Fluorescent conjugates of nucleosides or nucleotides,...

Details Fluorescent conjugates of nucleosides or nucleotides,... Fluorescent conjugates of nucleosides or nucleotides,...

2000-04-20

2002-01-22

Riley, Jezia (Department: 1656)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S022100, C536S025300, C536S025320, C435S006120, C540S549000, C534S015000

Reexamination Certificate

active

06340747

ABSTRACT:

The invention relates to novel fluorescent conjugates of nucleosides or nucleotides which can be used especially for detecting, locating and/or isolating nucleic acids or molecules of biological or clinical interest which have a nucleoside structure or are capable of interacting with nucleic acids.
The following abbreviations will be used in the remainder of the description:
RNA: ribonucleic acid
DNA: deoxyribonucleic acid
A: adenosine
C: cytidine
G: guanosine
T: thymidine
U: uridine
I: inosine
suffix MP: monophosphate
suffix DP: diphosphate
suffix TP: triphosphate
prefix d: deoxy
A reaction for the enzymatic synthesis of DNA employs an RNA or DNA template, an oligonucleotide primer whose sequence is complementary to a segment of the template, an appropriate enzyme, and the four deoxynucleotides dATP, dCTP, dGTP and dTTP (or dUTP). Various enzymes are known, such as
E. coli
DNA polymerase, T7 DNA polymerase, the Klenow fragment of DNA polymerase, Taq DNA polymerase and a reverse transcriptase, to which may be added terminal nucleotidyl transferase, which has the particular characteristic of not requiring a template. The synthesis of RNA is carried out in a similar manner except that the required primers and templates are different and ribonucleotides (ATP, CTP, GTP and UTP) are used in the presence of RNA polymerases.
Nucleotides or polynucleotides can be labeled radioactively (
3
H,
32
P,
14
C,
35
S or
125
I).
The use of labeled molecules has the disadvantages conventionally associated with radioactive isotopes, namely the risks inherent in radioactivity as well as the limited storage and availability due to radioactive decay and radiolysis phenomena.
The chemical labeling of nucleotides or polynucleotides, which makes it possible to avoid these disadvantages, has been described in the literature. The biotin labeling of nucleotides derived from dUTP or UTP has been described in particular (Langer P. R., Waldrop A. A., Ward D. C., 1981, Proc. Natl. Acad. Sci. USA, 78, 6633-37). These are derivatives in which the biotin is bonded to the C-5 position of the pyrimidine ring by an alkylamine arm. These labeled nucleotides are incorporated in vitro into polynucleotides by the action of DNA or RNA polymerases (EP 0 063 879) and allow the calorimetric detection of nucleic acids by means of a dot-blot reaction (Leary J. J., Brigati D. J. and Ward D. D., 1983, Proc. Natl. Acad. Sci. USA, 80, 4045-49).
Other analogs of biotin-labeled nucleotides, based on derivatives of N-4-aminoalkyldeoxycytidine and N-6-aminoalkyldeoxyadenosine, are described in patent U.S. Pat. No. 4,828,979 and in Gebegehu G. G. et al., Nucleic Acids Res., 1987, 15, 4513-4534.
Derivatives of dUTP and of dATP substituted by biotin in the C-8 position, as well as the possibility of the C-7 substitution of a 7-deazapurine, are also described (EP 0 063 879).
The derivative bio-15-dGTP has also been described (Gilliam I. C. and Tener G. M., 1989, Nucleoside & Nucleotide, 8, 1453-62). Fluorescent derivatives such as fluorescein or rhodamine can be incorporated into a nucleic acid via a labeled nucleoside triphosphate (dATP, dUTP, dCTP) (WO 93 19206). A discussion about the position of substitutions on purines and pyrimidines and about the nature of the spacer arms which can be used to label dideoxynucleotides with fluorescein derivatives, although devoted to chain terminators for sequencing, gives an overview of the chemistry of labeled nucleotides (Confalone P. T., 1990, J. Heterocyclic Chem., 27, 31-46).
The combined use of nucleoside triphosphates labeled with different tracers (biotin-11-dUTP, dig-11-dUTP and FITC-11-dUTP) affords the simultaneous visualization of different sequences during a hybridization (REED T. et al., Proc. Natl. Acad. Sci. USA (1992), 89, 1388-1392).
The incorporation of fluorescein-labeled deoxynucleotides such as Fl-dUTP or Fl-dCTP is also effected by replacing only part of the natural nucleotide with the labeled compound: dCTP/Fl-dCTP≈2:1 (WO 93/19206). This same patent describes that, by choosing the enzyme and the experimental conditions, it is possible to replace the whole of a nucleotide with a labeled nucleotide.
The literature data show that the efficiency of incorporation of a tripbosphate conjugate is modified by coupling with a molecule such as biotin and to a lesser extent by the presence of the arm which allows the coupling.
For example, in a nick translation reaction in the presence of a DNA polymerase, the degrees of incorporation of various triphosphate analogs were compared with the natural nucleoside taken as a reference (100% incorporation) for the same reaction time (90 min) (Gebeyehu G. et al., Nucleic Acids Res., 1987, 15, 4525).
Molecules conjugated with nucleoside triphosphates (Goodchild, J. Bioconjugate Chem., 1990, p. 171; Kricka J., Non isotopic Blotting, and Sequencing, 1995, Academic Press, p. 47; Zhu Z. et al., Nucleic Acids Res., 1994, 3418-3422) are molecules of relatively small size (<800 Da) which are either neutral or negatively charged and have an essentially planar shape; consequently, their bulk is reduced but sufficient to disturb the enzymatic incorporation of the nucleoside triphosphate.
Novel conjugates of nucleosides or nucleotides have now been found which comprise:
a ribo- or deoxyribo-nucleoside or -nucleotide which is native, chemically modified or conjugated with one or more labeling molecules, in which at least one carbon atom of the ring, exocyclic nitrogen atom of the purine or pyrimidine ring or carbon atom of the pentofuranose ring may be involved in bonding with a fluorescent marker, and
at least one fluorescent marker bonded to said atom(s) and consisting of a rare earth cryptate.


REFERENCES:
patent: 5220012 (1993-06-01), Mathis et al.
patent: 5457185 (1995-10-01), Lehn et al.
patent: 5512493 (1996-04-01), Mathis et al.
patent: 5527684 (1996-06-01), Mabile et al.
patent: 0321353 (1989-06-01), None

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