. et al., In: Postlabelling Methods for Detection of DNA Adducts. Lyon, International Agency for Research on Cancer, 1993:105-114); these methods provide the potential for very sensitive detection although the techniques are very time-consuming and cumbersome. Capillary electrophoretic determinations of 8-oxodeoxyguanosine (Guarnieri C. et al., 1994, J. Chromatogr. B. 656: 209-213) and 8-oxoguanine (Poon, K. W. et al., 1995, Biochem. Soc. Trans. 23: 433s) have been described. To date, these techniques lack concentration sensitivity due to inherent lack of sensitivity of UV absorbence measurements.
SUMMARY OF THE INVENTION
According to the present invention there is provided an agent for use in a method of detection of 8-oxoguanine, 8-oxodeoxyguanosine, 8-oxoadenine and 8-oxodeoxyadenosine comprising a molecule which binds specifically to biotin and to at least one of 8-oxoguanine, 8-oxodeoxyguanosine, 8-oxoadenine and 8-oxodeoxyadenosine (the most common tautomeric forms thereof known as, respectively, 8-hydroxy guanine, 8-hydroxydeoxyguanosine, 8-hydroxyadenine and 8-hydroxydeoxyadenosine).
The purine base guanine bears little structural relation to the biotin molecule. However, although the exact nature of the binding of avidin to damaged DNA is currently unknown, the keto form of 8-oxoguanine, a damage product of oxidative DNA damage, has been found by the inventors to be structurally remarkably similar to biotin, and avidin, streptavidin and antibodies to biotin appear to have significant binding affinity for this oxidised base product.
Structural considerations strongly suggest that avidin will bind to 8-oxoadenine and 8-oxodeoxyadenosine since these compounds also possess the imidazilidone group.
By ‘binds specifically’ is meant that the agent has a specific affinity for a particular epitope or epitopes. Hence avidin binds specifically to at least biotin, 8-oxoguanine and 8-oxodeoxyguanosine, the molecules having the same or substantially similar epitopes. This specificity is analogous to an antibody specific to an epitope of a polypeptide also being specific to a mimotope (Geysen, H. M. et al., 1987, Journal of Immunological Methods, 102: 259-274) of the epitope, although the sequence of the mimotope may be different to that of the polypeptide.
The agent may have substantially the same binding specificity for biotin as avidin or streptavidin. It may also have substantially the same binding specificity for at least one of 8-oxoguanine, 8-oxodeoxyguanosine, 8-oxoadenine and 8-oxodeoxyadenosine as avidin or streptavidin.
By “the same binding specificity” is meant that the agent-for example a fragment, analogue, antibody or antigen binding fragment—is specific to substantially the same epitope or epitopes as avidin or streptavidin.
The agent may be selected from any one of the group of avidin and streptavidin.
The agent may comprise a fragment or an analogue of avidin or streptavidin. Avidin and streptavidin may be readily studied and altered to produce fragments of the molecule, for example fragments which comprise solely the biotin-binding part of the molecule. Analogues (to the molecules themselves or to fragments thereof) may also be produced. For example the analogues may have an altered binding specificity or binding affinity for the biotin molecule or for damaged DNA.
By “fragment or analogue of avidin or streptavidin” is meant a fragment or analogue of the avidin or streptavidin molecule which binds specifically to biotin and to at least one of 8-oxoguanine, 8-oxodeoxyguanosine, 8-oxoadenine and 8-oxodeoxyadenosine.
The present inventors have found that, remarkably, avidin, streptavidin and antibody specific to biotin may be used to detect 8-oxoguanine, 8-oxodeoxyguanosine, 8-oxoadenine and 8-oxodeoxyadenosine, and therefore damaged nucleic acids, in particular damaged DNA.
Avidin is a naturally occurring factor found in egg white, which has a remarkable affinity (K
a
=10
15
M
−1
) for the vitamin biotin (Bayer, E. A. and Ichek, M., 1990, Methods in Enzymol., 184: 49-67), and the bacterial analogue streptavidin, from
Streptomyces avidinii
, has a substantially similar binding affinity and specificity.
The biotin molecule (Green, N. M., 1972, Adv. Protein Chem., 29: 85-131) is largely hydrophobic and consists of a ureido group and an imidazilidone ring. It would appear that the whole molecule interacts with the avidin binding site, avidin comprising a stable tetramer with two-fold symmetry and containing four biotin binding sites arranged in two clusters. Although a wide range of compounds that are analogues of small fragments of the biotin molecule will bind to avidin, the binding of avidin to biotin appears to be of relatively high specificity since related compounds do not bind significantly at the
10
mM level (Green, 1972, supra).
Avidin is widely employed in both research and technology primarily as a secondary means of detection and amplification. It is frequently used to detect biotin in immunoassays where a primary antibody is either directly biotinylated or is bound by a secondary biotinylated antibody. It is also used where a target molecule, such as a base, is chemically modified by biotinylation to aid in its visualisation.
Avidin is not currently used for the direct detection of biomolecules other than biotin.
Alternatively, the agent may comprise an antibody or antigen binding fragment thereof.
Any antibody referred to herein may be a whole antibody or an antigen binding fragment thereof and may in general belong to any immunoglobulin class. Thus, for example, it may be an IgM, IgG or IgA, antibody. The antibody or fragment may be of animal, for example, mammalian origin and may be for example of murine, rat or human origin. It may be a natural antibody or a fragment thereof, or, if desired, a recombinant antibody fragment, i.e. an antibody o
Bracewell & Patterson LLP
Saunders David
Tung Mary Beth
University of Leicester
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