Modifying oligonucleotides with amino and thiol linkers remains the most common method of bioconjugation. At Link, we currently offer 18 different products that fall into this category with four more in the pipeline. One question we are often asked is “why are there so many amino and thiol linkers?”.
The first point to consider is where the functional group needs to be within the oligonucleotide. There are three possibilities; 5’-end, 3’-end or internally within the sequence.
The choice very much depends on the application of the oligonucleotide. For instance, you may require the introduction of an internal azide e.g. to allow further modification by click chemistry, in which case one of the base modified linkers (Figure 1) is necessary.
Figure 1: Base Modified Amino and Thiol Linkers
The functionality of the linker will depend on the reactive group on the required label. For instance an azide NHS ester would need an aminolinker. The type of base depends on where in the sequence the modification is required (i.e. which of the natural bases needs to be replaced by the base modified aminolinker) and the impact this will have during hybridisation. If the reporter has a thiol reactive group, 2191 is required.
The base modified linkers also allow multiple incorporations within a sequence either of multiple amino sites (useful in terms of triplex stabilisation) or the incorporation of amino and thiol linkers allowing orthogonal post labelling of multiple reporters.
Where conjugation is required at the 3’-end, an amino or thiol functionalised support is required (see Figure 2).
Figure 2: 3’-Amino and Thiol Linkers
The non-nucleosidic modifiers are particularly useful where the 3’-end of the oligonucleotide needs to be blocked to prevent elongation or degradation. Conversely where there is a need for elongation in the presence of the label, then the base amino modified linkers make this possible.
Where 5’-modification is required, it is feasible to use the base modified linkers shown in Figure 1 but in general one of the 5’-linkers shown in Figure 3 is incorporated into the oligonucleotide.
Figure 3: 5’-Amino and Thiol Linkers
Again the choice of linker depends on the final application of the oligonucleotide but the purification method of the amino or thio functionalised oligonucleotide must also be taken into consideration.
Shorter linkers are used where either there is a need for the label to be in close proximity to the oligonucleotide or where the proximity of the reporter has no adverse effect on the performance of the modified oligonucleotide in the final application. For instance the introduction of a dye onto a probe where a change in signal is detected when the oligonucleotide binds to a target DNA sequence or a protein.
Longer linkers are used where there is a need to separate the label and the oligonucleotide. For instance, when attaching oligonucleotides to solid surfaces such as arrays, where there is a need to create space to allow accessibility of the enzyme.
There is also the possibility of choosing a hydrophobic linker or a hydrophilic linker. In this case there are two points to consider; the end use of the modified oligonucleotide and the role of the linker itself, e.g. oligonucleotides modified with hydrophilic linkers used in solid-PCR reduce steric hindrance in comparison with the use of a hydrophobic linker. Another point to consider is the label being conjugated. For instance, conjugating a short amino modified oligonucleotide with thioctic acid NHS ester (2166) is very efficient and for the most part the nature of the linker is of low importance. However, the conjugation of an enzyme (e.g. HRP, alkaline phosphatase) to an oligonucleotide becomes more efficient when using a hydrophilic linker to modify the oligonucleotide. In this case it is thought that the amino functionality becomes more accessible since the linker is compatible with aqueous solutions.
DMT or MMT protection of the amino or thiol linker incorporated at the 5’-end of the oligonucleotide allows the possibility of ‘DMT ON’ purification either by cartridge; e.g. TOP cartridges (9003-9006) or by HPLC where the trityl group is removed either during or after HPLC purification.
In response to our customers' needs, we are expanding our current offering of aminolinkers. We have four more in the pipeline (see Figure 4).
Figure 4: Amino Linkers in the Pipeline
2373 is the analogous aminolink CPG to the backbone found in TAMRA CPG (2372 now sold as 2434 (standard loading) and 2435 (low loading)) and Dabcyl CPG (2374). This allows a user to introduce a dye for direct comparison to 2372 or 2374.
Compound 2535 is allows multiple additions of aminolinkers to an oligonucleotide. For instance multiple incorporations of a hapten such as DIG improve the sensitivity of the probe when used in in situ hybridisation assays. DIG is introduced to the multiple aminolinkers post synthetically by reaction with DIG NHS ester.
2532 is analogous to 2133 but offering TFA protection rather than MMT.
2534 expands the option of the length of aminolinker we offer.
Further details on these new products will be available soon. If you want to be notified as soon at the products are ready, then send us your details in this short form.