Frequently Asked Technical Question
QUESTION: What is the "T" in the structure of thiol-modifier C6?
RESPONSE:"T" is short for trityl or triphenylmethyl. This group is not significantly acid labile and requires an oxidative cleavage with silver nitrate to remove it. It is also susceptible to oxidative cleavage with the iodine oxidizer and, for maximum yield, the iodine concentration in that oxidizer should be 0.02M, which is now virtually standard.
REFERENCE(S):
Glen Research User Guide to Modification and Labelling, 1999, 24.
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 (Glen Gel-Pak™ 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 Glen Gel-Pak™ 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 (Glen Gel-Pak™ 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.
QUESTION: What is the best method to deprotected thio-modified oligos (10-1926)?
RESPONSE:The trityl group used to protect the thiol is not acid labile and therefore can not be removed on a DNA synthesizer using
the normal acid deprotection. Cleavage of the oligonucleotide from the support and removal of the base-protecting groups are carried out with ammonium hydroxide in the normal manner. If purification is desired, it should be done before removing the trityl group. The presence of the trityl group allows standard trityl-on reverse phase (RP) purification techniques to be used.
Final deblocking of the oligonucleotide involves cleavage of the trityl-sulfur bond. This is accomplished by oxidation with silver nitrate with the excess silver nitrate being precipitated with dithiothreitol (DTT). Excess DTT can be removed by extraction with ethyl acetate, by desalting or by ethanol precipitation.
Procedure
1. Deprotect with ammonium hydroxide in the normal manner.
2. Purify the trityl containing oligonucleotide by HPLC or Poly-Pak cartridge.
3. Evaporate the product solution to dryness.
4. Suspend the product in 0.1M triethylammonium acetate (TEAA), pH6.5 at a concentration of ~100 A260 units/mL.
5. Add 0.15 volumes of 1M aqueous silver nitrate solution, mix thoroughly, and react at room temperature for 30 min.
6. Add 0.20 volumes of 1M aqueous DTT solution, mix thoroughly, and leave at room temperature for 5 minutes.
7. Centrifuge the suspension to remove the silver DTT complex. Remove the supernatant. Wash the precipitate with 1 volume of 0.1M TEAA. Centrifuge and combine the supernatant with the first volume.
8. Proceed directly to the conjugation reaction. (If desired, excess DTT can be removed by ethyl acetate extraction. The free thiol oligonucleotide must be stored under an inert atmosphere to avoid oxidative dimerization to the disulfide.)
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