Preparation of Oligonucleotides Containing Abasic Sites

Background

Hydrolysis of nucleoside residues in DNA occurs naturally to generate abasic sites. Most commonly, dA sites are hydrolyzed causing depurination and leading to abasic residues. Although this process is slow under physiological conditions, the reaction is faster at lower pH and especially if the bases are already oxidatively damaged. Damaged bases are also removed enzymatically by the action of DNA N-glycosylases. The abasic residue (dR) exists predominantly in the cyclic form and the structure is shown (1) in Figure 1. The abasic site exhibits poor stability, especially in basic medium. This is caused by the instability of the aldehyde, the tautomeric form of the cyclic structure, to ß-elimination. This degradation reaction leads to chain scission at the abasic site with formation of a 5'-phosphate segment and a 3'-modified segment.

Because of the instability of the abasic residue, it has not been simple to prepare this variant by chemical synthesis. However, some excellent results have been generated using the stable dSpacer tetrahydrofuran analogue.^{1, 2} This variant, (2) in Figure 1, is missing the 1'-OH of dR and is stable during oligo synthesis, purification and storage. It is possible to produce the genuine abasic site enzymatically using N-uracil glycosylase to remove uracil base from a 2'-deoxyuridine residue. A potentially very useful chemical method was described by Rayner.³ In this method, the abasic site is protected with a photolabile 2-nitrobenzyl group, (3) in Figure 1, during oligonucleotide synthesis and purification. The 2-nitrobenzyl group is then eliminated by photolysis to produce the abasic site. As always, there is the concern of thymine dimer formation during phototolysis. A quick literature check shows that several other methods^{4, 5, 6, 7} have been used to generate abasic sites but in all cases to date the synthesis of the monomer is fairly challenging and, in our opinion, the subsequent chemistry to generate the abasic site is hardly routine.

FIGURE 1: STRUCTURES OF PRODUCTS
(1) Tautomers of Abasic Residue (dR)
(2) dSpacer	(3) Photolabile Residue
(4) Abasic Phosphoramidite

Synthesis

A new chemical method has been described⁸ which allows the generation of abasic sites in double and single stranded oligonucleotides using very mild specific conditions and with very low probability of side reactions. A protected 3-deoxyhexitol is used as the monomer, (4) in Figure 1. Following oligonucleotide synthesis under standard conditions, the silyl protecting groups of the residue, (1) in Figure 2, are removed with aqueous acid. (This can be done in conjunction with trityl removal in the last step of a DMT-on purification.) The diol, (2) in Figure 2, so formed is then treated with aqueous sodium periodate to form the aldehyde, (3) in Figure 2, plus formaldehyde. The aldehyde (3) then immediately cyclizes to its preferred structure, the abasic cyclic sugar (dR). The process is illustrated in Figure 2.

FIGURE 2: FORMATION OF ABASIC SITE WITHIN AN OLIGONUCLEOTIDE

Oligonucleotide Stability

With the availability of oligonucleotides containing abasic sites, detailed stability information is now available.⁸ The abasic site is stable almost indefinitely in 0.2M triethylammonium acetate buffer (pH6) at 5°C or less. However, the site is less stable at room temperature (half-life of around 30 days) and quite unstable at 55° (half-life of about 7 hours). Interestingly, the abasic site is completely degraded during evaporation to dryness.

Structural Characteristics

Melting behavior of oligonucleotides containing the abasic site was examined⁸ and it was found to behave like a complete mismatch opposite the 4 natural bases, with characteristics almost identical to those of dSpacer, which has been used extensively as a model abasic site. Other physical characteristics of oligonucleotides containing abasic sites have been examined, as well as their implication in DNA damage and repair.^{6, 7, 9}

CATALOG INFORMATION

References:

(1) M. Takeshita, C.N. Chang, F. Johnson, S. Will, and A.P. Grollman, J. Biol. Chem., 1987, 262, 10171-10179.

(2) M.W. Kalnik, C.N. Chang, A.P. Grollman, and D.J. Patel, Biochemistry, 1988, 27, 924-931.

(3) D. Péoc'h, A. Meyer, J.-L. Imbach, and B. Rayner, Tetrahedron Lett., 1991, 32, 207.

(4) A. Laayoun, J.L. Decout, E. Defrancq, and J. Lhomme, Tetrahedron Lett., 1994, 35, 4991-4994.

(5) R.S. Coleman and R.M. Pires, Nucleosides and Nucleotides, 1999, 18, 2141-2146.

(6) J.T. Hwang, K.A. Tallman, and M.M. Greenberg, Nucleic Acids Res., 1999, 27, 3805-10.

(7) M. Jourdan, J. Garcia, E. Defrancq, M. Kotera, and J. Lhomme, Biochemistry, 1999, 38, 3985-95.

(8) I.G. Shishkina and F. Johnson, Chem Res Toxicol, 2000, 13, 907-912.

(9) J. Lhomme, J.F. Constant, and M. Demeunynck, Biopolymers, 1999, 52, 65-83.