Since our amino linkers and spacers possess extraordinarily long arms, we are able to synthesize long tethers with fewer incorporation's of charge from phosphodiester bonds. It has been shown that tethers of immobilized oligonucleotides with low negative charge and having a length of at least 40 atoms are optimal for obtaining the highest hybridization yields (Shchepinov et al., Nucleic Acids Res., 1997, 25, 1155). Using our longest spacer and amino linker we can synthesize a 72-atom tether possessing one negative charge. In order to match this length we would need 6 couplings of triethyleneglycol (TEG) spacers plus one C6 amino-linker coupling or 3 couplings of hexaethyleneglycol (HEG) spacers plus one C12 amino-linker coupling.
It is reasoned that a decreased negative charge on the tether reduces unfavorable electrostatic repulsion to the target DNA , thus maximizing the extent of hybridization. In our example, our tether has the least negative charge and possesses the same optimal length as the others. Furthermore, the synthesis of our tether requires only 2 couplings to attain the desired length, versus 7 couplings for the TEG-based tether and 4 couplings for the HEG-based tether. Fewer couplings not only ensure maximal synthetic yield.
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