UC Irvine

DNA polymerase catalyzes the correct replication and repair of DNA, an essential step in the life cycle of all organisms. The enzyme incorporates incoming deoxynucleotide triphosphates (dNTPs) into a nascent DNA strand that is complementary to a single-stranded DNA (ssDNA) template. This capability makes DNA polymerases workhorses for molecular biology and biotechnology. Single-molecule studies can identify the dynamics of DNA polymerase structural conformations, which are otherwise lost through averaging in ensemble populations. Therefore, the Weiss and Collins labs (UCI) collaboratively developed a single-walled carbon nanotube field-effect transistor (nanocircuit) to translate enzyme motions into electronic signals. These nanocircuits may illuminate hidden conformational transitions of DNA polymerase activity and reveal new mechanisms and dynamics during dNTP incorporation. The goal of this work is to apply this approach to understand the conformational dynamics of the thermostable DNA polymerase from Thermus aquaticus (Taq) and processive DNA polymerase from the Bacillus subtilis bacteriophage phi29 (Φ29). The work here resolves subtle dynamics and transient intermediate states of DNA polymerase that modulate catalytic speed and molecular recognition during DNA synthesis. Additional efforts are focused on evaluating the performance of nanocircuits for direct electrical monitoring of polymerase in future DNA sequencing.

Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. In work reported here, pyrocinchonimide (Pci) undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. The Pci motif can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody-drug conjugates. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications.

In response to the ongoing Coronavirus disease (COVID-19) pandemic, my laboratory members and I investigated the antibody response of patients infected by SARS-CoV-2 for predicting disease trajectories. Using methods of enzyme-linked immunosorbent assay (ELISA) and coronavirus antigen microarray (COVAM) analysis, antibody epitopes were mapped in the plasma of COVID-19 patients (n = 186) experiencing a wide range of disease states. This work identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the intensive care unit (ICU), requirement for ventilators, or death. By combining a disease risk factor score with a test for anti-Ep9 antibodies, severe COVID-19 outcomes could be predicted with 13.4 likelihood ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic that could guide more effective therapeutic intervention.