Professional Education


  • Licenciado, Universidad De Chile (2011)
  • Doctor of Philosophy, McGill University (2020)
  • Ph.D., McGill University, Chemistry, Chemical Biology, Biophysics (2020)
  • B. Sc., University of Chile, Chemistry (2011)

Stanford Advisors


All Publications


  • Unifying Mechanism for Thiol-Induced Photoswitching and Photostability of Cyanine Dyes JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Gidi, Y., Payne, L., Glembockyte, V., Michie, M. S., Schnermann, M. J., Cosa, G. 2020; 142 (29): 12681–89

    Abstract

    Cyanines (Cy3, Cy5, Cy3B) are the most utilized dyes for single-molecule fluorescence and localization-based super-resolution imaging. These modalities exploit cyanines' versatile photochemical behavior with thiols. A mechanism reconciling seemingly divergent results and enabling control over cyanine photoreactivity is however missing. Utilizing single-molecule fluorescence on Cy5 and Cy5B, transient-absorption spectroscopy, and DFT modeling on a range of cyanine dyes, herein we show that photoinduced electron transfer (PeT) from a thiolate to Cy in their triplet excited state and then triplet-to-singlet intersystem crossing in the nascent geminate radical pair are crucial steps. Next, a bifurcation occurs, yielding either back electron transfer and regeneration of ground state Cy, required for photostabilization, or Cy-thiol adduct formation, necessary for super-resolution microscopy. Cy regeneration via photoinduced thiol elimination is favored by adduct absorption spectra broadening. Elimination is also shown to occur through an acid-catalyzed reaction. Overall, our work provides a roadmap for designing fluorophores, photoswitching agents, and triplet excited state quenchers for single-molecule and super-resolution imaging.

    View details for DOI 10.1021/jacs.0c03786

    View details for Web of Science ID 000555420600018

    View details for PubMedID 32594743

  • Hepatitis C Virus Helicase Binding Activity Monitored through Site-Specific Labeling Using an Expanded Genetic Code ACS INFECTIOUS DISEASES Ablenas, C. J., Gidi, Y., Powdrill, M. H., Ahmed, N., Shaw, T. A., Mesko, M., Gotte, M., Cosa, G., Pezacki, J. 2019; 5 (12): 2118–26

    Abstract

    The mechanism of unwinding catalyzed by the hepatitis C virus nonstructural protein 3 helicase (NS3h) has been a subject of considerable interest, with NS3h serving as a prototypical enzyme in the study of helicase function. Recent studies support an ATP-fueled, inchworm-like stepping of NS3h on the nucleic acid that would result in the displacement of the complementary strand of the duplex during unwinding. Here, we describe the screening of a site of incorporation of an unnatural amino acid in NS3h for fluorescent labeling of the enzyme to be used in single-molecule Förster resonance energy transfer (FRET) experiments. From the nine potential sites identified in NS3h for incorporation of the unnatural amino acid, only one allowed for expression and fluorescent labeling of the recombinant protein. Incorporation of the unnatural amino acid was confirmed via bulk assays to not interfere with unwinding activity of the helicase. Binding to four different dsDNA sequences bearing a ssDNA overhang segment of varying length (either minimal 6 or 7 base length overhang to ensure binding or a long 24 base overhang) and sequence was recorded with the new NS3h construct at the single-molecule level. Single-molecule fluorescence displayed time intervals with anticorrelated donor and acceptor emission fluctuations associated with protein binding to the substrates. An apparent FRET value was estimated from the binding events showing a single FRET value of ∼0.8 for the 6-7 base overhangs. A smaller mean value and a broad distribution was in turn recorded for the long ssDNA overhang, consistent with NS3h exploring a larger physical space while bound to the DNA construct. Notably, intervals where NS3h binding was recorded were exhibited at time periods where the acceptor dye reversibly bleached. Protein induced fluorescence intensity enhancement in the donor channel became apparent at these intervals. Overall, the site-specific fluorescent labeling of NS3h reported here provides a powerful tool for future studies to monitor the dynamics of enzyme translocation during unwinding by single-molecule FRET.

    View details for DOI 10.1021/acsinfecdis.9b00220

    View details for Web of Science ID 000503114600018

    View details for PubMedID 31640339

  • A High-Throughput Image Correlation Method for Rapid Analysis of Fluorophore Photoblinking and Photobleaching Rates ACS NANO Sehayek, S., Gidi, Y., Glembockyte, V., Brandao, H. B., Francois, P., Cosa, G., Wiseman, P. W. 2019; 13 (10): 11955–66

    Abstract

    Super-resolution fluorescence imaging based on localization microscopy requires tuning the photoblinking properties of fluorescent dyes employed. Missing is a rapid way to analyze the blinking rates of the fluorophore probes. Herein we present an ensemble autocorrelation technique for rapidly and simultaneously measuring photoblinking and bleaching rate constants from a microscopy image time series of fluorescent probes that is significantly faster than individual single-molecule trajectory analysis approaches. Our method is accurate for probe densities typically encountered in single-molecule studies as well as for higher density systems which cannot be analyzed by standard single-molecule techniques. We also show that we can resolve characteristic blinking times that are faster than camera detector exposure times, which cannot be accessed by threshold-based single-molecule approaches due to aliasing. We confirm this through computer simulation and single-molecule imaging data of DNA-Cy5 complexes. Finally, we demonstrate that with sufficient sampling our technique can accurately recover rates from stochastic optical reconstruction microscopy super-resolution data.

    View details for DOI 10.1021/acsnano.9b06033

    View details for Web of Science ID 000492801600104

    View details for PubMedID 31513377

  • Efficient One-Step PEG-Silane Passivation of Glass Surfaces for Single-Molecule Fluorescence Studies ACS APPLIED MATERIALS & INTERFACES Gidi, Y., Bayram, S., Ablenas, C. J., Blum, A., Cosa, G. 2018; 10 (46): 39505–11

    Abstract

    Surface passivation to inhibit nonspecific interactions is a key requirement for in vitro single-molecule fluorescent studies. Although the standard passivation methods involve the covalent attachment of poly(ethylene glycol) (PEG) in two steps preferably over quartz surfaces, this protocol and improvements thereon require extensive labor and chemicals. Herein, we report an efficient one-step surface grafting of PEG-silane that yields enhanced passivation, as evidenced by reduced nonspecific interactions, over the conventional method at a minimal time and reagent cost and on glass surfaces. Our method is rooted in a mechanistic understanding of the silane reaction with the silanol groups on the glass surface. Single-molecule fluorescence studies with fluorescently tagged proteins and DNA on PEG-silane-functionalized glass surfaces validate the enhanced performance of the method. Combined with atomic force microscopy surface characterization, our study further illustrates that few remaining pinhole defects, plausibly from defects on the glass, on PEG-silane glass-coated surfaces account for the minimal background, where typically no more than one molecule is nonspecifically attached in a given diffraction-limited spot on the surface.

    View details for DOI 10.1021/acsami.8b15796

    View details for Web of Science ID 000451496000013

    View details for PubMedID 30346695

  • Intense White Molecular Fluorescence from Naphthoxazole-Quinoline Derivatives PHOTOCHEMISTRY AND PHOTOBIOLOGY Zanocco, R. P., Valdebenito, S., Gidi, Y., Zapata-Torres, G., Lemp, E., Nonell, S., Zanocco, A. L. 2018; 94 (6): 1092–99

    Abstract

    Naphthoxazole derivatives are small heterocyclic compounds endowed with outstanding fluorescence properties. In this work, we report a detailed study of the intense white light fluorescence observed in naphthoxazole-quinoline dyads in solvent mixtures including at least a strong hydrogen bonding solvent. The same phenomenon was also studied in inclusion complexes naphthoxazole derivatives-sulfonated-βCD either in aqueous solution as well as in solid phase. A novel mechanism of white molecular fluorescence generation based on solvent-to-fluorophore proton transfer facilitated by ground state hydrogen bonding was characterized. The emission combines both, a blue charge transfer fluorescence emitted by the locally excited singlet state along with a red-shifted emission from a proton transfer complex.

    View details for DOI 10.1111/php.12970

    View details for Web of Science ID 000450018200003

    View details for PubMedID 29964295

  • Tris-N-Nitrilotriacetic Acid Fluorophore as a Self-Healing Dye for Single-Molecule Fluorescence Imaging JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Glembockyte, V., Wieneke, R., Gatterdam, K., Gidi, Y., Tampe, R., Cosa, G. 2018; 140 (35): 11006–12

    Abstract

    The photostability of fluorescent labels comprises one of the main limitations in single-molecule fluorescence (SMF) and super-resolution imaging. An attractive strategy to increase the photostability of organic fluorophores relies on their coupling to photostabilizers, e.g., triplet excited state quenchers, rendering self-healing dyes. Herein we report the self-healing properties of trisNTA-Alexa647 fluorophores (NTA, N-nitrilotriacetic acid). Primarily designed to specifically label biomolecules containing an oligohistidine tag, we hypothesized that the increased effective concentration of Ni(II) triplet state quenchers would lead to their improved photostability. We evaluated photon output, survival time, and photon count rate of different Alexa647-labeled trisNTA constructs differing in the length and rigidity of the fluorophore- trisNTA linker. Maximum photon output enhancements of 25-fold versus Alexa647-DNA were recorded for a short tetraproline linker, superseding the solution based photostabilization by Ni(II). Steady-state and time-resolved studies illustrate that trisNTA self-healing role is associated with a dynamic excited triplet state quenching by Ni(II). Here improved photophysical/photochemical properties require for a judicious choice of linker length and rigidity, and in turn a balance between rapid dynamic triplet excited state quenching versus dynamic/static singlet excited state quenching. TrisNTA fluorophores offer superior properties for SMF allowing specific labeling and increased photostability, making them ideal candidates for extended single-molecule imaging techniques.

    View details for DOI 10.1021/jacs.8b04681

    View details for Web of Science ID 000444219100015

    View details for PubMedID 30085664

  • DNA Nanotubes with Hydrophobic Environments: Toward New Platforms for Guest Encapsulation and Cellular Delivery ADVANCED HEALTHCARE MATERIALS Rahbani, J. F., Vengut-Climent, E., Chidchob, P., Gidi, Y., Tuan Trinh, Cosa, G., Sleiman, H. F. 2018; 7 (6): e1701049

    Abstract

    Natural systems combine different supramolecular interactions in a hierarchical manner to build structures. In contrast, DNA nanotechnology relies almost exclusively on DNA base pairing for structure generation. Introducing other supramolecular interactions can expand the structural and functional range of DNA assemblies, but this requires an understanding of the interplay between these interactions. Here, an economic strategy to build DNA nanotubes functionalized with lipid-like polymers is reported. When these polymers are linked to the nanotube using a spacer, they fold inside to create a hydrophobic environment within the nanotube; the nanotube can encapsulate small molecules and conditionally release them when specific DNA strands are added, as monitored by single-molecule fluorescence microscopy. When the polymers are directly linked to the nanostructure without spacers, they interact intermolecularly to form a network of DNA bundles. This morphological switch can be directly observed using a strand displacement strategy. The two association modes result in different cellular uptake behavior. Nanotubes with internal hydrophobic association show dye-mediated mitochondrial colocalization inside cells; while the bundles disassemble into smaller polymer-coated structures that reduce the extent of nonspecific cellular uptake. This approach uncovers parameters to direct the hierarchical assembly of DNA nanostructures, and produces promising materials for targeted drug delivery.

    View details for DOI 10.1002/adhm.201701049

    View details for Web of Science ID 000428311900012

    View details for PubMedID 29356412

  • Conformational Changes Spanning Angstroms to Nanometers via a Combined Protein-Induced Fluorescence Enhancement-Forster Resonance Energy Transfer Method JOURNAL OF PHYSICAL CHEMISTRY B Gidi, Y., Goette, M., Cosa, G. 2017; 121 (9): 2039–48

    Abstract

    Förster resonance energy transfer (FRET)-based single-molecule techniques have revolutionized our understanding of conformational dynamics in biomolecular systems. Recently, a new single-molecule technique based on protein-induced fluorescence enhancement (PIFE) has aided studies in which minimal (<3 nm) displacements occur. Concerns have been raised regarding whether donor fluorophore intensity (and correspondingly fluorescence quantum yield Φf) fluctuations, intrinsic to PIFE methods, may adversely affect FRET studies when retrieving the donor-acceptor dye distance. Here, we initially show through revisions of Förster's original equation that distances may be calculated in FRET experiments regardless of protein-induced intensity (and Φf) fluctuations occurring in the donor fluorophore. We additionally demonstrate by an analysis of the recorded emission intensity and competing decay pathways that PIFE and FRET methods may be conveniently combined, providing parallel complementary information in a single experiment. Single-molecule studies conducted with Cy3- and ATTO647N-labeled RNA structures and the HCV-NS5B polymerase protein undergoing binding dynamics along the RNA backbone provide a case study to validate the results. The analysis behind the proposed method enables for PIFE and FRET changes to be disentangled when both FRET and PIFE fluctuate over time following protein arrival and, for example, sliding. A new method, intensity-FRET, is thus proposed to monitor conformational changes spanning from angstroms to nanometers.

    View details for DOI 10.1021/acs.jpcb.6b11495

    View details for Web of Science ID 000396296000004

    View details for PubMedID 28177636

  • Stepwise growth of surface-grafted DNA nanotubes visualized at the single-molecule level NATURE CHEMISTRY Hariri, A. A., Hamblin, G. D., Gidi, Y., Sleiman, H. F., Cosa, G. 2015; 7 (4): 295-300

    Abstract

    DNA nanotubes offer a high aspect ratio and rigidity, attractive attributes for the controlled assembly of hierarchically complex linear arrays. It is highly desirable to control the positioning of rungs along the backbone of the nanotubes, minimize the polydispersity in their manufacture and reduce the building costs. We report here a solid-phase synthesis methodology in which, through a cyclic scheme starting from a 'foundation rung' specifically bound to the surface, distinct rungs can be incorporated in a predetermined manner. Each rung is orthogonally addressable. Using fluorescently tagged rungs, single-molecule fluorescence studies demonstrated the robustness and structural fidelity of the constructs and confirmed the incorporation of the rungs in quantitative yield (>95%) at each step of the cycle. Prototype structures that consisted of up to 20 repeat units, about 450 nm in contour length, were constructed. Combined, the solid-phase synthesis strategy described and its visualization through single-molecule spectroscopy show good promise for the production of custom-made DNA nanotubes.

    View details for DOI 10.1038/NCHEM.2184

    View details for Web of Science ID 000351756200008

    View details for PubMedID 25803467

  • Naphthoxazole-Based Singlet Oxygen Fluorescent Probes PHOTOCHEMISTRY AND PHOTOBIOLOGY Ruiz-Gonzalez, R., Zanocco, R., Gidi, Y., Zanocco, A. L., Nonell, S., Lemp, E. 2013; 89 (6): 1427–32

    Abstract

    In this study, we report the synthesis and photochemical behavior of a new family of photoactive compounds to assess its potential as singlet oxygen ((1)O2) probes. The candidate dyads are composed by a (1)O2 trap plus a naphthoxazole moiety linked directly or through an unsaturated bond to the oxazole ring. In the native state, the inherent great fluorescence of the naphthoxazole moiety is quenched; but in the presence of (1)O2, generated by the addition and appropriate irradiation of an external photosensitizer, a photooxidation reaction occurs leading to the formation of a new chemical entity whose fluorescence is two orders of magnitude higher than that of the initial compound, at the optimal selected wavelength. The presented dyads outperform the commonly used indirect fluorescent (1)O2 probes in terms of fluorescence enhancement maintaining the required specificity for (1)O2 detection in solution.

    View details for DOI 10.1111/php.12106

    View details for Web of Science ID 000326507100022

    View details for PubMedID 23730728