Team:Cooper Union/TdT project

De novo Enzyme Mediated Oligonucleotide Synthesizer

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 wants to focus on the ways to minimize the time and money required for DNA synthesis and to allow the labs to produce oligonucleotides easily without ordering. Here we introduce the De novo Enzyme Mediated Oligonucleotide Synthesizer (DEMOS); a programmble enzyme based, template-free, synthesizer for nucleic acid polymers. Terminal Deoxynucleotidyl Transferase, also referred as TdT, is an enzyme that has the ability to add nucleotides to single stranded oligos, while other DNA enzymes can only add a nucleotide to double stranded ones. Our improved system is based on a theoretical model for template-free synthesis of DNA that proposed deoxynucletotiode triphospate substrates containing a reversible protective group on the 3' hydroxy group. In that system, the 3' protective group was an Acetyl group, with reversibility being achieved by a altering pH and thereby activating a Our system simplifies this novel approach by proposing heat and ultraviolet light labile reversible 3' protective groups. We first tested our system with commercially available heat labile

Improvements to the De novo Oligonucleotide Synthesizer

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.

Our eventual goal will be to a novel microfluidic de novo synthesizer that will allow laboratories, from academia and commercial biotech, to DIYBio community labs to rapidly and economically synthesize any strand of DNA. 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.

Potential Errors in the Synthesis System

During each cycle there are independent sources of error that can affect the overall efficiency of the reaction. The most obvious errors include: 1) Incomplete enzymatic addition of 3' reversibly protected dNTPs by TdT during the nucleotide incorporation step. 2) Incomplete hydrolysis of 3' protective group during heating or other treatment. 3) Incomplete removal of unincorporated 3' reversibly protected nucleotides after each incorporation step. 4) Spontaneous hydrolysis of 3' protective groups prior to intended deprotection. Each error, if not sufficiently addressed would compound the likelihood of misincorporated nucleotides and a reduction of overall yield.

For example, during error scenario 1, oligonucletide chains that have not had a 3' reversibly protective group nucleotide added during an incorporation step, will, during the subsequent step after deprotection, be potential substrates for the addition of the next incoming nucleotide. This would lead to the base deletion. During error scenario 2, the failure to remove a protective group would lead to a population of oligonucleotides that would fail to add a nucleotide during the next subsequent addition of incoming nucleotides, leading to a base deletion at this step. For error scenario 3, the presence of unremoved, unreacted nucletides would lead to misincorporation and hence base substitutions during each subsequent stage of oligonucleotide elongation. During Scenario 4, the presence of dNTPs without 3' protective groups would lead to the incorporation of multiple dNTPs, resulting in homopolymeric stretches of nucleotides in the growing oligonucleotide chain. This would lead to insertion mutations. For a truly reliable system to be built, these and potentially other sources of error will need to be adequately countered. References

Minhaz Ud-Dean, S.M. (2008) A Theoretical Model for Template-Free Synthesis of Long DNA Sequence. Syst. Synth. Biol. 2:67-73

Koukhareva, I. and Lebedev, A. (2009) 3'-Protected 2'-Deoxynucleoside 5'-Triphosphates as a Novel Tool for Heat-Triggered Activation of PCR. Anal Chem. 81(12):4955-4962

Kuan, W.L., Joy, J. Mee, N.F., Perlyn, K.Z., Wen, T.S., Nguen, T., James, J., Chai, E., Flotow, H., Crasta, S., Chua, K., Peng, N.S. and Hill, J. (2010) Generation of Active Bovine Terminal Deoxynucleotidyl Transferase (TdT) in E. coli. Biochemistry Insights. 3: 41-46.

Boule, J.B., Rougeon, F.Papanicolaou, C. (2001) Terminal Deoxynucleotidyl Transferase Indiscriminately Incorporates Ribonucleotides and Deoxyribonucleotides. J. Biol. Chem. 276, 33: 31388-31393.

Motea, E.A. and Berdis, A.J. (2010) Terminal Deoxynucleotidyl Transferase: The Story of a Misguided DNA Polymerase. Biochim. Biophys. Acta. 1804, 5: 1151-1166.