De novo synthesis is a way of creating DNA oligonucleotides without the need of a template strand. Since the conventional method is expensive, time consuming, and inefficient, our team has focused on minimizing the time and money required for DNA synthesis while allowing labs to produce oligonucleotides easily without ordering. Here we introduce the De novo Enzyme Mediated Oligonucleotide Synthesizer (DEMOS); a programmable enzyme based, template-free, synthesizer for nucleic acid polymers. Our eventual goal will be to build a microfluidic de novo synthesizer that will allow laboratories, from academia to DIYBio community labs to rapidly and economically synthesize any strand of DNA for their projects. We hope that this system will become the key platform that bridges the in silico to in vitro gap in the design-test-build cycle of DNA synthesis and experimentation.

Our system is fundamentally based on two key technologies, one practical and the other theoretical:

1. The development of deoxynucleotide triphosphate (dNTP) substrates with 3' reversible protective groups for "sequencing by synthesis" and "hot start" PCR technologies
2. The published theoretical model of directed template-free synthesis of DNA using the enzyme terminal deoxynucleotidyl transferase (TdT). This enzyme that has the ability to add nucleotides to the 3' ends of DNA, preferable 3' overhangs, in a template-free manner.

In the general scheme shown below in Figure 1, an incoming 3'-RPdNTP (reverse protective group dNTP) is added to a free 3'OH of a growing DNA chain by the enzyme TdT at 37 degrees Celsius. This is followed by a wash step where unincorporated 3'RPdNTPs and PPi (pyrophosphate) are removed. For heat labile 3'-RPdNTP’s deprotection is achieved by raising the temperature to 95 degrees Celsius, while photolabile 3'-RPdNTP’s are deprotected by pulses of ultra-violet light. This is followed by another wash step where the decoupled protective groups are removed, thereby resetting the system to begin another cycle of addition.



The deprotection reaction of the 3'-RPdNTP’s is shown in Figure 2a. The structures of two heat labile 3' protective group dNTPs that are commercially available from TriLink Biotechnologies (www.trilinkbiotech.com) are shown in Figure 2b . For our proof of concept experiments, 3'-TBE-dNTP’s were used, as it has a faster rate of deprotection at 95 degrees Celsius; 3'-TBE-dNTP t 1/2 + 5 minutes, 3'-THF-dNTP t 1/2= 90 minutes)



In the controllable system proposed by Ud-Dean, the reversibility of a 3' acetyl protective group was achieved by lowering pH during each cycle and thereby activating a deacetylase enzyme that is active at lower pH. During this step, TdT is also inactived by the shift to lower pH, thereby preventing the addition of extra nucleotides

Our system simplifies this novel approach by thermolabile reversible 3' protective groups, thereby combining the



In our present configuration, full length recombinant bovine TdT was used in the reaction. This particular enzyme denatures in the presence of elevated temperatures, thereby necessitating the repeated addition of enzyme during each step of nucleotide addition after blocking group removal via 95 degrees C heat pulse. Therefore it would be useful to either isolate a naturally occurring variant of TdT or similar enzyme, that is heat tolerant, most likely from a thermophylic organism, or engineer such a variant. Another potential course of improvement would be to construct truncated and other variants of engineered TdT that exhibit faster kinetics of modified nucleotide incorporation. Lastly, our present system used commercially available nucleotides that had heat labile 3' protective groups. By switching to photolabile nucleotides, the system should perform better by exhibiting an overall faster reaction cycle, since temperature ramp up and down cycles are avoided and no extra TdT enzyme would need to be added at the beginning of each cycle, as heat denaturation would also be avoided. We are presently exploring these improved features.