3'-Thiol Modifier C3 S-S CPG Column
3'-Thiol Modifier C3 S-S CPG Column
3'-Thiol Modifier C3 S-S CPG Column, 1000 Å, 200 nmol (Pack of 4), ALL-FIT
Key featuresShow Hide
- Incorporates a thiol reactive functional group for conjugation.
- Shorter C3 spacer.
- CPG has long-chain alkylamino succinyl linker.
Incorporation of a thiol reactive functional group at specific sites within an oligonucleotide allows for subsequent post-synthesis conjugation of the oligo with a number of different moieties such as fluorescent markers and biotin, depending on the application. Such labels need to be reactive towards the incorporated functional group: for example, thiols will react with iodoacetate and maleimide derivatives to form thioether linkages.
The 3'-thiol-modifier CPGs with C3 and C6 S-S linkers can be used to introduce a 3'-thio functionality, with subsequent cleavage of the disulphide linkage affording the free thiol. This is then available for conjugation.
|LK2126||Thiol-Modifier-C6 S-S CE Phosphoramidite|
|LK2166||Thioctic Acid NHS Ester|
|LK2187||(Hydrophilic) S-Bz TEG-CE Phosphoramidite|
|LK2361||3'-Thiol-Modifier-C3 S-S CPG|
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.
Unlike terminal amino linkers, which are generally designed for specific 3'- or 5'-modification, the various available terminal thiol linkers can often be used for both, depending on the sequence.
LK2187 is used for 5’-modification. The benzoyl protecting group was selected such that the free thiol is formed during oligonucleotide deprotection, but adding TCEP to the deprotection solution is recommended to prevent dimerisation of the oligo via a disulphide bridge. Therefore there is no need for the use of silver salts as is required to remove the trityl group when using LK2125. The lack of the disulphide bridge, as is present in LK2126, means better stability hence there is no drop in this amidite’s performance even after being on the synthesiser for 48h.
5’-Modification can also be achieved using either LK2125 or LK2126 using the standard synthesis cycle. The latter offers the advantage of avoiding the use of silver nitrate in the final deprotection. Both LK2126 and LK2361 can be used to achieve 3’-modification. To do this either use the LK2361 support as standard or add LK2126 as the first monomer to any support and continue the sequence from there. 3’-Thiol-modified oligos are particularly useful in cases where a different label is desired for the 5’-terminus, in which case an amino linker is used to achieve the latter.
Whereas an amino modifier is used to add a label via an active ester, thiol modification allows labelling via a haloacetamide or maleamide derivatised label.
Terminal modification (3’ or 5’) with a thiol for gold or silver conjugation is easily achieved with the aforementioned modifications or LK2166 can be added post-synthetically to an amino-functionalised oligo.
Use of Thiol Modifier Phosphoramidites
For all, prepare the amidite solution 5-10min before placing on the synthesiser to ensure complete dissolution.
A minimum coupling time of 5min (300s) is recommended for thiol modifiers. For LK2187 use 0.3M BTT or 0.25M DCI in acetonitrile. Do not use ETT as this reacts with the Bz-S group. A 15min (900s) coupling time is recommended.
LK2125 – It is recommended the final cycle oxidation is carried out with 0.02M (LK4132/LK4330) iodine to minimise oxidative cleavage of the trityl-S linkage.
LK2126 – For 5’-modification 0.02M iodine is used in the final cycle. For 3’-modification it is used in all oxidation steps.
Deprotection & Purification
LK2125 – The trityl group used to protect the thiol is not acid labile and therefore cannot be removed on a DNA synthesiser during the detritylation step. Cleavage of the oligonucleotide from the support and removal of the base protecting groups are carried out with ammonium hydroxide solution or AMA if fast deprotecting amidites are used. If purification is desired, it is best done before removing the trityl group. The presence of the trityl group allows standard DMT-ON RP-HPLC purification techniques to be used. Note this is not suitable for cartridge purification since, as previously mentioned, the trityl group is not acid labile.
Final deblocking of the oligonucleotide involves cleavage of the trityl-sulphur bond. This is achieved with silver nitrate, the excess silver being precipitated with TCEP. Excess TCEP is removed by desalting or by ethanol precipitation.
- Deprotect with ammonium hydroxide solution or AMA.
- Purify the trityl-containing oligo by HPLC.
- Evaporate the product solution to dryness.
- Suspend the product in 0.1M triethylammonium acetate (TEAA), pH 6.5 at a concentration of approximately 100 ODs/ml.
- Add 0.15 volumes of 1M aqueous silver nitrate solution, mix thoroughly, and leave to react at room temperature for 30min.
- Add 0.20 volumes of 1M aqueous TCEP solution, mix thoroughly, and leave at room temperature for 5min.
- Centrifuge the suspension to remove the silver-TCEP complex. Remove the supernatant. Wash the precipitate with 1 volume of 0.1M TEAA. Centrifuge and combine the supernatant with the first wash. (Alternatively, vortex the suspension and apply to a desalting column equilibrated with conjugation buffer.)
- Remove the excess TCEP from the supernatant by desalting using a NAP-25 column and proceed directly to the conjugation reaction.
LK2126 – 5’-Modification:
(a) Typical Protocol – DMT OFF synthesis:
- Add the Thiol-Modifier C6 S-S at the 5’-terminus of the oligo in the automated DMT OFF synthesis mode. The DMTr release from the last cycle can be used to determine coupling efficiency.
- Deprotect the oligo using ammonium hydroxide solution or AMA. AMA for 2h at RT works best, however other modifications in the oligo must also be compatible.
- Isolate, desalt, and, if necessary, purify by HPLC.
- Evaporate to dryness.
- Add 200μl of 87mM TCEP in water (29mg/ml) and allow to stand for 1h.
- Desalt. The thiol-modified oligo is ready for use.
(b) Typical Protocol – DMT ON synthesis
- Add the Thiol-Modifier C6 S-S at the 5’-terminus of the oligo in the automated DMT ON synthesis mode.
- Carry out deprotection in AMA for 2h at RT.
- Purify the trityl-containing oligo on a cartridge and evaporate the solution to dryness.
- Cleave the disulphide linkage using 87mM TCEP in water for 1h at room temperature.
- Desalt the oligo.
LK2126 – 3’-Modification: Similar procedures as used for LK2361 are used, noting strategies above.
LK2187 - Treat the column with 20% DEA/MeCN prior to deprotection. This is extremely important since the free thiol is readily capped by acrylonitrile if the cyanoethyl protection is not removed prior to cleavage and deprotection of the nucleobases.
For deprotection in AMA, the temperature and time will depend on the other modifications and protecting group chemistry of the other amidites. Typically this would be AMA, 10min at 65˚C.
The addition of 100mM TCEP to the deprotection solution has been shown to prevent the formation of the dimerised oligonucleotide via the disulphide bridge.
Immediate conjugation is recommended otherwise treatment with TCEP to cleave the disulphide bridge is required.
Storage & Stability
The oils are stored dry in a freezer at –10 to –30°C. Stability in solution is 2-3 days.
Thiol-modified oligos are best kept dry under an inert atmosphere in the Tr-S or S-S state until ready to use. Treat with TCEP immediately before use. Note even if the oligo has previously been treated with TCEP (or DTT) this will readily form a disulphide bridge to form the dimerised oligo, therefore TCEP treatment is required before use.
Oligonucleotide Modification with Thioctic Acid NHS Ester (LK2166) and Conjugation to Gold & Silver
A published protocol for preparing an oligo modified with thioctic acid at the 3’-end is freely available.1 This simple procedure utilises our 3’-Amino-Modifier C7 CPG product (LK2350), however either of the methods described below are applicable.
These procedures assume synthesis of the oligonucleotide on a 1μmol scale. If synthesis on an alternative scale is used then adjust the amounts of reagents accordingly. Expected yield from either conjugation method is approx. 50%.
(a) In-solution conjugation with 5’-amino-modified oligo:
- Prepare 0.1M Sodium Bicarbonate solution, pH ~8.0: Weigh sodium bicarbonate solution (0.84g, 0.01mol) into a 250ml beaker. Add water (90ml) and mix thoroughly. Transfer to a 100ml volumetric flask and make up to a final volume of 100ml.
- Prepare 0.1M Sodium Carbonate solution pH ~12: Weigh sodium carbonate monohydrate solution (1.24g, 0.01mol) into a 250ml beaker. Add water (90ml) and mix thoroughly. Transfer to a 100ml volumetric flask and make up to a final volume of 100ml.
- Prepare 0.1M Sodium carbonate/bicarbonate buffer: Place 0.1M sodium bicarbonate solution (10ml) into a screw top bottle. Add 0.1M sodium carbonate solution until the pH reaches 9.75.
- Prepare 0.1M TEAA: Place 2M TEAA (50ml) into a 2L beaker. Add water (950ml) and mix thoroughly. Adjust the pH to 7.0 - 7.2 (add acetic acid to lower and triethylamine to raise the pH). Filter directly into a 1L Duran bottle.
- Prepare 80mM thioctic acid/DMSO: Add DMSO (120μl) to a 2.5 - 3mg sample of thioctic acid NHS ester (LK2166).
- Dry the oligonucleotide(s) on a freeze drier or evaporator.
- To each of the oligonucleotides, add 0.1M sodium carbonate/bicarbonate buffer pH 9.75 (75μl).
- Add thioctic acid/DMSO solution (30μl).
- Allow to stand overnight.
- Prepare a G25 column prepared with 0.1M TEAA for each of the oligos.
- Label a 2ml sample tube for each of the oligonucleotide.
- Place the tubes from Step 11 into the larger centrifuge tubes used for G25 columns.
- Place a G25 column (prepared in step 10) on top of the sample tubes
- Add the oligonucleotide/thioctic acid mixture to the top of the G25 column and centrifuge at 9000rpm.
- The oligonucleotide is now ready for analysis or purification.
(b) On-column conjugation with 5’-amino-modified oligo
On-column conjugation can be achieved using a trityl-protected amino-modifier. Note, however, that removal of the MMTr-protecting group on the synthesiser is slow, inefficient and difficult to measure. TFA-protected amino-modifiers are incompatible with on-column conjugations as the TFA group is not removed until cleavage and deprotection of the oligo.
- Preparation of Modified Oligo: Synthesise the 5’-amino-modified oligo (DMT OFF).
- Wash the synthesis column with approx. 2ml of Cap Mix B (10% methylimidazole/pyridine/THF) (LK4122) for 2-3 min.
- Wash the column with 10ml of HPLC-grade acetonitrile.
- Preparation of Thioctic Acid NHS Ester: Dissolve 20mg (66μmol) of LK2166 in 1ml of acetonitrile.
- Conjugation: Pass the amino-modified oligo with the thioctic acid NHS ester solution prepared in Step 4 by means of two syringes.
- Incubate at room temperature overnight.
- Wash the column with 10ml of HPLC-grade acetonitrile.
- Blow the column dry with argon.
- Cleave and deprotect the oligo using the conditions recommended for the modifier and bases used.
A similar protocol can be applied to on-column labelling at the 3'-end using LK2350. In this case, after oligo synthesis, the Fmoc protection is removed by washing the column with 20% piperidine/MeCN. Leave for 5min then wash with MeCN. The above conjugation protocol can then be followed from Step 2 onwards.
Modified oligonucleotides are purified, if required, by standard methods such as RP-HPLC.
Storage & Stability
This product is stored dry in a freezer at –20°C. Solutions in acetonitrile must be prepared fresh for immediate use and not stored.
Gold & Silver Bioconjugation Protocols
Customers wishing to use oligonucleotides modified with LK2166 to conjugate to gold or silver nanoparticles should refer to reference 1 for applicable methods.
Use of Thiol-Modifier CPG (2361)
Use as per unmodified nucleoside supports. However, non-nucleosidic modifications are slow to detritylate and require an initial detritylation prior to use in synthesis. In this case it is important not to use a cycle with an initial capping step. AMA deprotection will require dmf-G and Ac-C to be used.
0.02M iodine (LK4132 & LK4330) is used in all oxidation steps.
Cleavage & Deprotection
Deprotect the oligo using conditions for unmodified bases, e.g. AMA at room temperature for 2h or ammonium hydroxide solution at 55˚C for 4h. This is dependent on the protection on the standard nucleobases and other modifications in the oligo.
Desalt the deprotected oligo and evaporate to dryness. Purify the oligo if required (RP-HPLC or cartridge), desalt and dry.
Add 200μl of a solution of 87mM TCEP in water (29mg/ml) and leave (preferably with agitation) for 1h.
Pass the oligo down a G25 or Nap 10 column equilibrated with water or the buffer being used in the subsequent conjugation reaction. Where conjugation is required, it is recommended this is carried out immediately.
Storage & Stability
The supports are stored in a freezer at –10 to –30°C.
Thiol-modified oligos are best kept dry under an inert atmosphere in the Tr-S or S-S state until ready to use. Treat with TCEP immediately before use. Note even if the oligo has previously been treated TCEP (or DTT) this will readily form a disulphide bridge between two oligos, therefore TCEP treatment is required before use.
- Enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides, J.A. Dougan, C. Karlsson, W.E. Smith and D. Graham, Nucleic Acids Research, 35, 3668-3675, 2007.