The best enzyme for de novo DNA synthesis may be one that is denied all its enzymatic attributes. Yes, it acts on a specific substrate. Yes, it catalyzes a chemical reaction. No, it does not exit the reaction unchanged—at least not right away, and not without assistance. Then it’s discarded, which may sound wasteful, except that it may prove less toxic and more efficient than conventional reagents used to produce synthetic DNA.
This enzyme is called TdT, or terminal deoxynucleotidyl transferase. TdT would seem a natural for DNA synthesis because it is, after all, a naturally occurring enzyme that can write DNA from scratch rather than copy an existing DNA strand. In the immune systems of vertebrates, TdT randomly adds nucleotides to genes that make antibodies, allowing the genes to express a great diversity of proteins, including antibodies that can target novel antigens.
To harness TdT’s base-adding power—while inducing TdT to add selected bases rather than random bases—scientists based at the Department of Energy’s Joint BioEnergy Institute (JBEI) have created TdT–nucleotide conjugates. Each such conjugate is used to add a single nucleotide to a growing DNA primer.
Crucially, the TdT portion of the conjugate stays attached to the primer, shielding it from like TdT-nucleotide conjugates, which would keep adding the same nucleotide. The TdT portion of the conjugate is released only when the TdT-nucleotide link is deliberately cleaved. Then the TdT is discarded, leaving the DNA primer exposed, potentially, to another TdT-nucleotide conjugate, which may incorporate a different nucleotide. This cycle, repeated over and over with different TdT-nucleotide conjugates, may generate an increasingly lengthy DNA strand that possesses a desired nucleotide sequence.
The TdT–nucleotide conjugates were developed by Sebastian Palluk and Daniel Arlow, graduate students in the JBEI lab of Jay Keasling, Ph.D., a chemical and biological engineer who is also affiliated with Lawrence Berkeley National Laboratory and the University of California, Berkeley. According to these scientists, the TdT-nucleotide conjugates succeed where blocking groups fail.
Previous proposals for harnessing TdT raised the possibility of using “blocked” nucleotides, that is, nucleotides modified to incorporate blocking groups. After extending a DNA molecule, a blocked nucleotide’s blocker could be removed, permitting the next extension. Unfortunately, TdT’s catalytic site is too small to accept a blocked nucleotide.
“People have basically tried to 'dig a hole' in the enzyme by mutating it to make room for this blocking group,” Arlow said. “It's tricky because you need to make space for it but also not screw up the activity of the enzyme.”
Palluk and Arlow came up with a different approach. “Instead of trying to dig a hole in the enzyme, what we do is tether one nucleotide to each TdT enzyme via a cleavable linker,” Arlow continued. “That way, after extending a DNA molecule using its tethered nucleotide, the enzyme has no other nucleotides available to add, so it stops.”
Details appeared June 18 in the journal Nature Biotechnology, in an article entitled “De novo DNA Synthesis Using Polymerase-Nucleotide Conjugates.” The article describes how TdT may be conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that TdT may then incorporate into a DNA primer.
“After incorporation of the tethered dNTP, the 3′ end of the primer remains covalently bound to TdT and is inaccessible to other TdT–dNTP molecules,” the article’s authors write. “Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension.”
The scientists demonstrated that TdT–dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10–20 seconds, and that the scheme can be iterated to write a defined sequence. This approach, the scientists suggested, may form the basis of an enzymatic oligonucleotide synthesizer.
In their first trials—10 cycles using the engineered TdT enzyme to create a 10-base oligonucleotide—the scientists showed that their technique is nearly as accurate in each step of the synthesis as current techniques.
“When we analyzed the products using next-generation sequencing, we were able to determine that about 80% of the molecules had the desired 10-base sequence,” Arlow said. “That means, on average, the yield of each step was around 98%, which is not too bad for a first go at this 50-plus-year-old problem. We want to get to 99.9% in order to make gene-length DNA.”
Once they can reach 99.9% fidelity, they can synthesize a 1000-base-long molecule in one go with a yield of more than 35%, which is impossible with current chemical synthesis techniques, Palluk added.
Current DNA synthesis, which dates from 1981 and uses techniques from organic chemistry labs, is limited to directly producing so-called oligonucleotides about 200 bases long, because inevitable errors in the process lead to a low yield of correct sequences as the length increases. To assemble even a small gene, scientists must synthesize it piecemeal in segments about 200 bases long and then stitch them together. This is time consuming, often requires multiple attempts, and sometimes fails completely.
In addition, when synthetic DNA is ordered from commercial suppliers, the turnaround time for one small gene around 1500 bases long can be two weeks at a cost of $300, limiting the number of genetic tweaks researchers can afford to try and the speed with which they can experiment.
“By directly synthesizing longer DNA molecules, the need to stitch oligonucleotides together and the limitations arising from this tedious process could be reduced,” Palluk noted. “Our dream is to directly synthesize gene-length sequences and get them to researchers within few days.”