Glen Report 16.21: Thymine dimers - DNA lesions induced by sunlight cis-syn thymine dimer phosphoramidite now available

One of the major sources of DNA damage in all organisms is the UV component of sunlight. The predominant reaction induced by UV light on DNA is dimerization of adjacent pyrimidine bases leading to cyclobutane dimers (CPDs) and 6-4 photoproducts. The dimers formed in the most significant quantity are the cis-syn cyclobutane dimer of two thymine bases (1) and the corresponding 6-4 photoproduct (3). The trans-syn thymine dimer (2) is formed at a much lower level in single and double stranded DNA. In sunlight, the 6-4 photoproduct is converted to its Dewar isomer (4) by absorption of long-wave UV light at around 325nm.1

Figure 1: Photo-induced thymine dimers
Figure 1
Figure 2
(1) Cis-syn Thymine Dimer
(2) Trans-syn Thymine Dimer
Figure 3
Figure 4
(3) 6-4 Thymine Dimer
(4) Dewar Thymine Dimer

Although formed routinely, these dimer products are, fortunately for us, efficiently excised and repaired enzymatically (nucleotide excision repair) or the dimerization is reversed by photolase enzymes. These lesions have been connected to the formation of squamous cell carcinomas. In addition, humans who lack ability to repair CPD lesions with high efficiency may be genetically predisposed to Xeroderma Pigmentosa (XP), a disease characterized by extreme sensitivity to sunlight and high frequency of skin cancer. Polymerases encountering unrepaired CPD lesions are quite error-prone, presumably leading to incorrect base insertions and subsequent mutations.2

The literature covering the chemistry of thymidine dimers and other CPDs is replete with references from John-Stephen Taylor’s group at Washington University in St. Louis. Any article on thymine dimer phosphoramidites has to acknowledge the work of Professor Taylor and his co-workers. Their contribution3 to this field has been outstanding.

It has been clear to us for some time that researchers into DNA damage and repair would value the ability to produce oligonucleotides containing cis-syn thymine dimer at specific locations within the sequence. Unfortunately, the chemical processes required to produce cis-syn thymine dimer phosphoramidite are very tortuous4,5 and it was only recently that we were able to obtain this phosphoramidite in sufficient quantity to offer it for sale. Due to the strategy used to synthesize our version of this phosphoramidite, it has methyl protecting groups on phosphorus, rather than the more usual cyanoethyl. This monomer exhibits high purity, performance and stability but requires the use of thiophenoxide (or another phosphate demethylating reagent) prior to regular deprotection.

Cis-syn thymine dimer phosphor-amidite (5) is by far the most expensive product we have offered for sale. We are packaging it initially in standard ABI and Expedite vials, as well as amber V vials compatible with the Expedite synthesizer. Using this V vial containing 50 micromoles, it is possible to prepare 6 oligos on a 0.2 µmole scale each containing a single insertion, or 4 oligos on a 1 µmole scale. Similar results should be possible using the LV cycles in Applied Biosystems instruments. Given the high price of this phosphoramidite, if only one or two oligos are required, it makes sense to have a custom oligo house prepare them. A custom oligo house may also wish to group oligos for several clients for most eficient use of this phosphoramidite. Contact us for the names of custom oligo services active in preparing oligos containing cis-syn thymine dimer.

Figure 5
Figure 2: Cis-syn thymine dimer
Cis-syn Thymine Dimer Phosphoramidite (5)

References

  1. C.A. Smith and J.-S. Taylor, J Biol Chem, 1993, 268, 11143-51.
  2. H. Ling, F. Boudsocq, B.S. Plosky, R. Woodgate, and W. Yang, Nature, 2003, 424, 1083-7.
  3. L. Sun, et al., Biochemistry, 2003, 42, 9431-7, and references cited therein.
  4. J.-S. Taylor, I. Brockie, and C. O’Day, J Am Chem Soc, 1987, 109, 6735-6742.
  5. T. Murata, S. Iwai, and E. Ohtsuka, Nucleic Acids Research, 1990, 18, 7279-86.

Product Information

Cis-syn Thymine Dimer Phosphoramidite (11-1330)