Synthesis and Applications of Error Free DNA Using Polymerase-Enabled Rapid, Frugal Error Correcting Technology (PERFECT) PCR

Enzymes act as catalysts and play a crucial role in biology by regulating biological reactions. Enzymes convert substrates into specific products which are essential for processes like energy production, DNA replication, nutrient metabolism, etc. Enzymes functionality is complex as they are affected by factors such as pH, temperature, and substrate/product concentrations. The first aim focused on identifying, expressing, and purifying thermostable enzymes to selectively eliminate incorrect DNA templates, thereby enhancing accuracy of the synthesized de novo DNA template. We identified that the Endonuclease NucS (NucS) enzyme from hyperthermophilic archaeon Pyrococcus kukulkanii (Pkl) survived up to 105 degrees C temperatures. The gene was codon optimized and expressed in Escherichia coli BL21(DE3) and purified through his-tag purification. We analyzed the enzyme's activity, kinetics, thermal stability, and optimal concentrations for DNA template error corrections. The second aim focused on integrating the NucS enzyme into PCR to establish a Polymerase-Enabled Rapid Error Correcting Technology (PERFECT) PCR platform for synthesizing accurate and error-free DNA. While a fast and cost-effective method to synthesize DNA involves using overlapping oligonucleotides, these oligonucleotides are prone to errors due to limitations in phosphoramidite synthesis chemistry. Enzymatic error correction is the most widely used approach to reducing error rates. However, it cannot be integrated with the PCR amplification step and must be performed sequentially, leading to a time-consuming, multi-step process. Currently, no method exists to reduce oligonucleotide-based errors during PCR. The PERFECT PCR platform addresses this knowledge gap by incorporating the error correction step directly into the PCR amplification process. This integration enabled error correction during each PCR cycle, allowing multiple rounds of correction and ultimately providing a more economical and faster approach to error-free DNA synthesis compared to conventional enzymatic error correction strategies. The third aim focused on using the PERFECT PCR technology for applications such as DNA based data storage and synthesis of important therapeutics like spike protein of SARS-CoV-2 virus. DNA-based data storage offers unprecedented storage density and durability compared to traditional media, but it faces challenges in long access latency and limited encoding efficiency. Current DNA storage methods achieve only 8% of the theoretical maximum storage density (TMSD) due to biological constraints and error rates. The PERFECT PCR technique reached 94% of TMSD, representing a twelve-fold improvement over current systems and approaching the theoretical limit for existing sequencing technologies. By addressing the core challenges of latency, density, and accuracy, our approach brings DNA-based data storage significantly closer to practical, large-scale implementation. We further used the PERFECT PCR technology for synthesis of 3.8 kb SARS CoV-2 spike protein DNA sequence. We successfully synthesize the 3.8 kb gene with an error rate of 0.2 errors/kb. In addition, the accurate DNA synthesis methods described here may be applied to de novo genome assembly and therefore have broad implications for the synthesis and analysis of biological systems. Throughout this dissertation, the overarching theme is to integrate thermostable enzymes into PCR to eliminate incorrect templates thereby synthesizing error free DNA template for accurate and efficient DNA synthesis for data storage, biological system, and therapeutic applications.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

Texas A&M University

Year dc:date.issued

2025

Author dc:creator

• Sabnis, Rushant

Advisor dc:contributor.advisor

• Sun, Qing

Committee members dc:contributor.committeemember

• Wu, Hung-Jen
• Defigueiredo, Paul
• Han, Arum
• Jayaraman, Arul

• Engineering, Chemical

Handle dc:identifier.uri

https://hdl.handle.net/1969.1/1597810

OAI record oai:identifier

https://oaktrust.library.tamu.edu/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Aoaktrust.library.tamu.edu%3A1969.1%2F1597810