Bz-Thiol-dT Linker: History and Applications

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During recent customer visits, our BzS-dT amidite (2191) has met with great interest and many customers have asked for further information on this product.

 

2191

 

The reason for developing this was simply that we were asked the following question: Is it possible to add multiple additions of enzymes (e.g. HRP or Alkphos) to an oligonucleotide with no adverse effect to the hybridisation properties?

The most obvious solution was to attach one enzyme at the 3’-end and one at the 5’-end using e.g. our thiol S-S C6 amidite (2126) in combination with spacer 18 amidite (2129) (see Figure 1).

 

2126

2129

 

Figure 1. 3’-5’ Enzyme-Oligonucleotide-Enzyme Conjugate

While this has the potential to obtain enhanced signal, we looked at solutions to be able to add 3 enzymes. Triple-labelling an oligonucleotide is widely used and is generally employed as a means of increasing signal or binding affinity. For instance 5’/3’/internal biotinylated oligos are often used for this reason e.g. in pyrosequencing (see Figure 2).

 

Figure 2. 3’-Internal-5’ Biotinylated Oligonucleotide

 

Based on this, we developed our BzS-dT amidite (2191). However, enzymes are significantly larger than biotin and other haptens, therefore, in this case the BzS-dT incorporations are not in the centre of the oligo but at the 5’-end, each of which is separated by at least one HEG spacer (2129). This separates the enzymes from the oligo and adds sufficient space to enable conjugation of the enzyme at each site (see Figure 3). Using this method, one customer has added 6 HRP enzymes to their oligonucleotide.

 

Figure 3. 5’ Multiple HRP-Oligonucleotide Conjugate

 

The use of BzS-dT (2191) is however not limited to enzyme conjugations, but is applicable to the coupling of any thiol-reactive species (e.g. gold or silver surfaces, maleimides and haloacetamides) - but with the added advantage over other thiol modifiers in that this is easily incorporated within the oligo sequence.

Thiol modifiers are usually protected as disulphides (e.g. 2126) or trityl (e.g. 2125), but for 2191 benzoyl protection was chosen because this is easily cleaved during oligo deprotection and, provided the solution contains TCEP or DTT, the possibility of dimerization to form disulphide bridges is minimised. This enables conjugation to the thiol-modified oligonucleotide directly after deprotection.

When used in conjunction with other modifiers it is possible to incorporate multiple labels to the oligonucleotide. For instance, 5’-6-FAM (2134), internal ROX-dT (2191 coupled to ROX-maleimide), and 3’-Alexa 770 (Amino-C7 (2350) coupled to Alexa 770 NHS ester).

 

2125

 

2134

 

2350

 

In summary, although our BzS-dT amidite was developed to provide a specific solution to one of our customers, it is clear that this product has wider use as an efficient means of enabling conjugations within an oligonucleotide using thiol reactive labels.

If you would like to evaluate this thiol modifier along with our other thiol modifiers, we currently have a special offer in which you can purchase all our thiol modifiers at a significantly reduced cost. Click to view further information and order online.

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