Erik A Rodriguez - Academia.edu (original) (raw)
Papers by Erik A Rodriguez
Journal of Back and Musculoskeletal Rehabilitation, Apr 11, 2001
The alpha-subunit of the nicotinic acetylcholine receptor (alphaAChR) contains a binding site for... more The alpha-subunit of the nicotinic acetylcholine receptor (alphaAChR) contains a binding site for alpha-bungarotoxin (alpha-BTX), a snake-venom-derived alpha-neurotoxin. Previous studies have established that the segment comprising residues 173-204 of alphaAChR contains the major determinant interacting with the toxin, but the precise boundaries of this determinant have not been clearly defined to date. In this study, we applied NMR dynamic filtering to determine the exact sequence constituting the major alphaAChR determinant interacting with alpha-BTX. Two overlapping synthetic peptides corresponding to segments 179-200 and 182-202 of the alphaAChR were complexed with alpha-BTX. HOHAHA and ROESY spectra of these complexes acquired with long mixing times highlight the residues of the peptide that do not interact with the toxin and retain considerable mobility upon binding to alpha-BTX. These results, together with changes in the chemical shifts of the peptide protons upon complex formation, suggest that residues 184-200 form the contact region. At pH 4, the molecular mass of the complex determined by dynamic light scattering (DLS) was found to be 11.2 kDa, in excellent agreement with the expected molecular mass of a 1:1 complex, while at pH >5 the DLS measurement of 20 kDa molecular mass indicated dimerization of the complex. These results were supported by T(2) measurements. Complete resonance assignment of the 11.2 kDa complex of alpha-BTX bound to the alphaAChR peptide comprising residues 182-202 was obtained at pH 4 using homonuclear 2D NMR spectra measured at 800 MHz. The secondary structures of both alpha-BTX and the bound alphaAChR peptide were determined using 2D (1)H NMR experiments. The peptide folds into a beta-hairpin conformation, in which residues (R)H186-(R)V188 and (R)Y198-(R)D200 form the two beta-strands. Residues (R)Y189-(R)T191 form an intermolecular beta-sheet with residues (B)K38-(B)V40 of the second finger of alpha-BTX. These results accurately pinpoint the alpha-BTX-binding site on the alphaAChR and pave the way to structure determination of this important alphaAChR determinant involved in binding acetylcholine and cholinergic agonists and antagonists.
The Journal of the Acoustical Society of America, 2019
The goal of this project is to facilitate the delivery of topical drugs into the cornea and anter... more The goal of this project is to facilitate the delivery of topical drugs into the cornea and anterior segment of the eye using therapeutic ultrasound which could present a promising treatment for keratoconus and other corneal diseases. Each cornea is dissected and placed in a diffusion cell. smURFP-blue, a blue fluorescent chromophore, was used as the drug. The experimental groups of corneas were treated with 1 and 0.8 W/cm2 continuous ultrasound for 5 min at frequencies of 400 kHz and 600 kHz, respectively, then left in the diffusion cell for another 55 min. Fluorescence images of the fixed corneas were obtained to determine the relative amount of smURFP-blue that remained in the tissue. Safety of ultrasound application was tested by comparing the damage in the corneal layers. Spectroscopy measurements indicated no statistical difference in the presence of the chromophore in the receiver compartment in ultrasound- and sham-treatment groups. Preliminary results showed greater fluores...
Microscopy and Microanalysis, 2015
RNA, Aug 13, 2007
The incorporation of unnatural amino acids into proteins is a valuable tool for addition of bioph... more The incorporation of unnatural amino acids into proteins is a valuable tool for addition of biophysical probes, bio-orthogonal functionalities, and photoreactive cross-linking agents, although these approaches often require quantities of protein that are difficult to access with chemically aminoacylated tRNAs. THG73 is an amber suppressor tRNA that has been used extensively, incorporating over 100 residues in 20 proteins. In vitro studies have shown that the Escherichia coli Asn amber suppressor (ENAS) suppresses better than THG73. However, we report here that ENAS suppresses with <26% of the efficiency of THG73 in Xenopus oocytes. We then tested the newly developed Tetrahymena thermophila Gln amber suppressor (TQAS) tRNA library, which contains mutations in the second to fourth positions of the acceptor stem. The acceptor stem mutations have no adverse effect on suppression efficiency and, in fact, can increase the suppression efficiency. Combining mutations causes an averaging of suppression efficiency, and increased suppression efficiency does not correlate with increased DG of the acceptor stem. We created a T. thermophila opal suppressor, TQOpS9, which shows ;50% suppression efficiency relative to THG73. The TQAS tRNA library, composed of functional suppressor tRNAs, has been created and will allow for screening in eukaryotic cells, where rapid analysis of large libraries is not feasible.
Optics Express, 2019
Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microsco... more Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microscopy. However, a systematic study of the efficiency of ND-TPE to guide the selection of fluorophore excitation wavelengths is missing. We measured the relative non-degenerate two-photon absorption cross-section (ND-TPACS) of several commonly used fluorophores (two fluorescent proteins and three small-molecule dyes) and generated 2-dimensional ND-TPACS spectra. We observed that the shape of a ND-TPACS spectrum follows that of the corresponding degenerate two-photon absorption cross-section (D-TPACS) spectrum, but is higher in magnitude. We found that the observed enhancements are higher than theoretical predictions.
Biochemical Society Transactions, 2020
Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deepe... more Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide....
Nature Communications, 2020
Genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensors are powerful tools ... more Genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensors are powerful tools to illuminate spatiotemporal regulation of cell signaling in living cells, but the utility of the red spectrum for biosensing was limited due to a lack of bright and stable red fluorescent proteins. Here, we rationally improve the photophysical characteristics of the coral-derived fluorescent protein TagRFP-T. We show that a new single-residue mutant, super-TagRFP (stagRFP) has nearly twice the molecular brightness of TagRFP-T and negligible photoactivation. stagRFP facilitates significant improvements on multiple green-red biosensors as a FRET acceptor and is an efficient FRET donor that supports red/far-red FRET biosensing. Capitalizing on the ability of stagRFP to couple with multiple FRET partners, we develop a novel multiplex method to examine the confluence of signaling activities from three kinases simultaneously in single living cells, providing evidence for a role of Src family ...
International Journal of Biological Macromolecules, 2020
Nanoparticles are excellent imaging agents for cancer, but variability in chemical structure, rac... more Nanoparticles are excellent imaging agents for cancer, but variability in chemical structure, racemic mixtures, and addition of heavy metals hinders FDA approval in the United States. We developed a small ultra-red fluorescent protein, named smURFP, to have optical properties similar to the small-molecule Cy5, a heptamethine subclass of cyanine dyes (Ex/Em = 642/670 nm). smURFP has a fluorescence quantum yield of 18% and expresses so well in E. coli, that gram quantities of fluorescent protein are purified from cultures in the laboratory. In this research, the fluorescent protein smURFP was combined with bovine serum albumin into fluorescent protein nanoparticles. These nanoparticles are fluorescent with a quantum yield of 17% and 12-14 nm in diameter. The far-red fluorescent protein nanoparticles noninvasively image tumors in living mice via the enhanced permeation and retention (EPR) mechanism. This manuscript describes the use of a new fluorescent protein nanoparticle for in vivo fluorescent imaging. This protein nanoparticle core should prove useful as a biomacromolecular scaffold, which could bear extended chemical modifications for studies, such as the in vivo imaging of fluorescent protein nanoparticles targeted *
![Research paper thumbnail of A fluorescent, [18F]-positron-emitting agent for imaging PMSA allows genetic reporting in adoptively-transferred, genetically-modified cells](https://attachments.academia-assets.com/90654345/thumbnails/1.jpg)
](ACS Chemical Biology, 2019
Clinical trials that involve genome-edited-cells are growing in popularity, where CART immunother... more Clinical trials that involve genome-edited-cells are growing in popularity, where CART immunotherapy and CRISPR editing are more recognized strategies. Genetic reporters are needed to localize the molecular events that these cells cause in patients. Specifically, a non-immunogenic genetic reporter is urgently needed as current reporters are immunogenic due to derivation from non-human sources. Prostate-specific membrane antigen (PSMA) is potentially non-immunogenic due to its natural, low-level expression in select tissues (self-MHC display). PSMA overexpression on human prostate adenocarcinoma is also visible with certain contrast. We exploit these properties in a transduced, two-component, Human-Derived, Genetic, Positron-emitting and Fluorescent (HD-GPF) reporter system. Mechanistically analogous to the luciferase/luciferin
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2017
An agent for visualizing cells by positron emission tomography is described and used to label red... more An agent for visualizing cells by positron emission tomography is described and used to label red blood cells. The labeled red blood cells are injected systemically so that intracranial hemorrhage can be visualized by positron emission tomography (PET). Red blood cells are labeled with 0.3 µg of a positron-emitting, fluorescent multimodal imaging probe, and used to non-invasively image cryolesion induced intracranial hemorrhage in a murine model (BALB/c, 2.36 × 10(8) cells, 100 µCi, <4 mm hemorrhage). Intracranial hemorrhage is confirmed by histology, fluorescence, bright-field, and PET ex vivo imaging. The low required activity, minimal mass, and high resolution of this technique make this strategy an attractive alternative for imaging intracranial hemorrhage. PET is one solution to a spectrum of issues that complicate single photon emission computed tomography (SPECT). For this reason, this application serves as a PET alternative to [(99m)Tc]-agents, and SPECT technology that i...
Nature Methods, 2016
In August of this year we lost Roger Y. Tsien, one of the scientific community’s leading lights. ... more In August of this year we lost Roger Y. Tsien, one of the scientific community’s leading lights. Roger was a Nobel laureate in chemistry, a pioneer of chemical biology, a friend to many, an inspiration to many more, and a mentor to a lucky few. As some of these lucky few, we would like to honor Roger by remembering both his accomplishments and what made him such an admirable mentor. Roger was a scientist through and through, with an early passion and aptitude for chemistry that led him to win the Westinghouse Science Talent Search at the age of 16. A defining feature of Roger’s illustrious career was his ability to approach biological problems with the mindset of an organic chemist. As a PhD candidate at the University of Cambridge in the early 1970s, he recognized that a thoughtfully designed Ca2+-chelating dye molecule had the potential to enable the visualization of brain activity at an unprecedented scale. Like so many of his ideas, this was at least a decade ahead of its time. It was only with Roger’s 1985 Journal of Biological Chemistry publication on the fura-2 and indo-1 Ca2+ dyes that the rest of the scientific community began to comprehend his vision. The impact of this paper cannot be overstated—as of 2014 it was the 42nd most cited paper of all time. This early success perfectly captures the fearless trailblazing spirit, boundless curiosity, and breadth of knowledge that remained key traits throughout his career. Roger continued to make contributions toward solving a dizzying array of problems, typically by first inventing a tool that changed the impossible to the possible. Concepts and inventions attributable to Roger include sequencing by synthesis, fast two-photon scanning, acetylation to improve compound permeability, voltage-sensing dyes, caged second messengers, and tags for chemical labeling of proteins. That is quite a list, and it does not even include his contributions to the understanding and use of fluorescent proteins, for which he shared the 2008 Nobel Prize in Chemistry with Osamu Shimomura and Martin Chalfie. Roger repeatedly applied his unique combination of chemical knowledge, biological interests, and deft imagination to create breakthroughs. He had far more original ideas than one person could hope to pursue, and he loved to share them. Infamously, he was never shy about offering insightful and constructive comments during research seminars or pointed criticisms when warranted. Roger inspired a generation of researchers to follow the trails that he blazed in a myriad of directions. He let many of his trainees build their careers on the foundation of their work in his lab, giving him the perfect excuse to launch his research in a new direction. Perhaps most important, he inspired his trainees to proudly assume the mantle of ‘tool builder’ even though he recognized, with humility, that many in the biology community at the time considered technology development to be of secondary importance. One of Roger’s many legacies was raising tool development to a first-class discipline and spurring the establishment of high-profile journals, such as Nature Methods. Roger led by example and bestowed on his trainees a clear sense of their purpose in the grand scheme of science and a roadmap for making contributions to promote scientific progress. We remember Roger fondly as a kind and generous mentor with infectious enthusiasm. His eyes would sparkle and his mouth would form a mischievous smile when he began discussing his latest, exciting research idea. Roger also liked to share amusing stories that illustrated the challenges of pursuing ideas before others could appreciate them, and the rewards that ensued when others acknowledged the accomplishment. Although we know Roger best for his scientific pursuits, he had a full range of interests outside the lab. Roger loved to travel with his wife Wendy, he kept up an active lifestyle, and he was a passionate and skilled pianist. After Roger’s stroke in 2013, he requested the most strenuous regimen of physical therapy available. With incredible willpower, he overcame paralysis of half his body and recovered the ability to walk on his own. Just as Roger pursued his ideas to their fullest potential, he lived his life to the fullest. With Roger’s passing, we have lost a close mentor and colleague, and the scientific world has lost one of its most creative and sharpest thinkers. In his Nobel biography, Roger humbly stated that “development of successful techniques to address important problems allows lesser mortals to exert a widespread beneficial impact for at least a few years.” As no lesser mortal, Roger developed techniques with beneficial impacts that have pushed the frontiers of research forward for three decades and are certain to live eternally in the world of scientific research. Erik A Rodriguez1, Nathan C Shaner2, Michael Z Lin3 & Robert E Campbell4
Trends in biochemical sciences, Feb 1, 2016
Over the past 20 years, protein engineering has been extensively used to improve and modify the f... more Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
Nature Methods, 2016
Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because less light is scatte... more Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because less light is scattered, absorbed, or reemitted by endogenous biomolecules. A new class of FP was developed from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, named small Ultra-Red FP (smURFP), covalently attaches biliverdin (BV) without a lyase, and has 642/670 nm excitation/emission peaks, a large extinction coefficient (180,000 M −1 cm −1) and quantum yield (18%), and comparable photostability to eGFP. SmURFP has significantly increased BV incorporation rate and protein stability compared to the bacteriophytochrome (BPH) FPs. BV supply is limited by membrane permeability, so expression of heme oxygenase-1 with heme precursors increases fluorescence of BPH/APCα FPs. SmURFP (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence in situ comparable to FPs from jellyfish/coral. A far-red/near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Photons Plus Ultrasound: Imaging and Sensing 2016, 2016
An optimal genetically-encoded probe for photoacoustic (PA) imaging should exhibit high optical a... more An optimal genetically-encoded probe for photoacoustic (PA) imaging should exhibit high optical absorption, low fluorescence quantum yield, and an absorption maxima within the near-infrared (NIR) window. One promising candidate is a newly engineered chromoprotein (CP), designated dark small ultra-red fluorescent protein (dsmURFP), which is based on a cyanobacterial phycobiliprotein. To optimize dsmURFP characteristics for PA imaging, we have developed a directed evolution method to iteratively screen libraries of protein variants with three different screening systems. Firstly, we took inspiration from dark-acceptor (also known as dark-quencher)-based Förster resonance energy transfer (FRET) constructs, and used dsmURFP as a dark acceptor from a mCardinal fluorescent donor. The rationale for this design was that the higher the extinction coefficient of the dsmURFP, the more the emission of the donor would be quenched. In addition, more energy transferred to the dark acceptor would lead to more thermoelastic expansion and a stronger PA signal. Three rounds of evolution using this first strategy resulted in dsmURFP1.3 that quenched the emission of mCardinal ~2-fold more efficiently than dsmURFP. Secondly, an absorption-based screening based on visual inspection of plates led to identification of the variant dsmURFP1.4, which exhibited a 2-fold higher absorbance and a 5 nm red shift. Thirdly, we developed a colony-based photoacoustic screening method. To demonstrate the utility of our optimized variants, we used photoacoustic imaging to visualize dsmURFP and its variants in phantom and in vivo experiments using chicken embryo models and murine bacterial bladder infection models.
![Research paper thumbnail of New dioxaborolane chemistry enables [18F]-positron-emitting, fluorescent [18F]-multimodality biomolecule generation from the solid-phase](https://attachments.academia-assets.com/90654340/thumbnails/1.jpg)
](Bioconjugate chemistry, May 11, 2016
New protecting group chemistry is used to greatly simplify imaging probe production. Temperature ... more New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates biomolecule immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled biomolecule release from the solid-phase, separated from unlabeled biomolecule, and creates [18F]-trifluoroborate- biomolecules for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. Dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [18F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) antibody (mAb) that labels prostate tumors and Cetuximab (FDA approved), an anti-epidermal growth factor receptor (EGFR) mAb that labels lung adenocarcinoma tumors. Colocali...
RNA, 2007
The incorporation of unnatural amino acids site-specifically is a valuable technique for structur... more The incorporation of unnatural amino acids site-specifically is a valuable technique for structure–function studies, incorporation of biophysical probes, and determining protein–protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xe...
Proceedings of the National Academy of Sciences, 2006
Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for ... more Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure–function relationships, incorporating biophysical probes, and elucidating protein–protein interactions. In higher eukaryotic cells, the methodology is currently limited to incorporation of a single UAA in response to a stop codon, which is known as nonsense suppression. Frameshift suppression is a unique methodology for incorporating UAAs in response to quadruplet codons, but currently, it is mostly limited to in vitro protein translation systems. Here, we evaluate the viability of frameshift suppression in Xenopus oocytes. We demonstrate UAA incorporation by using yeast phenylalanine frameshift suppressor (YFFS) tRNAs that recognize two different quadruplet codons (CGGG and GGGU) in vivo . Suppression efficiency of the YFFS tRNAs increases nonlinearly with the amount of injected tRNA, suggesting a significant competition with endogenous, triplet-recognizing tRNA. Both...
Biophysical Journal, 2009
Biophysical Journal, 2009
Journal of Back and Musculoskeletal Rehabilitation, Apr 11, 2001
The alpha-subunit of the nicotinic acetylcholine receptor (alphaAChR) contains a binding site for... more The alpha-subunit of the nicotinic acetylcholine receptor (alphaAChR) contains a binding site for alpha-bungarotoxin (alpha-BTX), a snake-venom-derived alpha-neurotoxin. Previous studies have established that the segment comprising residues 173-204 of alphaAChR contains the major determinant interacting with the toxin, but the precise boundaries of this determinant have not been clearly defined to date. In this study, we applied NMR dynamic filtering to determine the exact sequence constituting the major alphaAChR determinant interacting with alpha-BTX. Two overlapping synthetic peptides corresponding to segments 179-200 and 182-202 of the alphaAChR were complexed with alpha-BTX. HOHAHA and ROESY spectra of these complexes acquired with long mixing times highlight the residues of the peptide that do not interact with the toxin and retain considerable mobility upon binding to alpha-BTX. These results, together with changes in the chemical shifts of the peptide protons upon complex formation, suggest that residues 184-200 form the contact region. At pH 4, the molecular mass of the complex determined by dynamic light scattering (DLS) was found to be 11.2 kDa, in excellent agreement with the expected molecular mass of a 1:1 complex, while at pH >5 the DLS measurement of 20 kDa molecular mass indicated dimerization of the complex. These results were supported by T(2) measurements. Complete resonance assignment of the 11.2 kDa complex of alpha-BTX bound to the alphaAChR peptide comprising residues 182-202 was obtained at pH 4 using homonuclear 2D NMR spectra measured at 800 MHz. The secondary structures of both alpha-BTX and the bound alphaAChR peptide were determined using 2D (1)H NMR experiments. The peptide folds into a beta-hairpin conformation, in which residues (R)H186-(R)V188 and (R)Y198-(R)D200 form the two beta-strands. Residues (R)Y189-(R)T191 form an intermolecular beta-sheet with residues (B)K38-(B)V40 of the second finger of alpha-BTX. These results accurately pinpoint the alpha-BTX-binding site on the alphaAChR and pave the way to structure determination of this important alphaAChR determinant involved in binding acetylcholine and cholinergic agonists and antagonists.
The Journal of the Acoustical Society of America, 2019
The goal of this project is to facilitate the delivery of topical drugs into the cornea and anter... more The goal of this project is to facilitate the delivery of topical drugs into the cornea and anterior segment of the eye using therapeutic ultrasound which could present a promising treatment for keratoconus and other corneal diseases. Each cornea is dissected and placed in a diffusion cell. smURFP-blue, a blue fluorescent chromophore, was used as the drug. The experimental groups of corneas were treated with 1 and 0.8 W/cm2 continuous ultrasound for 5 min at frequencies of 400 kHz and 600 kHz, respectively, then left in the diffusion cell for another 55 min. Fluorescence images of the fixed corneas were obtained to determine the relative amount of smURFP-blue that remained in the tissue. Safety of ultrasound application was tested by comparing the damage in the corneal layers. Spectroscopy measurements indicated no statistical difference in the presence of the chromophore in the receiver compartment in ultrasound- and sham-treatment groups. Preliminary results showed greater fluores...
Microscopy and Microanalysis, 2015
RNA, Aug 13, 2007
The incorporation of unnatural amino acids into proteins is a valuable tool for addition of bioph... more The incorporation of unnatural amino acids into proteins is a valuable tool for addition of biophysical probes, bio-orthogonal functionalities, and photoreactive cross-linking agents, although these approaches often require quantities of protein that are difficult to access with chemically aminoacylated tRNAs. THG73 is an amber suppressor tRNA that has been used extensively, incorporating over 100 residues in 20 proteins. In vitro studies have shown that the Escherichia coli Asn amber suppressor (ENAS) suppresses better than THG73. However, we report here that ENAS suppresses with <26% of the efficiency of THG73 in Xenopus oocytes. We then tested the newly developed Tetrahymena thermophila Gln amber suppressor (TQAS) tRNA library, which contains mutations in the second to fourth positions of the acceptor stem. The acceptor stem mutations have no adverse effect on suppression efficiency and, in fact, can increase the suppression efficiency. Combining mutations causes an averaging of suppression efficiency, and increased suppression efficiency does not correlate with increased DG of the acceptor stem. We created a T. thermophila opal suppressor, TQOpS9, which shows ;50% suppression efficiency relative to THG73. The TQAS tRNA library, composed of functional suppressor tRNAs, has been created and will allow for screening in eukaryotic cells, where rapid analysis of large libraries is not feasible.
Optics Express, 2019
Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microsco... more Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microscopy. However, a systematic study of the efficiency of ND-TPE to guide the selection of fluorophore excitation wavelengths is missing. We measured the relative non-degenerate two-photon absorption cross-section (ND-TPACS) of several commonly used fluorophores (two fluorescent proteins and three small-molecule dyes) and generated 2-dimensional ND-TPACS spectra. We observed that the shape of a ND-TPACS spectrum follows that of the corresponding degenerate two-photon absorption cross-section (D-TPACS) spectrum, but is higher in magnitude. We found that the observed enhancements are higher than theoretical predictions.
Biochemical Society Transactions, 2020
Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deepe... more Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide....
Nature Communications, 2020
Genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensors are powerful tools ... more Genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensors are powerful tools to illuminate spatiotemporal regulation of cell signaling in living cells, but the utility of the red spectrum for biosensing was limited due to a lack of bright and stable red fluorescent proteins. Here, we rationally improve the photophysical characteristics of the coral-derived fluorescent protein TagRFP-T. We show that a new single-residue mutant, super-TagRFP (stagRFP) has nearly twice the molecular brightness of TagRFP-T and negligible photoactivation. stagRFP facilitates significant improvements on multiple green-red biosensors as a FRET acceptor and is an efficient FRET donor that supports red/far-red FRET biosensing. Capitalizing on the ability of stagRFP to couple with multiple FRET partners, we develop a novel multiplex method to examine the confluence of signaling activities from three kinases simultaneously in single living cells, providing evidence for a role of Src family ...
International Journal of Biological Macromolecules, 2020
Nanoparticles are excellent imaging agents for cancer, but variability in chemical structure, rac... more Nanoparticles are excellent imaging agents for cancer, but variability in chemical structure, racemic mixtures, and addition of heavy metals hinders FDA approval in the United States. We developed a small ultra-red fluorescent protein, named smURFP, to have optical properties similar to the small-molecule Cy5, a heptamethine subclass of cyanine dyes (Ex/Em = 642/670 nm). smURFP has a fluorescence quantum yield of 18% and expresses so well in E. coli, that gram quantities of fluorescent protein are purified from cultures in the laboratory. In this research, the fluorescent protein smURFP was combined with bovine serum albumin into fluorescent protein nanoparticles. These nanoparticles are fluorescent with a quantum yield of 17% and 12-14 nm in diameter. The far-red fluorescent protein nanoparticles noninvasively image tumors in living mice via the enhanced permeation and retention (EPR) mechanism. This manuscript describes the use of a new fluorescent protein nanoparticle for in vivo fluorescent imaging. This protein nanoparticle core should prove useful as a biomacromolecular scaffold, which could bear extended chemical modifications for studies, such as the in vivo imaging of fluorescent protein nanoparticles targeted *
ACS Chemical Biology, 2019
Clinical trials that involve genome-edited-cells are growing in popularity, where CART immunother... more Clinical trials that involve genome-edited-cells are growing in popularity, where CART immunotherapy and CRISPR editing are more recognized strategies. Genetic reporters are needed to localize the molecular events that these cells cause in patients. Specifically, a non-immunogenic genetic reporter is urgently needed as current reporters are immunogenic due to derivation from non-human sources. Prostate-specific membrane antigen (PSMA) is potentially non-immunogenic due to its natural, low-level expression in select tissues (self-MHC display). PSMA overexpression on human prostate adenocarcinoma is also visible with certain contrast. We exploit these properties in a transduced, two-component, Human-Derived, Genetic, Positron-emitting and Fluorescent (HD-GPF) reporter system. Mechanistically analogous to the luciferase/luciferin
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2017
An agent for visualizing cells by positron emission tomography is described and used to label red... more An agent for visualizing cells by positron emission tomography is described and used to label red blood cells. The labeled red blood cells are injected systemically so that intracranial hemorrhage can be visualized by positron emission tomography (PET). Red blood cells are labeled with 0.3 µg of a positron-emitting, fluorescent multimodal imaging probe, and used to non-invasively image cryolesion induced intracranial hemorrhage in a murine model (BALB/c, 2.36 × 10(8) cells, 100 µCi, <4 mm hemorrhage). Intracranial hemorrhage is confirmed by histology, fluorescence, bright-field, and PET ex vivo imaging. The low required activity, minimal mass, and high resolution of this technique make this strategy an attractive alternative for imaging intracranial hemorrhage. PET is one solution to a spectrum of issues that complicate single photon emission computed tomography (SPECT). For this reason, this application serves as a PET alternative to [(99m)Tc]-agents, and SPECT technology that i...
Nature Methods, 2016
In August of this year we lost Roger Y. Tsien, one of the scientific community’s leading lights. ... more In August of this year we lost Roger Y. Tsien, one of the scientific community’s leading lights. Roger was a Nobel laureate in chemistry, a pioneer of chemical biology, a friend to many, an inspiration to many more, and a mentor to a lucky few. As some of these lucky few, we would like to honor Roger by remembering both his accomplishments and what made him such an admirable mentor. Roger was a scientist through and through, with an early passion and aptitude for chemistry that led him to win the Westinghouse Science Talent Search at the age of 16. A defining feature of Roger’s illustrious career was his ability to approach biological problems with the mindset of an organic chemist. As a PhD candidate at the University of Cambridge in the early 1970s, he recognized that a thoughtfully designed Ca2+-chelating dye molecule had the potential to enable the visualization of brain activity at an unprecedented scale. Like so many of his ideas, this was at least a decade ahead of its time. It was only with Roger’s 1985 Journal of Biological Chemistry publication on the fura-2 and indo-1 Ca2+ dyes that the rest of the scientific community began to comprehend his vision. The impact of this paper cannot be overstated—as of 2014 it was the 42nd most cited paper of all time. This early success perfectly captures the fearless trailblazing spirit, boundless curiosity, and breadth of knowledge that remained key traits throughout his career. Roger continued to make contributions toward solving a dizzying array of problems, typically by first inventing a tool that changed the impossible to the possible. Concepts and inventions attributable to Roger include sequencing by synthesis, fast two-photon scanning, acetylation to improve compound permeability, voltage-sensing dyes, caged second messengers, and tags for chemical labeling of proteins. That is quite a list, and it does not even include his contributions to the understanding and use of fluorescent proteins, for which he shared the 2008 Nobel Prize in Chemistry with Osamu Shimomura and Martin Chalfie. Roger repeatedly applied his unique combination of chemical knowledge, biological interests, and deft imagination to create breakthroughs. He had far more original ideas than one person could hope to pursue, and he loved to share them. Infamously, he was never shy about offering insightful and constructive comments during research seminars or pointed criticisms when warranted. Roger inspired a generation of researchers to follow the trails that he blazed in a myriad of directions. He let many of his trainees build their careers on the foundation of their work in his lab, giving him the perfect excuse to launch his research in a new direction. Perhaps most important, he inspired his trainees to proudly assume the mantle of ‘tool builder’ even though he recognized, with humility, that many in the biology community at the time considered technology development to be of secondary importance. One of Roger’s many legacies was raising tool development to a first-class discipline and spurring the establishment of high-profile journals, such as Nature Methods. Roger led by example and bestowed on his trainees a clear sense of their purpose in the grand scheme of science and a roadmap for making contributions to promote scientific progress. We remember Roger fondly as a kind and generous mentor with infectious enthusiasm. His eyes would sparkle and his mouth would form a mischievous smile when he began discussing his latest, exciting research idea. Roger also liked to share amusing stories that illustrated the challenges of pursuing ideas before others could appreciate them, and the rewards that ensued when others acknowledged the accomplishment. Although we know Roger best for his scientific pursuits, he had a full range of interests outside the lab. Roger loved to travel with his wife Wendy, he kept up an active lifestyle, and he was a passionate and skilled pianist. After Roger’s stroke in 2013, he requested the most strenuous regimen of physical therapy available. With incredible willpower, he overcame paralysis of half his body and recovered the ability to walk on his own. Just as Roger pursued his ideas to their fullest potential, he lived his life to the fullest. With Roger’s passing, we have lost a close mentor and colleague, and the scientific world has lost one of its most creative and sharpest thinkers. In his Nobel biography, Roger humbly stated that “development of successful techniques to address important problems allows lesser mortals to exert a widespread beneficial impact for at least a few years.” As no lesser mortal, Roger developed techniques with beneficial impacts that have pushed the frontiers of research forward for three decades and are certain to live eternally in the world of scientific research. Erik A Rodriguez1, Nathan C Shaner2, Michael Z Lin3 & Robert E Campbell4
Trends in biochemical sciences, Feb 1, 2016
Over the past 20 years, protein engineering has been extensively used to improve and modify the f... more Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
Nature Methods, 2016
Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because less light is scatte... more Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because less light is scattered, absorbed, or reemitted by endogenous biomolecules. A new class of FP was developed from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, named small Ultra-Red FP (smURFP), covalently attaches biliverdin (BV) without a lyase, and has 642/670 nm excitation/emission peaks, a large extinction coefficient (180,000 M −1 cm −1) and quantum yield (18%), and comparable photostability to eGFP. SmURFP has significantly increased BV incorporation rate and protein stability compared to the bacteriophytochrome (BPH) FPs. BV supply is limited by membrane permeability, so expression of heme oxygenase-1 with heme precursors increases fluorescence of BPH/APCα FPs. SmURFP (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence in situ comparable to FPs from jellyfish/coral. A far-red/near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Photons Plus Ultrasound: Imaging and Sensing 2016, 2016
An optimal genetically-encoded probe for photoacoustic (PA) imaging should exhibit high optical a... more An optimal genetically-encoded probe for photoacoustic (PA) imaging should exhibit high optical absorption, low fluorescence quantum yield, and an absorption maxima within the near-infrared (NIR) window. One promising candidate is a newly engineered chromoprotein (CP), designated dark small ultra-red fluorescent protein (dsmURFP), which is based on a cyanobacterial phycobiliprotein. To optimize dsmURFP characteristics for PA imaging, we have developed a directed evolution method to iteratively screen libraries of protein variants with three different screening systems. Firstly, we took inspiration from dark-acceptor (also known as dark-quencher)-based Förster resonance energy transfer (FRET) constructs, and used dsmURFP as a dark acceptor from a mCardinal fluorescent donor. The rationale for this design was that the higher the extinction coefficient of the dsmURFP, the more the emission of the donor would be quenched. In addition, more energy transferred to the dark acceptor would lead to more thermoelastic expansion and a stronger PA signal. Three rounds of evolution using this first strategy resulted in dsmURFP1.3 that quenched the emission of mCardinal ~2-fold more efficiently than dsmURFP. Secondly, an absorption-based screening based on visual inspection of plates led to identification of the variant dsmURFP1.4, which exhibited a 2-fold higher absorbance and a 5 nm red shift. Thirdly, we developed a colony-based photoacoustic screening method. To demonstrate the utility of our optimized variants, we used photoacoustic imaging to visualize dsmURFP and its variants in phantom and in vivo experiments using chicken embryo models and murine bacterial bladder infection models.
Bioconjugate chemistry, May 11, 2016
New protecting group chemistry is used to greatly simplify imaging probe production. Temperature ... more New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates biomolecule immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled biomolecule release from the solid-phase, separated from unlabeled biomolecule, and creates [18F]-trifluoroborate- biomolecules for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. Dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [18F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) antibody (mAb) that labels prostate tumors and Cetuximab (FDA approved), an anti-epidermal growth factor receptor (EGFR) mAb that labels lung adenocarcinoma tumors. Colocali...
RNA, 2007
The incorporation of unnatural amino acids site-specifically is a valuable technique for structur... more The incorporation of unnatural amino acids site-specifically is a valuable technique for structure–function studies, incorporation of biophysical probes, and determining protein–protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xe...
Proceedings of the National Academy of Sciences, 2006
Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for ... more Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure–function relationships, incorporating biophysical probes, and elucidating protein–protein interactions. In higher eukaryotic cells, the methodology is currently limited to incorporation of a single UAA in response to a stop codon, which is known as nonsense suppression. Frameshift suppression is a unique methodology for incorporating UAAs in response to quadruplet codons, but currently, it is mostly limited to in vitro protein translation systems. Here, we evaluate the viability of frameshift suppression in Xenopus oocytes. We demonstrate UAA incorporation by using yeast phenylalanine frameshift suppressor (YFFS) tRNAs that recognize two different quadruplet codons (CGGG and GGGU) in vivo . Suppression efficiency of the YFFS tRNAs increases nonlinearly with the amount of injected tRNA, suggesting a significant competition with endogenous, triplet-recognizing tRNA. Both...
Biophysical Journal, 2009
Biophysical Journal, 2009