Bioorthogonal Probes

Diel-alder reaction consists of a 4 + 2 cycloaddition between a diene and an alkene, often called dienophile. Diene modified oligonucleotides can be prepared by solid-phase synthesis using a 3,5-hexadiene phosphoramidite derivative, which could be incorporated into the oligo at the 5' end.

The Trans-Cyclooctene-Tetrazine click chemistry is a very powerful tool in catalyst‐free bioconjugation chemistry. The reaction follows an inverse‐electron demand Diels‐Alder cycloaddition reaction of trans‐cyclooctenes (TCO) with tetrazines. This bioorthogonal reaction possesses an exceptionally high reaction speed and nearly quantitative yield under physiocological conditions with reaction rate constants 2000 M-1 s−1 (in 9:1 methanol/water). The extremely fast kinetics and selectivity enables the conjugation of two low abundance biopolymers in an aqueous and otherwise complex chemical environment through the formation of a stable dihydropyridazine. This bioorthogonal reaction possesses extreme selectivity and biocompatibility, such that the complimentary reagents can form covalent bonds within richly functionalized biological systems, in some cases, living organisms.

The reaction between an aldehyde or ketone and a hydrazide or hydrazine functional group to form a hydrzone bond has been frequently used for bioconjugation reaction. Aldehyde, hydrazide modified oligonucleotides can be prepared by solid-phase synthesis or functionalized post-synthetically.

The chemoselective conjugation of an aminooxy modified oligo with an aldehyde is similar to that of the reaction of a hydrazide with an aldehyde, except instead of giving a hydrazone bond it yields an oxime linkage. Aminooxy modified oligonucleotides can be prepared by solid-phase synthesis using a 5'-aminooxy modifier 11. This modifier is based on a tetraethylene glycol linkage for improved solubility and for reducing the potential negative impact on hybridization of the oligo. The oxime formed from the reaction of alkyloxyamines with aldehydes creates a stable covalent bond.