Solid-Phase Purification of Synthetic DNA Sequences Serge L. Beaucage, Ph.D. Chief, Laboratory of Biological Chemistry FDA-CDER
The one who follows the crowd will usually go no further than the crowd. Those who walk alone are likely to find themselves in places no one has ever been before. Albert Einstein
Synthetic DNA/RNA Sequence Applications DNA primers for PCR amplification DNA primers for DNA sequencing DNA probes for diagnostic applications DNA sequences for total gene assembly and gene therapy applications DNA sequences for gene editing (CRISPR/cas9) DNA sequences for therapeutic applications (antisense and steric blockers, DNA aptamers, DNA decoys) RNA sequences for therapeutic applications: shrna, ribosymes, sirna, mirna, RNA aptamers, ncrnas
Solid-Phase DNA Synthesis
Synthetic DNA Purification Methods Method High-throughput Scalable Cost effective Affinity chromatography of 5 - biotinylated DNA sequences using NeutrAvidin-coated microspheres Potentially No No RP-HPLC-based hydrophobic interaction No Yes No HPLC-based ion-exchange/ion-pair No Yes No Enzymatic hydrolysis of shorter than full-length nucleic acid sequences Catching shorter than full-length sequences through a polymerization process Potentially No No Potentially Yes Yes Polyacrylamide gel electrophoresis Potentially No Yes
High-Throughput, Efficient and Scalable Solid-Phase Purification of Synthetic DNA Sequences Grajkowski, A., Cieślak, J. and Beaucage, S. L. J. Org. Chem., 2016, 81, 6165 6175
The Concept
Preparation of the Capture Support: Si ONH 2
Synthesis of the Capture Linker
Synthesis of the 5 -Functionalized Deoxyribonuclosides and Their 3 -Phosphoramidite Derivatives
Synthesis of the 5 -Functionalized DNA Sequences a CLF, capture linker function; PS, phosphorothioate diester; P, phosphate diester
Solid-Phase purification of the DNA sequence 10a A: RP-HPLC analysis of unpurified 5 - functionalized DNA sequence 10a B: RP-HPLC analysis of the solid-phase capture of 10a C:RP-HPLC and PAGE analysis of the released DNA sequence 12a from the capture support
Solid-Phase purification of the DNA sequence 10b A: RP-HPLC analysis of unpurified 5 - functionalized DNA sequence 10b B: RP-HPLC analysis of the solid-phase capture of 10b C:RP-HPLC and PAGE analysis of the released DNA sequence 12b from the capture support
Solid-Phase purification of the DNA sequence 10c A: RP-HPLC analysis of unpurified 5 - functionalized DNA sequence 10c B: RP-HPLC analysis of the solid-phase capture of 10c C:RP-HPLC and PAGE analysis of the released DNA sequence 12c from the capture support
Solid-Phase purification of the DNA sequence 10d A: RP-HPLC analysis of unpurified 5 - functionalized DNA sequence 10d B: RP-HPLC analysis of the solid-phase capture of 10d C:RP-HPLC and PAGE analysis of the released DNA sequence 12d from the capture support
Solid-Phase purification of the DNA sequence 10e A: RP-HPLC analysis of unpurified 5 - functionalized DNA sequence 10e B: RP-HPLC analysis of the solid-phase capture of 10e C:RP-HPLC and PAGE analysis of the released DNA sequence 12e from the capture support
Solid-Phase purification of the DNA sequence 10f A: RP-HPLC analysis of unpurified 5 - functionalized DNA sequence 10f B: RP-HPLC analysis of the solid-phase capture of 10f C:RP-HPLC and PAGE analysis of the released DNA sequence 12f from the capture support
Scalability of the Solid-Phase Purification of DNA Sequence 10a (10-Fold Scale-up Purification) A: RP-HPLC analysis of unpurified 5 - functionalized DNA sequence 10a (10 µmol) C: RP-HPLC and PAGE analysis of the released DNA sequence 12a from the linearly scaled-up capture support. D: Photograph of the solid-phase purified DNA sequence 10a after precipitation in THF.
Collaborators Andrzej Grajkowski Jacek Cieślak