A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis

Abstract

Background: The quality of chemically synthesized oligonucleotides falls with the length of the oligonucleotide, not least due to depurinations and premature termination during production. This limits the use of long oligonucleotides in assays where long high-quality oligonucleotides are needed (e.g. padlock probes). Another problem with chemically synthesized oligonucleotides is that secondary structures contained within an oligonucleotide reduce the efficiency of HPLC and/or PAGE purification. Additionally, ligation of chemically synthesized oligonucleotides is less efficient than the ligation of enzymatically produced DNA molecules.

Preparation of 5′-O-(1-Thiotriphosphate)-Modified Oligonucleotides Using Polymerase-Endonuclease Amplification Reaction (PEAR)

“…However, these methods might not be suited for large-scale production of oligonucleotides, since the yields of reactions are largely limited by the concentrations of templates and primers added. On the other hand, isothermal reactions such as exponential amplification reactions (EXPAR) [16] and rolling circle replication (RCR) [17]–[18] have been developed for amplifying oligonucleotides, but they have not been validated for preparing oligonucleotides with modified groups. Previously, we developed a new thermal cyclic reaction, polymerase-endonuclease amplification reaction (PEAR) [19], and demonstrated the use of it for large-scale enzymatic production of a natural ASODN.…”

Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides

“…Moreover, in contrast to enzymatic production proposed before 9–11,13 , the MOSIC method does not rely on addition of any synthetic primers, neither for amplification, nor for the digestion. In fact, regardless of whether amplification is done in vitro or in vivo by E. coli /phage, all the components necessary for mass production of MOSIC ODNs, can be readily grown in bacterial cultures.…”

“…10,13 These methods rely on the ligation of small ODN circles that subsequently act as templates for amplification by a strand displacing phi29 polymerase. The result is a long single strand containing the complement to the circle sequence repeated a large number of times, a so-called tandem repeat.…”

Enzymatic preparation of multimilligram amounts of pure single-stranded DNA samples for material and analytical sciences

“…We experienced that if such a ligation event occurs in (or nearby) a restriction site, the amplified DNA is only partially digested or, in the worst-case scenario not at all, due to template length inhomogeneity at the 5' terminus. Such inhomogeneity in oligonucleotide length generally increases with the length of commercial synthetic DNA oligonucleotides due to decreasing coupling efficiencies of automated DNA synthesizers and loss of resolution during PAGE purification [47]. The ligation site was therefore placed several nucleotides away from both restriction sites.…”