Modern methods in medicine and synthetic biology require very long DNA strands, in particular with the advent of the increasingly popular CRISPR/Cas9 system in recent years. This technique makes it easier than before to manipulate whole genomes even in living organisms. Therefore, finding new ways for efficient gene syntheses and the recently shown synthesis of small genomes becomes more and more important.
Currently, available methods involve as a first step, solid-phase DNA synthesis. While this is a very high yielding procedure, the length of the oligonucleotides generated via solid-phase synthesis is limited to approximately 150 nucleotides.
Multiple steps of enzymatic ligation reactions are necessary to generate DNA strands up to several thousand nucleotides in length. While enzymatic ligations are very efficient reactions in terms of conversion, this methodology has its drawbacks. In general, it is an expensive, time-consuming process, which requires multiple purification steps for the intermediates and gives an overall low yield of the final length oligonucleotide. Aligning several oligonucleotides via short connecting splint strands as templates is still a challenge and can lead to cross-ligation as a side reaction. An alternative, sequence-specific method of aligning several dozen or even hundreds of strands is the use of DNA nanostructures. In particular, the DNA origami method has proven itself useful in creating large nanostructures with very good yields by hybridizing hundreds of different short strands, the staple strands, to a long single-stranded scaffold, which is normally derived from a bacteriophage vector.
While there have been attempts to connect the short staple strands in the origami enzymatically, due to the compact form of the nanostructure, this approach gives unsatisfying results. Recently, we were able to show that replacing the continuous scaffold strand of an origami by a series of 100mers and assembling the origami structure with the help of 20 staple strands is possible. Chemical condensing agents are a cheap alternative to enzymes, are well known and have been used to generate phosphodiester bonds between different oligonucleotides. They are also small enough to penetrate the structure. However chemical ligations are rather slow and give incomplete conversions.
In this study, we report the parallel ligation of six 5′-phosphorylated 100mers with a chemical condensing agent in a DNA origami in contrast to established methods in linear duplexes. The idea was to combine the sequence-specific alignment of several oligonucleotides in a DNA nanostructure and the use of chemical ligation methods to induce the formation of new phosphodiester bonds. We tested two different chemical ligation agents, EDC ((1-[(3-dimethylamino)propyl]-3-ethylcarbodiimide) and Cyanogen Bromide (BrCN). While a ligation with BrCN is very fast with reaction times of under 5 minutes, the ligation reaction to the 600mer was incomplete. In contrast, a ligation reaction in the origami with EDC leads to a conversion of 7% to the final length product within 3 days at 8 °C. The product could be isolated by gel electrophoresis or by HPLC (high-performance liquid chromatography). A control experiment in a linear DNA duplex with EDC did not give the desired product, the 600mer.
This demonstrates that the chemical ligation is in a packed nanostructure, where the reacting strands are held in close proximity, is favored. Furthermore, a one-step, six-fold enzymatic ligation in a linear duplex gave the 600mer, but a broad product distribution was detected and purification of the 600mer was not possible. This probably was caused by cross-ligation reactions. At last, the compatibility of the 600mer, which was obtained by the EDC-mediated reaction, with enzymes, was shown in a polymerase chain reaction (PCR). So, the obtained 600mer could, in theory, be used for further biological applications, involving enzymes.
While there is still much work to do, particularly in speeding up the reaction and increasing the still-moderate conversion, these experiments could be a first step in creating a method for one-pot parallel ligation reactions of numerous oligonucleotides and present a cheap alternative to established enzymatic techniques.
The study, Enzyme-Free Ligation of 5′-Phosphorylated Oligodeoxynucleotides in a DNA Nanostructure was recently published in the journal Chemistry & Biodiversity. This work was led by Markus Kramer from the University of Stuttgart.