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*****Glen Research Glen Report*****
New Product - Universal Tosyl Phosphoramidite
The reactivity of the tosyl functional group in biomolecule conjugation has now been demonstrated1 in a number of publications. One of the first uses of a tosyl phosphoramidite was in the work by Herrlein, Nelson and Letsinger2 in the early 1990s. They demonstrated the template-dependent chemical ligation of a 3’-phosphorothioate and a 5’-tosyl oligo to join two synthetic oligonucleotides together. This methodology was later used by Letsinger to demonstrate3 the tolerance of the Taq polymerase in efficiency of amplification. In Letsinger’s work, a 5’-tosyl phosphoramidite was prepared that lacked a DMT purification handle and was not extendable in oligonucleotide synthesis. In recent work by Mirkin et al., a Universal Tosyl Phosphoramidite containing a DMT group was used4 to prepare 3’-tosyl oligonucleotides. Antibodies were then coupled to the oligonucleotides via nucleophilic displacement of the tosyl group by an amine or thiol functional group on the antibody.
The Universal Tosyl Phosphoramidite can be used to insert a tosyl group at the 3’ or 5’ terminus of an oligonucleotide. The tosyl group can be displaced by nucleophilic substitution using, e.g., an amino tag, on the synthesis column. Alternatively, the oligonucleotide can be cleaved and deprotected using UltraMild chemistry to yield the fully-deprotected tosyl oligonucleotide. The tosyl group can then be displaced in solution with a protein, antibody, or even an amino-modified nucleic acid of interest.
As described above4, the Mirkin group has used the Universal Tosyl Phosphoramidite to functionalize the 3’ terminus of oligonucleotides destined for conjugation with gold nanoparticles. These oligonucleotides are modified at the 5’ terminus with disulfide residues prepared using Dithiol Phosphoramidite (DTPA) or using a Trebler phosphoramidite followed by Thiol-Modifier C6 S-S. Modified oligonucleotides attached to gold nanoparticles are described as “barcodes” and are used in a nanoparticle-based biobarcode assay.
Glen Research now offers this Universal Tosyl Phosphoramidite that can be coupled either at the 5’ or the 3’ end of an oligo and conveniently purified via standard reverse phase purification. This phosphoramidite also potentially may allow the introduction of other nucleophiles (azido, amino, cyano, etc.) via nucleophilic displacement of the tosyl group on the support. The use of UltraMild monomers is recommended in the synthesis of tosyl oligos, along with DTPA monomer for subsequent gold attachment chemistry.
Preparation and Use of Tosylated Oligonucleotides
Add simple description for on-column and solution conjugations.
Conjugation of 3’-Tosylated 5’-Disulfide Oligonucleotides (Barcodes) to Gold Nanoparticles
Tosyl-oligonucleotide gold conjugates were prepared by addition of 1 O.D. of the barcode to 1mL of 30nm gold particles. The mixture was allowed to stand at room temperature for 24 hours. Following this initial incubation period, 10% SDS was introduced to a final concentration of 0.1%, followed by addition of sodium chloride to a final concentration of 0.1M using a 1M salt solution. The mixture was then allowed to stand at room temperature for 48 hours. The conjugates were harvested by centrifugation at 6800 rpm for 15 minutes using an Eppendorf bench top centrifuge, washed twice with nanopure water, and then finally suspended in nanopure water and refrigerated.
General Method for Preparation of Biobarcode Probes Co-loaded with Antibodies
The co-loaded probes were prepared by concentration of 3.0mL of the tosyl-oligonucleotide gold conjugates down to 60 µL by centrifugation and removal of the supernatant. To this concentrate was added 20 µL of a 0.2% Tween20 solution followed by 10 µg of the desired protein in 20µL of PBS buffer, pH 7.4. (In the case of PSA detection4, Human Kallikrein 3/PSA Biotinylated Affinity Purified PAb, Goat IgG was used.) To this mixture, 100µL of a 0.2M borate buffer solution at pH 9.5 was added. The mixture was allowed to react at 37 ºC for 24 hours at 550 rpm on an Eppendorf Thermomixer R. To this mixture 10µL of a 10% BSA solution was added and allowed to react for an additional 24 hours under the previous conditions. The probes were harvested by centrifugation at 5800 rpm for 15 minutes followed by washes using a pH 7.4 PBS buffer containing 0.1% BSA, 0.025% Tween20 (assay buffer), and finally re-suspended in 3mL of the assay buffer and refrigerated.
1. P.A. Bertin, et al., J Amer Chem Soc, 2006, 128, 4168-9.
2. M.K. Herrlein, J.S. Nelson, and R.L. Letsinger, J. Amer Chem. Soc., 1995, 117, 10151-10152.
3. R.L. Letsinger, T.F. Wu, and R. Elghanian, Nucleos Nucleot, 1997, 16, 643-652.
4. C.S. Thaxton, et al., Proc Natl Acad Sci U S A, 2009, 106, 18437-42.
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