Catalog Number: 10-1936-xx

Description: Thiol-Modifier C6 S-S

Frequently Asked Technical Question

QUESTION: Do you have a biotin phosphoramidite containing a disulfide linker which can be cleaved later with DTT to release the DNA from a streptavidin support?

RESPONSE:No. However, this can be produced on the synthesizer by adding to the 5'- terminus first 5'-thiol-modifier C6 S-S (10-1936) followed by BioTEG phosphoramidite (10-1955). This should generate a biotinylated primer with a long spacer arm containing the disulfide linkage which can be cleaved later with DTT.

QUESTION: What is the best method to make peptide-oligonucleotide conjugates?

RESPONSE:It would seem that the best method to make peptide-oligo conjugates would be to use Fmoc chemistry and synthesize the peptide off an oligo synthesized on amino-CPG. However, deprotection of peptides synthesized using Fmoc chemistry requires 50% TFA and t-boc synthesized peptides require HF both of which would severely damage if not completely hydrolyze the oligo.

The best and most straight foward method is to use a heterobifunctional crosslinking reagent to link a synthetic peptide, containing an N-terminal lysine, to a 5'-Thiol modified oligo or conversely a 5'-amino modified oligo to a cysteine containing peptide . A good crosslinking reagent is N-Maleimido-6-aminocaproyl- (2'-nitro,4'-sulfonic acid)-phenyl ester . Na + (mal-sac-HNSA) from Bachem Bioscience (cat. # Q-1615). Reaction of this crosslinker with an amino group releases the dianion phenolate, 1-hydroxy-2-nitro -4-benzene sulfonic acid a yellow chromophore. The chromophore allows both quantitation of the coupling reaction as well as act as an aid in monitoring the seperation of "activated peptide" from free crosslinking reagent using gel filtration.

Method A: Couple Peptide Amine To Oligo Thiol (Note peptide MW must be > 5,000 to be excluded from desalting column). This method best for oligo-enzyme conjugation.

Step 1: Synthesize a peptide with an N-terminal, or internal, lysine (The epsilon amino group is more reactive than an alpha amino group).

Step 2: Synthesize an oligonucleotide with a 5' Thiol group.

Step 3: React peptide with excess mal-sac-HNSA (pH 7.5 Sodium phosphate)

Step 4: Seperation of peptide-mal-sac conjugate from free crosslinker and buffer exchange (pH 6.0 Sodium phosphate) using a gel filtration column (NAP or eq.). Note peptide must be large enough to seperate from the free linker which can be visualized as a yellow band. Do not collect yellow band with peptide.

Step 5: Activate thiol modified oligo, desalt and buffer exchange (pH 6 Sodium phosphate) on NAP 5 column.

Step 6: React acitvated peptide with Thiol modified oligo.

Step 7: Purify Peptide-Oligo conjugate by ion exchange chromatography on Nucleogen DEAE-500-10 or eq. Elution order: free peptide, peptide-oligo, free oligo.

Method B: Couple Oligo Amine To Peptide Cysteine (Note oligos > 15mers are excluded from desalting column). Use above procedure switching oligo for peptide.

Step 1: Synthesize a peptide with an N-terminal, or internal, cysteine

Step 2: Synthesize an oligonucleotide with a 5' amino modifier.

Step 3: Purify oligo Trityl-on by RP HPLC or cartridge.

Step 4: React oligo with excess mal-sac-HNSA (pH 7.5 Sodium phosphate)

Step 5: Seperation of oligo-mal-sac conjugate from free crosslinker and buffer exchange (pH 6 Sodium phosphate) using a gel filtration column (NAP or eq.). Note oligo must be large enough to seperate from the free linker which can be visualized as a yellow band. Do not collect yellow band with oligo.

Step 6: Dissolve peptide in pH 6.0 Sodium phosphate buffer and react with activated oligo.

Step 7: Purify Peptide-Oligo conjugate by ion exchange chromatography on Nucleogen DEAE-500-10 or eq. Elution order: free peptide, peptide-oligo, free oligo.

DILUTION/COUPLING DATA

The table below shows pack size data and, for solutions, dilutions and approximate couplings based on normal priming procedures. Please link for more detailed usage information with the various synthesizers.

11/04/2011 | http://www.glenres.com/ProductFiles/10-1936.html