Attachment of oligonucleotides to solid supports through schiff base type linkages for capture and detection of nucleic acids

#The present invention generally relates to the chemistry of the attachment of oligonucleotides to solid supports. More particularly the present invention relates to linking oligonucleotides to solid supports through a Schiff base type covalent linkage for capture and detection of single- and double stranded DNA and RNA targets.

#The detection and quantification of very small quantifies of nucleic acids plays an important role in the biological, forensic and medical sciences. Typically nucleic acids in samples are detected by hybridization to a complementary oligonucleotide containing more than 8 contiguous nucleotides. To provide a signal proportional to the target-oligonucleotide hybrid, typically either the target or the second probe contains a signal generating label, such as a radioactive-, fluorescent-, chemiluminescent-moiety or an enzyme (such as horseradish peroxidase) that through its catalytic activity yields a detectable product. The prior art is well developed in this regard and numerous methods are available for the detection and quantification of signal in the nucleic acid field.

#Following the hybridization of the capturing and labeled oligonucleotide to the target nucleic acid it is necessary to separate the signal generating duplex from unreacted target and labeled oligonucleotide. This can usually be accomplished because either the target, or more typically the capturing oligonucleotide has been immobilized on a solid support thereby allowing the isolation of the hybrid free from unhybridized molecules. In a “sandwich assay” variation, an oligonucleotide is immobilized to a solid support and is used to capture a target. The captured target is detected by hybridization with a second labeled oligonucleotide, that has a different sequence than the capturing oligonucleotide.

#FIG. 1 is a graph showing in three dimensions the optimization of oligonucleotide attachment via semicarbazone bonds to a glass surface as a function of concentration of the oligonucleotide and pH of the media.

#FIG. 2 is a graph showing the attachment of oligonucleotide to the glass surface as expressed in units of fmol/spot as a function of time.

#FIG. 3 is a graph showing the efficiency of oligonucleotide attachment and efficiency of hybridization as a function of oligonucleotide concentration applied on each spot.

#Derivatized Supports

#As is noted in the Summary, in accordance with the present invention one of the solid support or the oligonucleotide (ODN) contains a nucleophilic amino group while the other contains an aromatic or heteroaromatic aldehyde or ketone capable of reacting with the nucleophilic amino group to form a Schiff base-type covalent linkage that attaches the ODN to the solid support, in a reasonably fast, high yield reaction resulting in high concentrations of the ODN per unit surface of the solid support, bound thereto by a stable covalent bond. In order to have these properties the nucleophilic amino group preferably and ideally has a pKa less than 7.0 In the preferred embodiments the nucleophilic amino (NH2) group is covalently linked to the solid support while the aromatic aldehyde or ketone (preferably aldehyde) is linked to the ODN.

#Thus, the solid supports used in the preferred embodiments of the invention contain the nucleophilic NH2 group, as a primary amine (R′—NH2), or as a hydrazinyl,