Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1990 Sep 25;18(18):5419–5423. doi: 10.1093/nar/18.18.5419

Site specific functionalization of oligonucleotides for attaching two different reporter groups.

S Agrawal 1, P C Zamecnik 1
PMCID: PMC332219  PMID: 2216715

Abstract

The synthesis of an oligonucleotide functionalized to attach two different reporter groups at specific internucleotide linkages is described. To incorporate the amine specific reporter group the internucleotide linkage is modified to phosphoramidate (N-1-aminoalkyl) and for a thiol specific reporter group the internucleotide linkage is modified to a phosphorothioate diester. The synthetic cycle for introducing the modified internucleotide linkages at specific sites can be carried out using an automated DNA synthesizer. Combination of reporter groups have been attached successfully.

Full text

PDF
5419

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agrawal S., Christodoulou C., Gait M. J. Efficient methods for attaching non-radioactive labels to the 5' ends of synthetic oligodeoxyribonucleotides. Nucleic Acids Res. 1986 Aug 11;14(15):6227–6245. doi: 10.1093/nar/14.15.6227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Agrawal S., Mayrand S. H., Zamecnik P. C., Pederson T. Site-specific excision from RNA by RNase H and mixed-phosphate-backbone oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1401–1405. doi: 10.1073/pnas.87.4.1401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Allen D. J., Darke P. L., Benkovic S. J. Fluorescent oligonucleotides and deoxynucleotide triphosphates: preparation and their interaction with the large (Klenow) fragment of Escherichia coli DNA polymerase I. Biochemistry. 1989 May 30;28(11):4601–4607. doi: 10.1021/bi00437a014. [DOI] [PubMed] [Google Scholar]
  4. Ansorge W., Sproat B., Stegemann J., Schwager C., Zenke M. Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis. Nucleic Acids Res. 1987 Jun 11;15(11):4593–4602. doi: 10.1093/nar/15.11.4593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Arai H., Emson P. C., Agrawal S., Christodoulou C., Gait M. J. In situ hybridization histochemistry: localisation of vasopressin mRNA in rat brain using a biotinylated oligonucleotide probe. Brain Res. 1988 Aug;464(1):63–69. doi: 10.1016/0169-328x(88)90019-8. [DOI] [PubMed] [Google Scholar]
  6. Beck S., Pohl F. M. DNA sequencing with direct blotting electrophoresis. EMBO J. 1984 Dec 1;3(12):2905–2909. doi: 10.1002/j.1460-2075.1984.tb02230.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bischoff R., Coull J. M., Regnier F. E. Introduction of 5'-terminal functional groups into synthetic oligonucleotides for selective immobilization. Anal Biochem. 1987 Aug 1;164(2):336–344. doi: 10.1016/0003-2697(87)90502-1. [DOI] [PubMed] [Google Scholar]
  8. Brumbaugh J. A., Middendorf L. R., Grone D. L., Ruth J. L. Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5610–5614. doi: 10.1073/pnas.85.15.5610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cardullo R. A., Agrawal S., Flores C., Zamecnik P. C., Wolf D. E. Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8790–8794. doi: 10.1073/pnas.85.23.8790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Caruthers M. H., Barone A. D., Beaucage S. L., Dodds D. R., Fisher E. F., McBride L. J., Matteucci M., Stabinsky Z., Tang J. Y. Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method. Methods Enzymol. 1987;154:287–313. doi: 10.1016/0076-6879(87)54081-2. [DOI] [PubMed] [Google Scholar]
  11. Chollet A., Kawashima E. H. Biotin-labeled synthetic oligodeoxyribonucleotides: chemical synthesis and uses as hybridization probes. Nucleic Acids Res. 1985 Mar 11;13(5):1529–1541. doi: 10.1093/nar/13.5.1529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chu B. C., Orgel L. E. Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds. Nucleic Acids Res. 1988 May 11;16(9):3671–3691. doi: 10.1093/nar/16.9.3671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chu B. C., Wahl G. M., Orgel L. E. Derivatization of unprotected polynucleotides. Nucleic Acids Res. 1983 Sep 24;11(18):6513–6529. doi: 10.1093/nar/11.18.6513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Connolly B. A., Potter B. V., Eckstein F., Pingoud A., Grotjahn L. Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site. Biochemistry. 1984 Jul 17;23(15):3443–3453. doi: 10.1021/bi00310a010. [DOI] [PubMed] [Google Scholar]
  15. Connolly B. A. The synthesis of oligonucleotides containing a primary amino group at the 5'-terminus. Nucleic Acids Res. 1987 Apr 10;15(7):3131–3139. doi: 10.1093/nar/15.7.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Emson P. C., Arai H., Agrawal S., Christodoulou C., Gait M. J. Nonradioactive methods of in situ hybridization: visualization of neuroendocrine mRNA. Methods Enzymol. 1989;168:753–761. doi: 10.1016/0076-6879(89)68056-1. [DOI] [PubMed] [Google Scholar]
  17. Froehler B. C., Ng P. G., Matteucci M. D. Synthesis of DNA via deoxynucleoside H-phosphonate intermediates. Nucleic Acids Res. 1986 Jul 11;14(13):5399–5407. doi: 10.1093/nar/14.13.5399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ghosh S. S., Kao P. M., Kwoh D. Y. Synthesis of 5'-oligonucleotide hydrazide derivatives and their use in preparation of enzyme-nucleic acid hybridization probes. Anal Biochem. 1989 Apr;178(1):43–51. doi: 10.1016/0003-2697(89)90354-0. [DOI] [PubMed] [Google Scholar]
  19. Haralambidis J., Duncan L., Angus K., Tregear G. W. The synthesis of polyamide-oligonucleotide conjugate molecules. Nucleic Acids Res. 1990 Feb 11;18(3):493–499. doi: 10.1093/nar/18.3.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hodges R. R., Conway N. E., McLaughlin L. W. "Post-assay" covalent labeling of phosphorothioate-containing nucleic acids with multiple fluorescent markers. Biochemistry. 1989 Jan 10;28(1):261–267. doi: 10.1021/bi00427a036. [DOI] [PubMed] [Google Scholar]
  21. Jablonski E., Moomaw E. W., Tullis R. H., Ruth J. L. Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res. 1986 Aug 11;14(15):6115–6128. doi: 10.1093/nar/14.15.6115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jäger A., Levy M. J., Hecht S. M. Oligonucleotide N-alkylphosphoramidates: synthesis and binding to polynucleotides. Biochemistry. 1988 Sep 20;27(19):7237–7246. doi: 10.1021/bi00419a010. [DOI] [PubMed] [Google Scholar]
  23. Kaiser R. J., MacKellar S. L., Vinayak R. S., Sanders J. Z., Saavedra R. A., Hood L. E. Specific-primer-directed DNA sequencing using automated fluorescence detection. Nucleic Acids Res. 1989 Aug 11;17(15):6087–6102. doi: 10.1093/nar/17.15.6087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lamond A. I., Sproat B., Ryder U., Hamm J. Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2'-OMe RNA. Cell. 1989 Jul 28;58(2):383–390. doi: 10.1016/0092-8674(89)90852-0. [DOI] [PubMed] [Google Scholar]
  25. Landegren U., Kaiser R., Caskey C. T., Hood L. DNA diagnostics--molecular techniques and automation. Science. 1988 Oct 14;242(4876):229–237. doi: 10.1126/science.3051381. [DOI] [PubMed] [Google Scholar]
  26. Letsinger R. L., Bach S. A., Eadie J. S. Effects of pendant groups at phosphorus on binding properties of d-ApA analogues. Nucleic Acids Res. 1986 Apr 25;14(8):3487–3499. doi: 10.1093/nar/14.8.3487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Loke S. L., Stein C. A., Zhang X. H., Mori K., Nakanishi M., Subasinghe C., Cohen J. S., Neckers L. M. Characterization of oligonucleotide transport into living cells. Proc Natl Acad Sci U S A. 1989 May;86(10):3474–3478. doi: 10.1073/pnas.86.10.3474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Matayoshi E. D., Wang G. T., Krafft G. A., Erickson J. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science. 1990 Feb 23;247(4945):954–958. doi: 10.1126/science.2106161. [DOI] [PubMed] [Google Scholar]
  29. Matthews J. A., Kricka L. J. Analytical strategies for the use of DNA probes. Anal Biochem. 1988 Feb 15;169(1):1–25. doi: 10.1016/0003-2697(88)90251-5. [DOI] [PubMed] [Google Scholar]
  30. Murchie A. I., Clegg R. M., von Kitzing E., Duckett D. R., Diekmann S., Lilley D. M. Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules. Nature. 1989 Oct 26;341(6244):763–766. doi: 10.1038/341763a0. [DOI] [PubMed] [Google Scholar]
  31. Smith L. M., Fung S., Hunkapiller M. W., Hunkapiller T. J., Hood L. E. The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis. Nucleic Acids Res. 1985 Apr 11;13(7):2399–2412. doi: 10.1093/nar/13.7.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sproat B. S., Beijer B., Rider P., Neuner P. The synthesis of protected 5'-mercapto-2',5'-dideoxyribonucleoside-3'-O-phosphoramidites; uses of 5'-mercapto-oligodeoxyribonucleotides. Nucleic Acids Res. 1987 Jun 25;15(12):4837–4848. doi: 10.1093/nar/15.12.4837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sproat B. S., Beijer B., Rider P. The synthesis of protected 5'-amino-2',5'-dideoxyribonucleoside-3'-O-phosphoramidites; applications of 5'-amino-oligodeoxyribonucleotides. Nucleic Acids Res. 1987 Aug 11;15(15):6181–6196. doi: 10.1093/nar/15.15.6181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sproat B. S., Lamond A. I., Beijer B., Neuner P., Ryder U. Highly efficient chemical synthesis of 2'-O-methyloligoribonucleotides and tetrabiotinylated derivatives; novel probes that are resistant to degradation by RNA or DNA specific nucleases. Nucleic Acids Res. 1989 May 11;17(9):3373–3386. doi: 10.1093/nar/17.9.3373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stryer L. Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem. 1978;47:819–846. doi: 10.1146/annurev.bi.47.070178.004131. [DOI] [PubMed] [Google Scholar]
  36. Urdea M. S., Warner B. D., Running J. A., Stempien M., Clyne J., Horn T. A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes. Nucleic Acids Res. 1988 Jun 10;16(11):4937–4956. doi: 10.1093/nar/16.11.4937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zuckermann R., Corey D., Schultz P. Efficient methods for attachment of thiol specific probes to the 3'-ends of synthetic oligodeoxyribonucleotides. Nucleic Acids Res. 1987 Jul 10;15(13):5305–5321. doi: 10.1093/nar/15.13.5305. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES