Tandem oligonucleotide synthesis using linker phosphoramidites

Abstract

Multiple oligonucleotides of the same or different sequence, linked end-to-end in tandem can be synthesized in a single automated synthesis. A linker phosphoramidite [R. T. Pon and S. Yu (2004) Nucleic Acids Res., 32, 623-631] is added to the 5'-terminal OH end of a support-bound oligonucleotide to introduce a cleavable linkage (succinic acid plus sulfonyldiethanol) and the 3'-terminal base of the new sequence. Conventional phosphoramidites are then used for the rest of the sequence. After synthesis, treatment with ammonium hydroxide releases the oligonucleotides from the support and cleaves the linkages between each sequence. Mixtures of one oligonucleotide with both 5'- and 3'-terminal OH ends and other oligonucleotides with 5'-phosphorylated and 3'-OH ends are produced, which are deprotected and worked up as a single product. Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence. When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection. Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated.

Figure 1

A string of oligonucleotides can be prepared by a single automated tandem synthesis. After synthesis, the linker to the support and the linkers joining each oligonucleotide are cleaved so that a mixture of oligonucleotides is released.

Figure 2

Structures of linker phosphoramidite reagents have a cleavable linkage between the protected nucleoside and the phosphoramidite group.

Scheme 1

Synthesis scheme for preparing strings of oligonucleotides linked in tandem via linker phosphoramidite 4. For simplicity, only two oligonucleotides are shown. However, the number of oligonucleotides which can be produced is only limited by the maximum number of bases in the total string which can be produced.

Figure 3

Results from capillary gel electrophoresis of crude 20mer produced individually by a conventional synthesis (1× 20mer) and 2-fold (2×, equivalent to a 40mer), 3-fold (3×, equivalent to a 60mer), 4-fold (4×, equivalent to an 80mer), 5-fold (5×, equivalent to a 100mer) and 6-fold (6×, equivalent to a 120mer) tandem syntheses.

Figure 4

Tandem synthesis of duplex DNA fragments (T is the linker phosphoramidite 4d). (A) A 44 base-long synthesis of a 24/20mer duplex. (B) A 52 base-long synthesis of a 28/24mer duplex. (C) A 64 base-long synthesis of 34/30mer duplex. (D) A 76 base-long synthesis of a 40/36mer duplex.

Figure 5

PAGE of double-stranded oligonucleotides (crude products) made from the tandem syntheses in Figure 4. (A) Non-denaturing conditions (20% polyacrylamide, no urea, room temperature). (B) Denaturing conditions (24% polyacrylamide, 7 M urea, 50°C). Lane 1, 24/20mer duplex A; lane 2, single-stranded 24mer (top strand only); lane 3, 28/24mer duplex B; lane 4, single-stranded 28mer (top strand only); lane 5, 34/30mer duplex C; lane 6, single-stranded 34mer (top strand only); lane 7, 40/36mer duplex D; and lane 8, single-stranded 40mer (top strand only).

Figure 6

Capillary gel electrophoresis analyses of crude products from tandem oligonucleotide synthesis under non-denaturing conditions. (A–D) The duplexes A–D (dsDNA) whose structures depicted in Figure 4A–D are shown. In each panel, a separately synthesized single-stranded oligonucleotide (ssDNA) corresponding to the top strand of each duplex is also shown.