Construction of Long, Complex, and Diverse DNA Sequences

Author: Robinson, Noah Evan

Year: 2025

Degree: Dissertation (Ph.D.)

Advisor: Wang, Kaihang

Committee Members: Parker, Joseph; Wang, Kaihang; Qian, Lulu; Winfree, Erik; Newman, Dianne K.

Option: Bioengineering

Abstract

The DNA molecule encodes the information required for biological systems to carry out a broad range of functions. The understanding of this relationship has sparked inquiries across vast fields of biology and biological engineering as we read, edit, and write the genetic information of organisms. Great advancements have been made toward these pursuits, from revolutions in DNA reading with long read sequencing and the ability to generate terabytes of data from a single run to the breakthroughs in DNA editing with the major advancements in CRISPR/Cas technologies over the last decade. However, writing DNA, as the ability to construct DNA of any length, complexity, or diversity, lags significantly behind our capacity for reading and editing.

DNA oligo synthesis can only reach short lengths of a few hundred nucleotides of single stranded DNA. The field of DNA assembly develops the methods for stitching together DNA oligos and DNA fragments into larger constructs. The current field applies a broad range of approaches that each occupy their own niche due to their unique set of advantages and disadvantages. No existing technique is able to assemble a large number of DNA fragments simultaneously with high accuracy and without placing restrictions on the sequences being assembled. This is because all existing DNA assembly technologies rely on the information contained within the complementary sequences of the DNA molecules being constructed to direct the assembly.

To meet the demand for robust DNA assembly, we have developed a new DNA assembly technique named Sidewinder which separates the information that guides assembly from the final assembled sequence using DNA 3-Way junctions. We demonstrate the transformative nature of the Sidewinder technique with highly robust and accurate assembly of complex DNA sequences of both high GC and high repeats, a 40-piece multi-fragment assembly, a parallel construction of multiple distinct genes in the same reaction, and construction of a combinatorial library with a large number of diversified positions across the entire length of the gene for high coverage of a library of 442,368 variants.

Where Sidewinder excels at the assembly of oligos to the kilobase scale, we have made a series of advancements to an existing 2-Way junction assembly technique, USER cloning, for the accurate and efficient assembly of PCR-based DNA inputs. We characterize these improvements with a series of assemblies where we achieve an average accuracy over 95%, gain 3 orders of magnitude increase in yield of transformants, and conduct large multi-fragment assemblies beyond what was previously possible with the technique. We then interface these two state-of-the-art capacities for the rapid and efficient construction of a complex 10 kilobase sequence de novo and entirely cell-free.