Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-06-01
2001-04-17
Riley, Jezia (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C536S022100, C536S023100, C536S024300, C536S024330, C536S025300
Reexamination Certificate
active
06218530
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compounds and methods for detecting biomolecules, and in particular, the detection of nucleic acid and nucleic acid hybridization using photocleavable phosphoramidites.
BACKGROUND
Numerous methods have been developed for the incorporation of ligands into synthetic oligonucleotides using phosphoramidite chemistry. See S. L. Beaucage and P. I. Radhakrishnan,
Tetrahedron
49:1925 (1993). One approach is based on the incorporation of an aliphatic amino group into synthetic oligonucleotides by use of an amino-modifier phosphoramidites or modified CPGs. See S. Agrawal et al.,
Nucleic Acids Res.,
14: 6227 (1986). See P. S. Nelson et al.,
Nucleic Acids Res.
20:6253 (1992); P. S. Nelson et al.,
Nucleic Acids Res.
17:7179 (1989); P. S. Nelson et al.,
Nucleic Acids Res.
17: 7187 (1989). Such amino groups allow the post-synthetic addition of a variety of molecules including fluorescent markers, biotin, haptens or enzymes which can be used as hybridization probes for non-radioactive detection of specific target DNA/RNA or as sequencing primers. Amino-modified oligonucleotides have also been used for preparation of affinity matrices and as immobilized PCR primers.
One disadvantage of covalent labeling and immobilization of oligonucleotides is that the covalent bond is not selectively cleavable, and, therefore does not allow for the recovery/release of oligonucleotide or the removal of the label. In order to circumvent this problem, several approaches have been proposed based on chemical cleavage. These include the use of cleavable disulfide bonds or the introduction of acid cleavable linkers. See W Bannwarth and J. Wippler,
Helv. Chim. Acta
73:1139 (1990); B. D. Gildea et al.,
Tetrahedron Lett.
31:7095 (1990); E.Leikauf et al.,
Tetrahedron
51:3793 (1995); J. Olejnik et al.,
Nucleic Acids Res.
24: 361 (1996). These approaches, however, require the application of chemical reagents that are not suitable for many applications. They also necessitate removal of the excess of cleaving reagents and result in residual modification of the oligonucleotide.
SUMMARY OF THE INVENTION
The present invention relates to compounds and methods for detecting biomolecules, and in particular, the detection of nucleic acid and nucleic acid hybridization using photocleavable agents, including photocleavable detectable moieties, photocleavable nucleotides (e.g. photocleavable NTPs) and photocleavable phosphoramidites. It is not intended that the present invention be limited to the type of biomolecule detected. Both nucleic acids and proteins can be detected using the photocleavable agents of the present invention. The details regarding detection of various biomolecules (including proteins) are set forth in Attachments A, B, C, D and E hereto, all of which constitute the disclosure and specification of the present application.
In one embodiment, the present invention contemplates a photocleavable nucleotide. In a specific embodiment, the photocleavable nucleotide is a photocleavable dideoxynucleotide. The latter is useful in a variety of formats and assays, including but not limited to, nucleic acid sequencing.
In one embodiment, the present invention contemplates a composition, comprising a photocleavable aminotag phosphoramidite selected from the group consisting of compounds 6 and 7 of FIG.
2
B. From
FIG. 2B
, it should be clear that the present invention contemplates particular protecting groups (“PG”) suitable for such compounds, including but not limited to, 9-fluorenylmethoxycarbonyl (FMOC). In a preferred embodiment, the photocleavable aminotag phosphoramidite is incorporated into an oligonucleotide during synthesis at the 5′ end of an oligonucleotide.
In another embodiment, the present invention contemplates a composition, comprising a photocleavable sulfhydryltag phosphoramidite such as that shown in FIG.
4
. In a preferred embodiment, the photocleavable sulfhydryltag phosphoramidite is incorporated at the 5′ end of an oligonucleotide.
The present invention also contemplates methods of synthesis of photocleavable phosphoramidites and methods of synthesis of oligonucleotides containing such photocleavable phosphoramidites. Moreover, the present invention contemplates methods of incorporating detectable moieties or markers (“M”) to a phosphoramidite and photocleaving so as to release said marker.
In a further embodiment, the present invention contemplates oligonucleotides linked to a solid support or surface via a photocleavable linkage. In such an embodiment, the oligonucleotide can be released from the solid support (e.g. a bead) or a surface (e.g. a gold surface) by photocleavage.
REFERENCES:
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Agrawal, S., Christodoulu, C., and Gait, M., “Efficient Methods For Attaching Non-Radioactive Label To The 5′ Ends Of Synthetic Oligodeoxyribonucleotides,”Nucleic Acids Researchvol. 14 No. 15 pp. 6227-6245 (1986).
Bannwarth, W. and Wippler, J., “110. A new Combined Purification/Phosphorylation Procedure For Oligodeoxynucleotides,”Helvetica Chimica Acta, vol. 73, pp. 1139-1147 (1990).
Beaucage, S. and Radhakrishnan, P., “The Functionalization Of Oligonucleotides Via Phosphoramidite Derivatives,”Tetrahedronvol. 49, No. 10. pp. 1925-1963 (1993).
Gildea, B., Coull, J., and Koster, H., “A Versatile Acid-Labile Linker For Modification Of Synthetic Biomolecules,”Tetrahedron Letters, vol. 31, No. 49, pp. 7095-7098 (1990).
Leikauf, E., Barnekow, F., and Koster, H., “Heterobifunctional Trityl Derivatives As Linking Reagents For The Recovery Of Nucleric Acids After Labeling And Immobilization,”Tetrahedronvol. 51 No. 13, pp. 3793-3802 (1995).
Nelson, P., Frye R., and Edison, L., Bifunctional Oligonucleotide Probes Synthesized Using A Novel CPG Support Are Able To Dectect Single Base Pair Mutations Nucleic Acids Research, vol. 17 No. 18, pp. 7187-7195 (1989).
Nelson, P., Kent, M., and Muthini, S., “Oligonucleotide Labeling Methods 3. Direct Labeling Of Oligonucleotides Employing A Novel, Non-Nucleosidic, 2-Aminobutyl-1,3-Propanediol Backbone,”Nucleic Acids Researchvol. 20 No.23, pp. 6253-6259 (1992).
Nelson, P., Sherman-gold, R., and Leon, R., “A New And Versatile Reagent For Incorporating Mutiple Primary Aliphatic Amines Into Synthetic Oligonucleotides,”Nucleic Acids Researchvol. 17 No. 18, pp. 7179-7186 (1989).
Olejnik, J., Krymanska-Olejnik, E., and Rothschild, K., “Photocleavable Biotin Phosphoramidite For 5′-End-Labeling, Affinity Purification And Phosphorylation Of Synthetic Oligonucleotides,”Nucleic Acids Researchvol. 24 No. 2, pp. 361-366 (1996).
Olejnik Jerzy
Rothschild Kenneth J.
AmberGen Inc.
Medlen & Carroll LLP
Riley Jezia
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