A novel catechol-based universal support for oligonucleotide synthesis.
A novel universal support for deoxyribo- and ribonucleic acid synthesis has been developed. The support, constructed from 1,4-dimethoxycatechol, represents an improvement over existing universal supports because of its ability to cleave and deprotect under mild conditions in standard reagents. Because no nonvolatile additives are required for cleavage and deprotection, the synthesized oligonucleotides do not require purification prior to use in biochemical assays. Using reverse phase HPLC and electrospray mass spectroscopy, it was determined that oligonucleotides synthesized on the universal support (UL1) 3'-dephosphorylate quickly (9 h in 28-30% ammonium hydroxide (NH4OH) at 55 degrees C, 2 h in 28-30% NH4OH at 80 degrees C, or <1 h in ammonium hydroxide/methylamine (1:1) (AMA) at 80 degrees C). Oligonucleotides used as primers for the polymerase chain reaction (PCR) assay were found to perform identically to control primers, demonstrating full biological compatibility. In addition, a method was developed for sintering the universal support directly into a filter plug which can be pressure fit into the synthesis column of a commercial synthesizer. The universal support plugs allow the synthesis of high-quality oligonucleotides at least 120 nucleotides in length, with purity comparable to non-universal commercial supports and approximately 50% lower reagent consumption. The universal support plugs are routinely used to synthesize deoxyribo-, ribo-, 3'-modified, 5'-modified, and thioated oligonucleotides. The flexibility of the universal support and the efficiency of 3'-dephosphorylation are expected to increase the use of universal supports in oligonucleotide synthesis.
Stanford Genome Technology Center, Stanford University, Palo Alto, California 94304, USA. keith.anderson@stanford.edu
Keith M
KM
Anderson
Laurent
L
Jaquinod
Michael A
MA
Jensen
Nam
N
Ngo
Ronald W
RW
Davis
D002396:Catechols / Q000737:chemistry*; D006139:Guaiacol / Q000737:chemistry; D007700:Kinetics; D015394:Molecular Structure; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D010766:Phosphorylation; D016133:Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors
C527457:1,4-dimethoxycatechol; D002396:Catechols; D009841:Oligonucleotides; D006139:Guaiacol; C066344:3-methoxycatechol