Key featuresShow Hide
- Improves duplex stabilisation when used in place of dA. Forms an additional hydrogen bond with thymidine.
- Also destabilises A-G wobble mismatches, thus increasing specificity.
- Also known as 2,6-diaminopurine.
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During duplex hybridisation of unmodified oligos, A-T base pairs have two hydrogen bonds, whereas G-C base pairs have three. One of the simplest methods of improving duplex stabilisation is the use of 2-Amino-dA phosphoramidite (2,6-diaminopurine) in place of dA.(1) This forms an additional hydrogen bond with thymidine. However, this product also destabilises A-G wobble mismatches, thus increasing specificity.
- Oligonucleotides containing 2-aminoadenine and 5-methylcytosine are more effective as primers for PCR amplification than their non-modified counterparts, Y. Lebedev, N. Akopyants, T. Azhikina, Y. Shevchenko, V. Potapov, D. Stecenko, D. Berg and E. Sverdlov, Genetic Analysis – Biomolecular Engineering, 13, 15-21, 1996.
|LK2287||dU SynBase™ CPG 1000/110|
|LK2293||dI SynBase™ CPG 1000/110|
|LK2323||5-Me-dC SynBase™ CPG 1000/110|
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.
Couple the phosphoramidites LK2013, LK2016 and LK2017 using the standard method as recommended by synthesiser manufacturer with coupling times as per standard bases. 90s is recommended for 2529. A coupling time of 15min is recommended for LK2145. A coupling time of 3min is recommended for LK2164. The solid supports are used as per standard nucleoside supports.
For LK2013, LK2016, LK2017 and LK2529 standard oligonucleotide deprotection conditions can be applied when deprotecting an oligo containing these modifications, however LK2013, LK2016 and LK2529 are also compatible with AMA deprotection; typically 10min at 55oC.
LK2145 - Use AMA deprotection in conjunction with Ac-dC, rather than Bz-dC, or transamidation will occur. The supports are cleaved and deprotection carried out using the protocols required by the nucleobases.
LK2164 - Cleavage from support and primary deprotection is accomplished by treatment with ammonium hydroxide solution at room temperature for 24h.
After removing the solution1, the NPE groups are removed by treatment with 0.3M tetramethylguanidinium 2-nitrobenzaldoximate solution in water/dioxan (1:1) at 70°C for 48h. We find that this extended treatment is necessary to ensure complete removal of both of the NPE protecting groups.
It should be noted that this treatment generates a variety of low molecular weight by-products which are observed in the HPLC. Satisfactory results are obtained by de-salting the deprotection mixture into water using a NAP or G25 column before HPLC, from which the final oligonucleotide product can then be isolated.
Storage & Stability
Refrigerate dry compounds. Stability of phosphoramidites in solution is similar to standard dA, dC, dG and dT monomers.
- This is best achieved by removing the deprotection solution by G25 then freeze drying to remove the water. Heat cannot be used or the NPE groups would cleave.