De novo Bacteriochlorin with Orthogonal Bioconjugation Linkers for the Detection of Point Mutations in Cell-Free DNA
Faculty Mentor Information
Dr. Olya Mass, Boise State University
Presentation Date
7-16-2026
Abstract
The reliable detection of rare, single-nucleotide mutations—such as the EGFR L858R variant found in cell-free DNA—is often hampered by a fundamental trade-off between sensitivity and specificity. Current fluorescence-based probes (e.g., TaqMan, molecular beacons) rely on signal suppression, which generates high background noise and leaves a significant portion of target DNA unhybridized at optimal annealing temperatures. To overcome these limits, we propose a sensing platform based on signal generation rather than suppression. Specifically, we utilize irreversible DNA-templated ligation to shift the thermodynamic equilibrium toward a fully hybridized probe-target complex while generating a distinct fluorescence signal.
For our platform, we designed and synthesized a novel bacteriochlorin with orthogonal bioconjugation linkers and strong fluorescence in the low-noise infrared region. As an analytical model, we designed a dual oligonucleotide probe consisting of a discriminating probe (PD) and a non-discriminating probe (PN) targeting the EGFR L858R mutation site. Next, an NHS-ester functionalized bacteriochlorin-alkyne was covalently tethered to an amine-modified PD. To address the hydrophobicity of the bacteriochlorin, we investigated the effects of stoichiometry and organic cosolvents on the bioconjugation yield.
De novo Bacteriochlorin with Orthogonal Bioconjugation Linkers for the Detection of Point Mutations in Cell-Free DNA
The reliable detection of rare, single-nucleotide mutations—such as the EGFR L858R variant found in cell-free DNA—is often hampered by a fundamental trade-off between sensitivity and specificity. Current fluorescence-based probes (e.g., TaqMan, molecular beacons) rely on signal suppression, which generates high background noise and leaves a significant portion of target DNA unhybridized at optimal annealing temperatures. To overcome these limits, we propose a sensing platform based on signal generation rather than suppression. Specifically, we utilize irreversible DNA-templated ligation to shift the thermodynamic equilibrium toward a fully hybridized probe-target complex while generating a distinct fluorescence signal.
For our platform, we designed and synthesized a novel bacteriochlorin with orthogonal bioconjugation linkers and strong fluorescence in the low-noise infrared region. As an analytical model, we designed a dual oligonucleotide probe consisting of a discriminating probe (PD) and a non-discriminating probe (PN) targeting the EGFR L858R mutation site. Next, an NHS-ester functionalized bacteriochlorin-alkyne was covalently tethered to an amine-modified PD. To address the hydrophobicity of the bacteriochlorin, we investigated the effects of stoichiometry and organic cosolvents on the bioconjugation yield.