Nanopore sequencing of ALIEN DNA

Abstract

Nucleic acids in the forms of DNA and RNA carry the genetic information of all known living organisms, enabled by an aperiodic crystalline structure in which four nucleobases, adenine, cytosine, guanine, and thymine (A, C, G and T) are arranged sequentially along a repeating sugar-phosphate backbone. How and why life came to adopt these specific nucleobases for genetic information storage is a mystery, and there are strong suspicions that other nucleobases could form robust genetic information storage systems. A variety of artificial genetic systems have been synthesized, retaining the backbone of DNA but incorporating non-standard nucleobases in addition to (or replacing) A,C,G,T. These artificial systems have applications in molecular diagnostics and targeted medicine, but are largely incompatible with modern techniques of nucleic acid sequencing. By tackling the sequencing problem, we can help realize the full potential of these applications by increasing the effectiveness of artificial nucleic acid quality control and sample reproducibility. For my dissertation in the UW nanopore biophysics laboratory, I developed nanopore sequencing technology of an expanded eight-letter “hachimoji” DNA alphabet containing A,C,G,T as well as four additional hydrogen-bonding nucleobases B, P, S, and Z. In nanopore nucleic acid sequencing, a single nucleic acid molecule is pulled through a nanoscale pore by an applied electric field while a bound motor enzyme controls its motion. The the bases of the molecule block the electric current through the pore with magnitudes based on their chemical structure, enabling sequencing. This technique is advantageous for hachimoji DNA sequencing because the signal only depends on the chemical structure of the analyte, with no requirements related to interaction with biological systems present in other techniques. I begin in chapter 1 with an introduction into artificial genetic systems, their applications, and the specific difficulties involved in sequencing them. In chapter 2 I summarize the historical development of nanopore DNA sequencing, setting the stage for the techniques I developed in this work. In chapter 3 I characterize the readability of hachimoji DNA with nanopores. I also analyze the error modes in nanopore sequencing of hachimoji DNA that are due to interactions between the non-standard bases and the motor enzyme involved in sequencing. In chapter 4, I build off of the preliminary work in chapter 3 to demonstrate the first direct sequencing of an entirely synthetic DNA alphabet. These results provide insights into further development of artificial genetic systems, such as prioritizing compatibility with existing biomolecules. This work is also relevant to the study of “natural” non-standard bases that occur in biology and have relevance to human health. These include DNA methylation and RNA modifications that have so far proven difficult to interrogate.