5' Amino Modified Oligonucleotides
Amino-modified oligonucleotides have been routinely employed in solid support and label (or functionality) attachment chemistries. The 5′-terminus of the oligonucleotide is normally the target end for modification because of the ease of incorporation as the last step in automated synthesis. For several applications (see below) factors, such as sterics, electrostatic repulsion, binding kinetics and hybridization efficiency, require a longer distance between the oligo and the point of attachment. This is why our 5′-amino linkers are available in a variety of tethering arms, based on their length, charge density, hydrophobicity, flexibility, and multiplicity of amino groups on the tether. You can also create your own tether or a library of oligonucleotides with a differently tethered functionality to satisfy your particular needs.
Many factors come into play when designing oligonucleotides for immobilization on a solid-phase support for a multitude of applications and assays. Maximizing hybridization of target DNA/RNA to oligonucleotides covalently attached to the surface of a chip is key and ensures that accurate information is derived from the microarray assay. The parameters having the largest effect on hybridization yield are length, charge, and hydrophobicity of the tethers holding the probe oligonucleotide in place.
Long tethers increase accessibility for enzymes involved in nucleic acid processing events staged at the solid-phase support (Carmon et al., BioTechniques, 2002, 32, 410). The tethering arms of our “super-long” amino linkers are up to 39 atoms in length and possess no net charge. The tether lengths can be maximized by incorporating our longest spacer prior to amino modification at the cost of adding 1 negative charge (from a phosphodiester bond) for each 33-atom increase in length. Thus, we can make amino linkers with 72-atom, 105-atom, and 138-atom tethers adding just one negative charge per jump in length. Our spacers,
in combination with our amino linkers, may not only be used to alter the tether length, but also vary flexibility, hydrophobicity, and charge density on the tether to suit your individual requirements. You can see the incorporation of charge in a comparison of our tethers and ones based on triethyleneglycol and hexaethyleneglycol units.
Another parameter which has been shown to affect hybridization and the kinetics of target capture is the probe density at the array surface (Southern et al., Nat. Genet. Suppl., 1999, 21, 5). Controlling the electrostatic repulsion between probe strands and minimizing the conformation (flexibility) of the DNA strands are factors that directly affect the probe density. Our tethers, which can be made at variable lengths and flexibilities, may provide the ideal conditions for optimal probe coverage and hybridization kinetics by extending the oligonucleotide away from the crowded surface.
It has been shown that steric hindrance, rather than the efficiency of hybridization between the template and the immobilized oligonucleotide, is the primary reason for loss in solid-phase primer extension (Carmon et al., BioTechniques, 2002, 32, 410). This unfavorable interaction was minimized by increasing (optimizing) the tether length to 5-10 hexaethyleneglycol (HEG) units. However, by coupling HEG units sequentially to the 5′-end two adverse events occur. The overall synthetic yield of the primer is decreased, and a net negative charge (due to phosphodiester bonds) is incorporated within the tether. This negative charge density on the tether has been shown in a previous study to decrease the yield of hybridization due possibly to electrostatic repulsion to the target (Shchepinov et al., Nucleic Acids Res., 1997, 25, 1155). Our longest amino linker with a 39-atom tether is more than twice the length of a HEG unit and is slightly more rigid to ensure full arm extension of the tether. Therefore, when used together with our long spacers (up to 31 atoms long), fewer sequential additions to the 5′-end are needed to attain the optimal length tether (and less negative charge density) in the amino-modified oligonucleotides immobilized for use in solid-phase PCR. You can see a comparison of our tethers and ones based on triethyleneglycol and hexaethyleneglycol units.
In another example overcoming unfavorable sterics, increasing the tether length of primer oligos bound to Au particles resulted in lower primer surface coverage, which subsequently led to improved hybridization efficiencies (Nicewarner Peña et al., JACS, 2002, 25, 7314). Furthermore, enzymatic extension of the particle-bound primers attached by the longest (49-atom) tether in this study was as efficient as the solution-phase reaction. It is worth noting, however, that this tether possessed 7 negative charges from the nucleotides and C6 thiol spacer required to make it. The combination of our “super-long” thiol spacers might be beneficial for applications in such a setting.
Linkers of Triplex forming oligonucleotides (TFO)
In a study demonstrating triplex-directed alkylation of a minor groove site by a major groove binding TFO, the optimal length of flexible linker used to “wrap around” one DNA strand was found to be 50-58 atoms long (Lukhtanov et al., Nucleic Acids Res., 1997, 25, 5077). We can easily use a combination of our spacers and amino linkers to encompass this length, forming a tether that is flexible and available for conjugation to a groove binder or intercalator.
In another study variability in the tether length of camptothecin-TFO conjugates affected triplex-directed DNA cleavage by topoisomerase I (Arimondo et al., J. Biol. Chem., 2002, 277, 3132). In a one-step transformation we can synthesize a small library of amino modified oligonucleotides with varying tether lengths, from the same precursor (same sequence), used to test the selectivity and efficacy of cleavage in analogous systems.