Conjugation & Surface Attachment Design and Protocols

Conjugation & Surface Attachment Design / Protocol

Over the past 20 years, a wide variety of robust, publicly available protocols have been developed either to conjugate oligonucleotides to chemical moieties (e.g., to haptens, enzymes, fluorescent dyes) for use as probes, or to covalently attach oligonucleotides to a solid surface (e.g., glass slides) for use in DNA microarray applications. Many oligonucleotide-based assays actually combine both of these aspects in one package. Optimal design of such combination assays requires consideration of several different parameters.

DNA microarrays are excellent platforms for high-density screening applications, as a large number of different sequences can be immobilized to a planar surface for interrogation of a sample. Either amino- or thiol-end-modified oligonucleotides can be covalently attached to glass slides or silicon wafers that have been suitably modified chemically for that purpose. In addition, it is critical that the microarray be designed so that it has the appropriate oligo surface density to ensure sufficient hybridization between immobilized oligo probe and target occurs to obtain a good signal. In particular, optimal surface coverage decreases with increasing oligo length dependent, presumably due to steric hindrance (6).

Oligonucleotides attached to microspheres are used in a variety of assays requiring oligos that are immobilized on a solid support that can be freely suspended in solution. The use of magnetic microspheres with oligo-dT to capture mRNA from cell lysates is one well-known application. Another particularly interesting such application is termed “liquid assays”. Here a probe oligo is covalently attached to a polystyrene microsphere containing a fluorescent dye inside them. By using microspheres with different dyes, or dye combinations, and a unique oligo probe for each color microsphere, highly multiplexed, solution-based hybridization assays can be designed in convenient, microtiter-plate format. Such assays exhibit rapid hybridization times, which is an important advantage. It is important to remember, however, that careful optimization of hybridization conditions may need to be done to ensure robust, reproducible detection of all the sample targets being probed for. The targets may be an oligo, cDNA, PCR product, or even a protein, and are fluorescently labeled (with the label different from that of the fluorescent microsphere). After hybridization, the microspheres are typically assayed by flow cytometry, with the fluorescence of the microsphere identifying the probe and the simultaneous fluorescence of the target indicating hybridization (7,8).

While many chemical moieties are available as phosphoramidites, and so can be directly incorporated into an oligo during synthesis, others are not. The latter often have properties that are particularly useful for oligos slated for use as probes. For example, Alexa dyes (highly fluorescent) and digoxigenin (DIG--low background/high sensitivity in situ probes) are available only as NHS esters. Their incorporation into an oligonucleotide requires the presence of an primary amino group on the oligo for conjugation.

Generally speaking, conjugating an NHS-ester to an amine-modified oligo is an excellent way to generate a modified oligo. The resulting amide linkage between the modification and the oligo is very stable, and the modified oligo can be stored long term at -20C.

References

(1) Immobilization Chemistry. in Immobilization of DNA on Chips II, C. Whittman (Vol. Ed.). Springer-Verlag, Berlin, Heidelberg (2005): 51-53.
(2) Connolly, B.A., Rider, P. Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specfic probes. Nucleic Acids Res. (1985), 13: 4485-4502.
(3) Roger, Y-H., Jiang-Baucom, P., Huang, Z-J., Bogdanov, V., Anderson, S., Boyce-Jacino, M.T. Immobilization of oligonucleotides onto a glass support via disulfide bonds: A method for preparation of DNA microarrays. Anal. Biohem. (1999), 266: 23-30.
(4) Ackerson, C.J., Sykes, M.T., Kornberg, R.D. Defined DNA/nanoparticle conjugates. Proc. Natl. Acad. Sci. USA (2005), 102: 13383-13385.
(5) Li, Z., Jin, R., Mirkin, C.A., Letsinger, R.L. Multiple thiol-anchor capped DNA-gold nanoparticle conjugates Nucleic Acids Res. (2002), 30: 1558-1562.
(6) Guo, Z., Guilfoyle, R.A., Thiel, A.J., Wang, R., Smith, L.M. Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Res. (1994), 22: 5456-5465.
(7) Yang, L., Tran, D.K., Wang, X. BADGE, Beads Array for the Detection of Gene Expression, a high-throughput diagnostic bioassay. Genome Res. (2001), 11: 1888-1898.
(8) Defoort, J.P. Simulataneous detection of multiplex-amplified human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA using a flow cytometer microsphere-based hybridization assay. J. Clin. Microbiol. (2000), 39: 131-140.