Research High-throughput engineering of DNA aptamers for rapidly evolving viral variants Since the outbreak of COVID-19 in early 2020, WHO has declared five variants of concern (Alpha, Beta, Gamma, Delta and Omicron) whose transmissibility is higher than that of the original strain. As the coronavirus is rapidly evolving and new variants often lead to vaccine breakthrough infections, extended quarantine, and increased hospitalization, it is critical to have a modular technology that can be easily reprogrammed to diagnose and neutralize novel variants of concern once they are identified. DNA aptamers are DNA molecules that can bind various targets strongly and selectively, including small molecules, proteins, and even viruses. In this project, we developed a high-throughput aptamer engineering platform that combines rationally diversified libraries with a repurposed Illumina MiSeq next-generation sequencing (NGS) flow cell for massively parallel on-chip screening. Rather than performing de novo aptamer selection, our platform rapidly re-engineers existing aptamers to recognize evolving viral variants by simultaneously profiling thousands of sequence variants in a single experiment. Our end goal is to create a streamline process to generate new aptamers that can be used both as a sensor and even a treatment to battle emerging viral variants. Using SARS-CoV-2 spike protein as a model system, we successfully generated aptamer variants with substantially improved affinity toward the Delta variant, restored nanomolar binding to the Omicron’s XBB subvariant, and identified highly selective wild-type binders that were further developed into FRET-based sensors. More broadly, this platform provides a general strategy for rapidly adapting aptamers against evolving targets, offering a scalable alternative to repeated SELEX campaigns for future pathogen surveillance and diagnostic development. This project is in collaboration with Dr. Yi Lu in UT Chemistry. More details can be found in He et al., biorxiv, 2026. Non-FRET silver nanocluster reporters for nucleic acid detection Fluorescent reporters are essential components of modern molecular diagnostics, including PCR and CRISPR-based nucleic acid detection assays. Most current reporters rely on fluorescence resonance energy transfer (FRET), requiring a fluorophore and quencher to be chemically attached to opposite ends of an oligonucleotide. This dual-label design increases manufacturing cost and limits signal generation to fluorescence intensity changes. We developed Subak, a new class of DNA-templated silver nanocluster (AgNC) reporters that bypasses FRET entirely. Instead of switching fluorescence on or off, Subak undergoes a dramatic green-to-red fluorescence conversion upon nuclease cleavage through a transformation of the encapsulated silver nanocluster. This unique mechanism enables low-cost, ratiometric nucleic acid detection with a readily visible color change and provides a versatile alternative to conventional FRET reporters. Building on this platform, we further uncovered the molecular mechanism underlying the color transformation and engineered RNA-responsive variants (rSubak) for CRISPR/Cas13-based RNA detection, achieving amplification-free detection of viral RNAs with substantially improved sensitivity over commercial FRET substrates. Unlike conventional FRET reporters whose optical properties are determined by donor-acceptor distance, Subak exploits cleavage-induced structural reorganization of DNA-templated silver nanoclusters to generate a large emission color shift. Through site-specific cleavage experiments and electrospray ionization mass spectrometry, we demonstrated that the fluorescence transition is governed by changes in the silver coordination environment rather than direct enzyme-cluster interactions or simple cluster fragmentation. These mechanistic insights established design principles for engineering programmable non-FRET reporters and enabled the development of rSubak substrates for RNase and CRISPR/Cas13 assays. Beyond viral diagnostics, the Subak platform provides a general strategy for creating inexpensive, ratiometric fluorescent substrates for diverse enzyme activities and expands the toolbox of DNA-templated nanomaterials for molecular sensing and bioanalytical applications. This project is in collaboration with Dr. Jennifer Brodbelt in UT Chemistry. More details can be found in Hong et al., Nature Nanotechnology, 2024; Kim et al., biorxiv, 2026. Advanced multiplexed fluorescence lifetime imaging for live-cell analysis and retinal health assessment Fluorescence lifetime imaging microscopy (FLIM) reads out the average time a fluorophore spends in its excited state, which is an intrinsic property that is independent of excitation power and concentration, resists photobleaching, and separates dyes with overlapping spectra. We advances multiplexed FLIM through a shared toolkit of pulsed interleaved excitation, synchronized multi-channel time-resolved detection, and phasor unmixing driven by Gaussian Mixture Models, applied to two settings. In live cells, a pulsed interleaved excitation spectral FLIM (PIE-sFLIM) system uses a two-wavelength alternating laser scheme and a 16-channel spectral detector to image six fluorescent tags in a single hyperspectral snapshot, cleanly unmixing all six without prior knowledge of the fluorophores and greatly improving the speed needed to follow rapid cellular events. The same lifetime approach was applied to the rabbit retina. Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. We uses a custom two-photon fluorescence lifetime ophthalmoscope (2P-FLIO) to probe photoreceptors and retinal pigment epithelium (RPE), and revealed key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure. This project is in collaboration with Dr. Grady Rylander in UT BME. More details can be found in Nguyen et al.,Optics Express, 2024; Nguyen et al., Biomedical Optics Express, 2024. Measuring hybridization kinetics of single DNA molecules in live cells Single-molecule detection provides researchers with a unique method to probe kinetics of biomolecules in their native environment, without the need to synchronize the molecular states. However, current single-molecule measurements of DNA hybridization kinetics are mostly performed on a surface or inside an electrokinetic trap, which are not physiologically relevant conditions. Recently we demonstrate a time-resolved, 3D single-molecule tracking (3D-SMT) method that that can follow individual DNA molecules diffusing inside a mammalian cell and observe multiple annealing and melting events on the same molecules. By comparing the hybridization kinetics of the same DNA strand in vitro, we found the association constants can be 13- to 163-fold higher in the molecular crowding cellular environment. In contrast to other confocal-feedback 3D single-particle tracking demonstrations, we tracked single DNA reporter strands inside a live cell and measured their annealing-melting kinetics. Although camera-based techniques combined with point-spread function engineering can achieve 3D tracking in live cells, they do not offer any lifetime monitoring capability that can be used to reveal the molecular binding kinetics. While two-color colocalization and 2D tracking can provide a full dimerization kinetics analysis of G protein coupled receptor in live cells, the 2D-TIRF imaging method is not suitable to probe the binding kinetics of a biomolecule deep in cytoplasm. On the contrary, our 3D-SMT method uses multiple single-photon detectors or multiplexed pulsed laser illuminations to achieve spatial filtering, which not only allows for high-resolution 3D localization of single molecules in live cells, but enables simultaneous characterization of molecular binding state through a continuous lifetime measurement. The data acquired can be used to generate new models that can predict in-cellulo hybridization kinetics from sequence, study the molecular crowding inside cells and probe the cellular development and transition states. More details can be found in Chen et al., JACS, 2019. Deep Learning-assisted design of fluorescence nanobiosensors The ability to design and synthesize nanomaterials with specific photophysical properties is not only a great intellectual challenge, but also one with important practical consequences. To address this challenge, we are currently exploring a new class of biolabels termed few-atom noble metal nanoclusters. Noble metal nanoclusters are collections of small numbers of gold or silver atoms (2-30 atoms) with physical sizes close to the Fermi wavelength of an electron (~0.5 nm for gold and silver). Providing the missing link between atomic and nanoparticle behavior in noble metals, these nanoclusters exhibit optical, electronic, and chemical properties dramatically different from those of much larger nanoparticles or bulk materials. Among those water-soluble noble metal nanoclusters newly developed, DNA-templated silver nanoclusters (DNA/AgNCs) have attracted great interest in biosensing owing to a number of useful photophysical and photochemical properties. For instance, controlled conversion of DNA/AgNCs between bright and dark states by guanine proximity has led to the invention of a new molecular probe, termed a NanoCluster Beacon (NCB), that “lights up” upon binding with a DNA target. Not relying on Főrster energy transfer as the fluorescence on/off switching mechanism, NCBs have the potential to reach an ultrahigh signal-to-background (S/B) ratio in molecular sensing. Since the fluorescence enhancement is caused by intrinsic nucleobases, NCB detection is simple, inexpensive, and compatible with commercial DNA synthesizers. We hold 3 US patents on the silver nanocluster probes (10407715, 9499866, 8476014) and are currently collaborating with Dr. Jennifer Brodbelt in UT Chemistry and Dr. Minjun Kim at SMU in using mass spectrometry and nanopores to study DNA/AgNCs. More details can be found in Yeh et al., Nano Letters, 2010; Yeh et al., JACS, 2012; Obliosca et al., ACS Nano, 2014; Chen et al., JACS, 2015; Blevins et al., ACS Nano, 2019. We are currently using deep learning models to predictively design silver nanocluster sensors with desired colors, on/off ratios and photostabilities. This is achieved by taking advantage of repurposed next generation sequencing technique. We recently screened 40,000 fluorescent nanomaterial species and elucidated the key factors to achieve a high synthesis yield and the design rules for creating bright yellow or red nanomaterial fluorophores (Kuo et al., Adv. Mater., 2022). Deep and high-resolution 3D tracking of single particles using nonlinear and multiplexed illumination (TSUNAMI) Molecular trafficking within cells, tissues, and engineered 3D multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of ~15 μm. To achieve deep and high-resolution 3D tracking, we have developed a two-photon, 3D single-particle tracking (2P-3D-SPT) method capable of tracking particles at depths up to 200 μm in scattering samples with 22/90 [xy/z] nm spatial localization precision and 50 µs temporal resolution. At shallow depths the localization precision can be as good as 35 nm in all three dimensions. The approach is based on passive pulse splitters used for nonlinear microscopy to achieve spatiotemporally multiplexed 2P excitation and temporally demultiplexed detection to discern the 3D position of the particle. The z-tracking range is up to ± 50 μm (limited by the objective z-piezo stage) and the method enables simultaneous fluorescence lifetime measurements on the tracked particles. A major advantage of this method over previous tracking approaches is that it requires only one detector for SPT and is compatible with multi-color two-photon microscopy. We describe our approach and demonstrate its capabilities by tracking single fluorescent beads in aqueous solutions that include scattering, as well as tracking prescribed motions in these controlled environments. We then demonstrate tracking of EGFR (epidermal growth factor receptor) complexes tagged with fluorescent beads in tumor spheroids, demonstrating deep 3D SPT in multicellular models. We have coined this technique TSUNAMI (Tracking Single particles Using Nonlinear And Multiplexed Illumination; US patent 10281399). We are currently using TSUNAMI to study membrane receptor motion and drug delivery. More details can be found in Perillo et al., Nature Communications, 2015; Li et al., Cancer Cell, 2018; Liu, ACS Nano, 2020.
