Tandem Oligonucleotide Synthesis on Solid-Phase Supports for the Production of Multiple Oligonucleotides

Abstract

More than one oligonucleotide can be synthesized at a time by linking multiple oligonucleotides end-to-end in a tandem manner on the surface of a solid-phase support. The 5'-terminal hydroxyl position of one oligonucleotide serves as the starting point for the next oligonucleotide synthesis. The two oligonucleotides are linked via a cleavable 3'-O-hydroquinone-O,O'-diacetic acid linker arm (Q-linker). The Q-linker is rapidly and efficiently coupled to the 5'-OH position of immobilized oligonucleotides using HATU, HBTU, or HCTU in the presence of 1 equiv of DMAP. This protocol avoids introduction of phosphate linkages on either the 3'- or 5'-end of oligonucleotides. A single NH(4)OH cleavage step can simultaneously release the products from the surface of the support and each other to produce free 5'- and 3'-hydroxyl termini. Selective cleavage of one oligonucleotide out of two sequences has also been accomplished via a combination of succinyl and Q-linker linker arms. Tandem synthesis of multiple oligonucleotides is useful for producing sets of primers for PCR, DNA sequencing, and other diagnostic applications as well as double-stranded oligonucleotides. Tandem synthesis of the same sequence multiple times increases the yield of material from any single synthesis column for maximum economy in large-scale synthesis. This method can also be combined with reusable solid-phase supports to further reduce the cost of oligonucleotide production.

Tandem oligonucleotide synthesis using linker phosphoramidites

“…In our previous tandem oligonucleotide syntheses (1,2), we used simple dicarboxylic acids, such as succinic, diglycolic or Q-linker, to join one oligonucleotide to the next in tandem. However, this required using a second coupling chemistry, which was not practical on most automated synthesizers.…”

“…Tandem synthesis may also be advantageous to produce multiple copies of the same sequence for large-scale applications, although the reagents described in this manuscript introduce 5′-phosphate groups on some of the products. If 5′-non-phosphorylated oligonucleotides are required, tandem synthesis via ester linkages (2) and not phosphate linkages should be used.…”

“…Tandem oligonucleotide synthesis is a term we introduced (1,2) to describe the synthesis of multiple oligonucleotides joined, end-to-end, through a dicarboxylic acid linker arm (Figure 1). Tandem synthesis allows more than one oligonucleotide sequence or more than one copy of a single sequence to be produced in a single automated synthesis.…”

“…In our previous approach to tandem synthesis (1,2), we improved upon the tandem process by eliminating the issue of 3′-dephosphorylation. We did this by eliminating phosphoramidite coupling chemistry when adding a new oligonucleotide onto the 5′ end of an existing one.…”

Strategies Useful for the Chemical Synthesis of Oligonucleotides and Related Compounds

“…The process involves the following steps: (1) nucleoside of solid phase synthesis without lowering the average coupling yield. This approach was extended for use in tandem oligonucleotide synthesis, which simultaneously produces more than one oligomer on the surface of a support [152].…”