Ivan J. Dmochowski

Thermophilic Ferritin 24mer Assembly and Nanoparticle Encapsulation Modulated by Interdimer Electrostatic Repulsion

Co-reporter:Katherine W. Pulsipher, Jose A. Villegas, Benjamin W. Roose, Tacey L. Hicks, Jennifer Yoon, Jeffery G. Saven, and Ivan J. Dmochowski

Biochemistry July 18, 2017 Volume 56(Issue 28) pp:3596-3596

Publication Date(Web):June 22, 2017

DOI:10.1021/acs.biochem.7b00296

Protein cage self-assembly enables encapsulation and sequestration of small molecules, macromolecules, and nanomaterials for many applications in bionanotechnology. Notably, wild-type thermophilic ferritin from Archaeoglobus fulgidus (AfFtn) exists as a stable dimer of four-helix bundle proteins at a low ionic strength, and the protein forms a hollow assembly of 24 protomers at a high ionic strength (∼800 mM NaCl). This assembly process can also be initiated by highly charged gold nanoparticles (AuNPs) in solution, leading to encapsulation. These data suggest that salt solutions or charged AuNPs can shield unfavorable electrostatic interactions at AfFtn dimer–dimer interfaces, but specific “hot-spot” residues controlling assembly have not been identified. To investigate this further, we computationally designed three AfFtn mutants (E65R, D138K, and A127R) that introduce a single positive charge at sites along the dimer–dimer interface. These proteins exhibited different assembly kinetics and thermodynamics, which were ranked in order of increasing 24mer propensity: A127R < wild type < D138K ≪ E65R. E65R assembled into the 24mer across a wide range of ionic strengths (0–800 mM NaCl), and the dissociation temperature for the 24mer was 98 °C. X-ray crystal structure analysis of the E65R mutant identified a more compact, closed-pore cage geometry. A127R and D138K mutants exhibited wild-type ability to encapsulate and stabilize 5 nm AuNPs, whereas E65R did not encapsulate AuNPs at the same high yields. This work illustrates designed protein cages with distinct assembly and encapsulation properties.

Nanomolar small-molecule detection using a genetically encoded 129Xe NMR contrast agent

Co-reporter:B. W. Roose;S. D. Zemerov;I. J. Dmochowski

Chemical Science (2010-Present) 2017 vol. 8(Issue 11) pp:7631-7636

Publication Date(Web):2017/10/23

DOI:10.1039/C7SC03601A

Genetically encoded magnetic resonance imaging (MRI) contrast agents enable non-invasive detection of specific biomarkers in vivo. Here, we employed the hyper-CEST 129Xe NMR technique to quantify maltose (32 nM to 1 mM) through its modulation of conformational change and xenon exchange in maltose binding protein (MBP). Remarkably, no hyper-CEST signal was observed for MBP in the absence of maltose, making MBP an ultrasensitive “smart” contrast agent. The resonance frequency of 129Xe bound to MBP was greatly downfield-shifted (Δδ = 95 ppm) from the 129Xe(aq) peak, which facilitated detection in E. coli as well as multiplexing with TEM-1 β-lactamase. Finally, a Val to Ala mutation at the MBP–Xe binding site yielded 34% more contrast than WT, with 129Xe resonance frequency shifted 59 ppm upfield from WT. We conclude that engineered MBPs constitute a new class of genetically encoded, analyte-sensitive molecular imaging agents detectable by 129Xe NMR/MRI.

A cryptophane-based “turn-on” 129Xe NMR biosensor for monitoring calmodulin

Co-reporter:Brittany A. Riggle;Mara L. Greenberg;Yanfei Wang;Rebecca F. Wissner;Serge D. Zemerov;E. James Petersson

Organic & Biomolecular Chemistry 2017 vol. 15(Issue 42) pp:8883-8887

Publication Date(Web):2017/10/31

DOI:10.1039/C7OB02391J

We present the first cryptophane-based “turn-on” 129Xe NMR biosensor, employing a peptide-functionalized cryptophane to monitor the activation of calmodulin (CaM) protein in solution. In the absence of CaM binding, interaction between the peptide and cryptophane completely suppresses the hyperpolarized 129Xe-cryptophane NMR signal. Biosensor binding to Ca2+-activated CaM produces the expected 129Xe-cryptophane NMR signal.

Oligonucleotide modifications enhance probe stability for single cell transcriptome in vivo analysis (TIVA)

Co-reporter:S. B. Yeldell;B. K. Ruble;I. J. Dmochowski

Organic & Biomolecular Chemistry 2017 vol. 15(Issue 47) pp:10001-10009

Publication Date(Web):2017/12/06

DOI:10.1039/C7OB02353G

Single cell transcriptomics provides a powerful discovery tool for identifying new cell types and functions as well as a means to probe molecular features of the etiology and treatment of human diseases, including cancer. However, such analyses are limited by the difficulty of isolating mRNA from single cells within biological samples. We recently introduced a photochemical method for isolating mRNA from single living cells, Transcriptome In Vivo Analysis (TIVA). The TIVA probe is a “caged” polyU : polyA oligonucleotide hairpin designed to enter live tissue, where site-specific activation with 405 nm laser reveals the polyU-biotin strand to bind mRNA in a target cell, enabling subsequent mRNA isolation and sequencing. The TIVA method is well suited for analysis of living cells in resected tissue, but has not yet been applied to living cells in whole organisms. Adapting TIVA to this more challenging environment requires a probe with higher thermal stability, more robust caging, and greater nuclease resistance. In this paper we present modifications to the original TIVA probe with multiple aspects of enhanced stability. These newer probes utilize an extended 22mer polyU capture strand with two 9mer polyA blocking strands (“22/9/9”) for higher thermal stability pre-photolysis and improved mRNA capture affinity post-photolysis. The “22/9/9 GC” probe features a terminal GC pair to reduce pre-photolysis interactions with mRNA by more than half. The “PS-22/9/9” probe features a phosphorothioated backbone, which extends serum stability from <1 h to at least 48 h, and also mediates uptake into cultured human fibroblasts.

Controlling gold nanoparticle seeded growth in thermophilic ferritin protein templates

Co-reporter:Katherine W. Pulsipher, Stephanie Honig, Sunbin Deng, Ivan J. Dmochowski

Journal of Inorganic Biochemistry 2017 Volume 174(Volume 174) pp:

Publication Date(Web):1 September 2017

DOI:10.1016/j.jinorgbio.2017.06.012

•Gold nanoparticle (AuNP) seeded growth within thermophilic ferritin is explored.•AuCl3 yields larger, more monodisperse AuNPs than charge-complementary Au(en)23 +.•Gold complex charge, redox potential and sterics play a role in optimal AuNP growth.•Optimal balance of high Au incorporation efficiency and large NP size is presented.•WT open-pore ferritin yields larger AuNPs than mutant closed-pore ferritin.Ferritin protein cages provide templates for inorganic nanoparticle synthesis in more environmentally-friendly conditions. Thermophilic ferritin from Archaeoglobus fulgidus (AfFtn) has been shown to encapsulate pre-formed 6-nm gold nanoparticles (AuNPs) and template their further growth within its 8-nm cavity. In this study, we explore whether using a gold complex with electrostatic complementarity to the anionic ferritin cavity can promote efficient seeded nanoparticle growth. We also compare wt AfFtn and a closed pore mutant AfFtn to explore whether the ferritin pores influence final AuNP size.The choices of gold salt and ferritin pore size affect the efficiency of gold nanoparticle seeded growth within the protein template. The combination of AuCl3 and open-pore, wt ferritin yields monodisperse gold nanoparticle-protein assembly.Download high-res image (234KB)Download full-size image

An Expanded Palette of Xenon-129 NMR Biosensors

Co-reporter:Yanfei Wang and Ivan J. Dmochowski

Accounts of Chemical Research 2016 Volume 49(Issue 10) pp:2179

Publication Date(Web):September 19, 2016

DOI:10.1021/acs.accounts.6b00309

Molecular imaging holds considerable promise for elucidating biological processes in normal physiology as well as disease states, by determining the location and relative concentration of specific molecules of interest. Proton-based magnetic resonance imaging (1H MRI) is nonionizing and provides good spatial resolution for clinical imaging but lacks sensitivity for imaging low-abundance (i.e., submicromolar) molecular markers of disease or environments with low proton densities. To address these limitations, hyperpolarized (hp) 129Xe NMR spectroscopy and MRI have emerged as attractive complementary methodologies. Hyperpolarized xenon is nontoxic and can be readily delivered to patients via inhalation or injection, and improved xenon hyperpolarization technology makes it feasible to image the lungs and brain for clinical applications.In order to target hp 129Xe to biomolecular targets of interest, the concept of “xenon biosensing” was first proposed by a Berkeley team in 2001. The development of xenon biosensors has since focused on modifying organic host molecules (e.g., cryptophanes) via diverse conjugation chemistries and has brought about numerous sensing applications including the detection of peptides, proteins, oligonucleotides, metal ions, chemical modifications, and enzyme activity. Moreover, the large (∼300 ppm) chemical shift window for hp 129Xe bound to host molecules in water makes possible the simultaneous identification of multiple species in solution, that is, multiplexing. Beyond hyperpolarization, a 106-fold signal enhancement can be achieved through a technique known as hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST), which shows great potential to meet the sensitivity requirement in many applications.This Account highlights an expanded palette of hyper-CEST biosensors, which now includes cryptophane and cucurbit[6]uril (CB[6]) small-molecule hosts, as well as genetically encoded gas vesicles and single proteins. In 2015, we reported picomolar detection of commercially available CB[6] via hyper-CEST. Inspired by the versatile host–guest chemistry of CB[6], our lab and others developed “turn-on” strategies for CB[6]-hyper-CEST biosensing, demonstrating detection of protein analytes in complex media and specific chemical events. CB[6] is starting to be employed for in vivo imaging applications. We also recently determined that TEM-1 β-lactamase can function as a single-protein reporter for hyper-CEST and observed useful saturation contrast for β-lactamase expressed in bacterial and mammalian cells. These newly developed small-molecule and genetically encoded xenon biosensors offer significant potential to extend the scope of hp 129Xe toward molecular MRI.

Kinetics and Photochemistry of Ruthenium Bisbipyridine Diacetonitrile Complexes: An Interdisciplinary Inorganic and Physical Chemistry Laboratory Exercise

Co-reporter:Teresa L. Rapp, Susan R. Phillips, and Ivan J. Dmochowski

Journal of Chemical Education 2016 Volume 93(Issue 12) pp:2101-2105

Publication Date(Web):November 10, 2016

DOI:10.1021/acs.jchemed.6b00173

The study of ruthenium polypyridyl complexes can be widely applied across disciplines in the undergraduate curriculum. Ruthenium photochemistry has advanced many fields including dye-sensitized solar cells, photoredox catalysis, light-driven water oxidation, and biological electron transfer. Equally promising are ruthenium polypyridyl complexes that provide a sterically bulky, photolabile moiety for transiently “caging” biologically active molecules. Photouncaging involves the use of visible (1-photon) or near-IR (2-photon) light to break one or more bonds between ruthenium and coordinated ligand(s), which can occur on short time scales and in high quantum yields. In this work we demonstrate the use of a model “caged” acetonitrile complex, Ru(2,2′-bipyridine)2(acetonitrile)2, or RuMeCN in an advanced synthesis and physical chemistry laboratory. Students made RuMeCN in an advanced synthesis laboratory course and performed UV–vis spectroscopy and electrochemistry. The following semester students investigated RuMeCN photolysis kinetics in a physical chemistry laboratory. These two exercises may also be combined to create a 2-week module in an advanced undergraduate laboratory course.Keywords: Aqueous Solution Chemistry; Chromatography; Coordination Compounds; Crystal Field/Ligand Field Theory; Hands-On Learning/Manipulatives; Inorganic Chemistry; Interdisciplinary/Multidisciplinary; Laboratory Instruction; Physical Chemistry; Upper-Division Undergraduate;

Programming A Molecular Relay for Ultrasensitive Biodetection through 129XeNMR

Co-reporter:Yanfei Wang;Benjamin W. Roose;John P. Philbin;Jordan L. Doman ; Ivan J. Dmochowski

Angewandte Chemie International Edition 2016 Volume 55( Issue 5) pp:1733-1736

Publication Date(Web):

DOI:10.1002/anie.201508990

Abstract

A supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized ¹²⁹Xe NMR is reported. A cucurbit[6]uril (CB[6])-based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks the CB[6]–Xe interaction. The protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]- and carbonic anhydrase II (CAII)-binding domains were synthesized in one or two steps. X-ray crystallography confirmed TFG binding to Zn²⁺ in the deep CAII active-site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidin by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive ¹²⁹Xe NMR spectroscopy.

A Genetically Encoded -Lactamase Reporter for Ultrasensitive 129Xe NMR in Mammalian Cells

Co-reporter:Yanfei Wang;Benjamin W. Roose;Eugene J. Palovcak;Dr. Vincenzo Carnevale; Ivan J. Dmochowski

Angewandte Chemie International Edition 2016 Volume 55( Issue 31) pp:8984-8987

Publication Date(Web):

DOI:10.1002/anie.201604055

Abstract

Molecular imaging holds considerable promise for elucidating biological processes in normal physiology as well as disease states, but requires noninvasive methods for identifying analytes at sub-micromolar concentrations. Particularly useful are genetically encoded, single-protein reporters that harness the power of molecular biology to visualize specific molecular processes, but such reporters have been conspicuously lacking for in vivo magnetic resonance imaging (MRI). Herein, we report TEM-1 β-lactamase (bla) as a single-protein reporter for hyperpolarized (HP) ¹²⁹Xe NMR, with significant saturation contrast at 0.1 μm. Xenon chemical exchange saturation transfer (CEST) interactions with the primary allosteric site in bla give rise to a unique saturation peak at 255 ppm, well removed (≈60 ppm downfield) from the ¹²⁹Xe-H₂O peak. Useful saturation contrast was also observed for bla expressed in bacterial cells and mammalian cells.

Programming A Molecular Relay for Ultrasensitive Biodetection through 129XeNMR

Co-reporter:Yanfei Wang;Benjamin W. Roose;John P. Philbin;Jordan L. Doman ; Ivan J. Dmochowski

Angewandte Chemie 2016 Volume 128( Issue 5) pp:1765-1768

Publication Date(Web):

DOI:10.1002/ange.201508990

Abstract

A supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized ¹²⁹Xe NMR is reported. A cucurbit[6]uril (CB[6])-based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks the CB[6]–Xe interaction. The protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]- and carbonic anhydrase II (CAII)-binding domains were synthesized in one or two steps. X-ray crystallography confirmed TFG binding to Zn²⁺ in the deep CAII active-site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidin by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive ¹²⁹Xe NMR spectroscopy.

A Genetically Encoded -Lactamase Reporter for Ultrasensitive 129Xe NMR in Mammalian Cells

Co-reporter:Yanfei Wang;Benjamin W. Roose;Eugene J. Palovcak;Dr. Vincenzo Carnevale; Ivan J. Dmochowski

Angewandte Chemie 2016 Volume 128( Issue 31) pp:9130-9133

Publication Date(Web):

DOI:10.1002/ange.201604055

Abstract

Molecular imaging holds considerable promise for elucidating biological processes in normal physiology as well as disease states, but requires noninvasive methods for identifying analytes at sub-micromolar concentrations. Particularly useful are genetically encoded, single-protein reporters that harness the power of molecular biology to visualize specific molecular processes, but such reporters have been conspicuously lacking for in vivo magnetic resonance imaging (MRI). Herein, we report TEM-1 β-lactamase (bla) as a single-protein reporter for hyperpolarized (HP) ¹²⁹Xe NMR, with significant saturation contrast at 0.1 μm. Xenon chemical exchange saturation transfer (CEST) interactions with the primary allosteric site in bla give rise to a unique saturation peak at 255 ppm, well removed (≈60 ppm downfield) from the ¹²⁹Xe-H₂O peak. Useful saturation contrast was also observed for bla expressed in bacterial cells and mammalian cells.

A “Smart” 129Xe NMR Biosensor for pH-Dependent Cell Labeling

Co-reporter:Brittany A. Riggle; Yanfei Wang

Journal of the American Chemical Society 2015 Volume 137(Issue 16) pp:5542-5548

Publication Date(Web):April 7, 2015

DOI:10.1021/jacs.5b01938

Here we present a “smart” xenon-129 NMR biosensor that undergoes a peptide conformational change and labels cells in acidic environments. To a cryptophane host molecule with high Xe affinity, we conjugated a 30mer EALA-repeat peptide that is α-helical at pH 5.5 and disordered at pH 7.5. The 129Xe NMR chemical shift at room temperature was strongly pH-dependent (Δδ = 3.4 ppm): δ = 64.2 ppm at pH 7.5 vs δ = 67.6 ppm at pH 5.5, where Trp(peptide)–cryptophane interactions were evidenced by Trp fluorescence quenching. Using hyper-CEST NMR, we probed peptidocryptophane detection limits at low-picomolar (10–11 M) concentration, which compares favorably to other NMR pH reporters at 10–2–10–3 M. Finally, in biosensor-HeLa cell solutions, peptide–cell membrane insertion at pH 5.5 generated a 13.4 ppm downfield cryptophane-129Xe NMR chemical shift relative to pH 7.5 studies. This highlights new uses for 129Xe as an ultrasensitive probe of peptide structure and function, along with potential applications for pH-dependent cell labeling in cancer diagnosis and treatment.

Ruthenium-caged antisense morpholinos for regulating gene expression in zebrafish embryos

Co-reporter:Julianne C. Griepenburg, Teresa L. Rapp, Patrick J. Carroll, James Eberwine and Ivan J. Dmochowski

Chemical Science 2015 vol. 6(Issue 4) pp:2342-2346

Publication Date(Web):29 Jan 2015

DOI:10.1039/C4SC03990D

Photochemical approaches afford high spatiotemporal control over molecular structure and function, for broad applications in materials and biological science. Here, we present the first example of a visible light responsive ruthenium-based photolinker, Ru(bipyridine)2(3-ethynylpyridine)2 (RuBEP), which was reacted stoichiometrically with a 25mer DNA or morpholino (MO) oligonucleotide functionalized with 3′ and 5′ terminal azides, via Cu(I)-mediated [3+2] Huisgen cycloaddition reactions. RuBEP-caged circular morpholinos (Ru-MOs) targeting two early developmental zebrafish genes, chordin and notail, were synthesized and tested in vivo. One-cell-stage zebrafish embryos microinjected with Ru-MO and incubated in the dark for 24 h developed normally, consistent with caging, whereas irradiation at 450 nm dissociated one 3-ethynylpyridine ligand (Φ = 0.33) and uncaged the MO to achieve gene knockdown. As demonstrated, Ru photolinkers provide a versatile method for controlling structure and function of biopolymers.

Cucurbit[6]uril is an ultrasensitive 129Xe NMR contrast agent

Co-reporter:Yanfei Wang and Ivan J. Dmochowski

Chemical Communications 2015 vol. 51(Issue 43) pp:8982-8985

Publication Date(Web):21 Apr 2015

DOI:10.1039/C5CC01826A

A lack of molecular contrast agents has slowed the application of ultrasensitive hyperpolarized 129Xe NMR methods. Here, we report that commercially available cucurbit[6]uril (CB[6]) undergoes rapid xenon exchange kinetics at 300 K, and is detectable by Hyper-CEST NMR at 1.8 pM in PBS and at 1 μM in human plasma where many molecules, including polyamines, can compete with xenon for CB[6] binding.

Cryptophane-Folate Biosensor for 129Xe NMR

Co-reporter:Najat S. Khan, Brittany A. Riggle, Garry K. Seward, Yubin Bai, and Ivan J. Dmochowski

Bioconjugate Chemistry 2015 Volume 26(Issue 1) pp:101

Publication Date(Web):December 1, 2014

DOI:10.1021/bc5005526

Folate-conjugated cryptophane was developed for targeting cryptophane to membrane-bound folate receptors that are overexpressed in many human cancers. The cryptophane biosensor was synthesized in 20 nonlinear steps, which included functionalization with folate recognition moiety, solubilizing peptide, and Cy3 fluorophore. Hyperpolarized 129Xe NMR studies confirmed xenon binding to the folate-conjugated cryptophane. Cellular internalization of biosensor was monitored by confocal laser scanning microscopy and quantified by flow cytometry. Competitive blocking studies confirmed cryptophane endocytosis through a folate receptor-mediated pathway. Flow cytometry revealed 10-fold higher cellular internalization in KB cancer cells overexpressing folate receptors compared to HT-1080 cells with normal folate receptor expression. The biosensor was determined to be nontoxic in HT-1080 and KB cells by MTT assay at low micromolar concentrations typically used for hyperpolarized 129Xe NMR experiments.

Caging Metal Ions with Visible Light-Responsive Nanopolymersomes

Co-reporter:Julianne C. Griepenburg, Nimil Sood, Kevin B. Vargo, Dewight Williams, Jeff Rawson, Michael J. Therien, Daniel A. Hammer, and Ivan J. Dmochowski

Langmuir 2015 Volume 31(Issue 2) pp:799-807

Publication Date(Web):December 17, 2014

DOI:10.1021/la5036689

Polymersomes are bilayer vesicles that self-assemble from amphiphilic diblock copolymers, and provide an attractive system for the delivery of biological and nonbiological molecules due to their environmental compatibility, mechanical stability, synthetic tunability, large aqueous core, and hyperthick hydrophobic membrane. Herein, we report a nanoscale photoresponsive polymersome system featuring a meso-to-meso ethyne-bridged bis[(porphinato)zinc] (PZn2) fluorophore hydrophobic membrane solute and dextran in the aqueous core. Upon 488 nm irradiation in solution or in microinjected zebrafish embryos, the polymersomes underwent deformation, as monitored by a characteristic red-shifted PZn2 emission spectrum and confirmed by cryo-TEM. The versatility of this system was demonstrated through the encapsulation and photorelease of a fluorophore (FITC), as well as two different metal ions, Zn2+ and Ca2+.

Bacterial spore detection and analysis using hyperpolarized 129Xe chemical exchange saturation transfer (Hyper-CEST) NMR

Co-reporter:Yubin Bai, Yanfei Wang, Mark Goulian, Adam Driks and Ivan J. Dmochowski

Chemical Science 2014 vol. 5(Issue 8) pp:3197-3203

Publication Date(Web):23 May 2014

DOI:10.1039/C4SC01190B

Previously, we reported hyperpolarized 129Xe chemical exchange saturation transfer (Hyper-CEST) NMR techniques for the ultrasensitive (i.e., 1 picomolar) detection of xenon host molecules known as cryptophane. Here, we demonstrate a more general role for Hyper-CEST NMR as a spectroscopic method for probing nanoporous structures, without the requirement for cryptophane or engineered xenon-binding sites. Hyper-CEST 129Xe NMR spectroscopy was employed to detect Bacillus anthracis and Bacillus subtilis spores in solution, and interrogate the layers that comprise their structures. 129Xe–spore samples were selectively irradiated with radiofrequency pulses; the depolarized 129Xe returned to aqueous solution and depleted the 129Xe-water signal, providing measurable contrast. Removal of the outermost spore layers in B. anthracis and B. subtilis (the exosporium and coat, respectively) enhanced 129Xe exchange with the spore interior. Notably, the spores were invisible to hyperpolarized 129Xe NMR direct detection methods, highlighting the lack of high-affinity xenon-binding sites, and the potential for extending Hyper-CEST NMR structural analysis to other biological and synthetic nanoporous structures.

Engineering a well-ordered, functional protein-gold nanoparticle assembly

Co-reporter:Jasmina C. Cheung-Lau, Dage Liu, Katherine W. Pulsipher, Weiren Liu, Ivan J. Dmochowski

Journal of Inorganic Biochemistry 2014 130() pp: 59-68

Publication Date(Web):

DOI:10.1016/j.jinorgbio.2013.10.003

Direct Modulation of Microtubule Stability Contributes to Anthracene General Anesthesia

Co-reporter:Daniel J. Emerson ; Brian P. Weiser ; John Psonis ; Zhengzheng Liao ; Olena Taratula ; Ashley Fiamengo ; Xiaozhao Wang ; Keizo Sugasawa ; Amos B. Smith ; III; Roderic G. Eckenhoff

Journal of the American Chemical Society 2013 Volume 135(Issue 14) pp:5389-5398

Publication Date(Web):March 13, 2013

DOI:10.1021/ja311171u

Recently, we identified 1-aminoanthracene as a fluorescent general anesthetic. To investigate the mechanism of action, a photoactive analogue, 1-azidoanthracene, was synthesized. Administration of 1-azidoanthracene to albino stage 40–47 tadpoles was found to immobilize animals upon near-UV irradiation of the forebrain region. The immobilization was often reversible, but it was characterized by a longer duration consistent with covalent attachment of the ligand to functionally important targets. IEF/SDS-PAGE examination of irradiated tadpole brain homogenate revealed labeled protein, identified by mass spectrometry as β-tubulin. In vitro assays with aminoanthracene-cross-linked tubulin indicated inhibition of microtubule polymerization, similar to colchicine. Tandem mass spectrometry confirmed anthracene binding near the colchicine site. Stage 40–47 tadpoles were also incubated 1 h with microtubule stabilizing agents, epothilone D or discodermolide, followed by dosing with 1-aminoanthracene. The effective concentration of 1-aminoanthracene required to immobilize the tadpoles was significantly increased in the presence of either microtubule stabilizing agent. Epothilone D similarly mitigated the effects of a clinical neurosteroid general anesthetic, allopregnanolone, believed to occupy the colchicine site in tubulin. We conclude that neuronal microtubules are “on-pathway” targets for anthracene general anesthetics and may also represent functional targets for some neurosteroid general anesthetics.

Caged oligonucleotides for bidirectional photomodulation of let-7 miRNA in zebrafish embryos

Co-reporter:Julianne C. Griepenburg, Brittani K. Ruble, Ivan J. Dmochowski

Bioorganic & Medicinal Chemistry 2013 Volume 21(Issue 20) pp:6198-6204

Publication Date(Web):15 October 2013

DOI:10.1016/j.bmc.2013.04.082

Many biological functions of microRNA (miRNA) have been identified in the past decade. However, a single miRNA can regulate multiple gene targets, thus it has been a challenge to elucidate the specific functions of each miRNA in different locations and times. New chemical tools make it possible to modulate miRNA activity with higher spatiotemporal resolution. Here, we describe light-activated (caged) constructs for switching let-7 miRNA ‘on’ or ‘off’ with 365 nm light in developing zebrafish embryos.

Measuring Interactions between Polydimethylsiloxane and Serum Proteins at the Air–Water Interface

Co-reporter:Zhengzheng Liao, Wan-Ting Hsieh, Tobias Baumgart, and Ivan J. Dmochowski

Langmuir 2013 Volume 29(Issue 30) pp:9420-9427

Publication Date(Web):July 2, 2013

DOI:10.1021/la401619s

The interaction between synthetic polymers and proteins at interfaces is relevant to basic science as well as a wide range of applications in biotechnology and medicine. One particularly common and important interface is the air–water interface (AWI). Due to the special energetics and dynamics of molecules at the AWI, the interplay between synthetic polymer and protein can be very different from that in bulk solution. In this paper, we applied the Langmuir–Blodgett technique and fluorescence microscopy to investigate how the compression state of polydimethylsiloxane (PDMS) film at the AWI affects the subsequent adsorption of serum protein [e.g., human serum albumin (HSA) or immunoglobulin G (IgG)] and the interaction between PDMS and protein. Of particular note is our observation of circular PDMS domains with micrometer diameters that form at the AWI in the highly compressed state of the surface film: proteins were shown to adsorb preferentially to the surface of these circular PDMS domains, accompanied by a greater than 4-fold increase in protein found in the interfacial film. The PDMS-only film and the PDMS–IgG composite film were transferred to cover glass, and platinum–carbon replicas of the transferred films were further characterized by scanning electron microscopy and atomic force microscopy. We conclude that the structure of the PDMS film greatly affects the amount and distribution of protein at the interface.

Synthesis of Enantiopure, Trisubstituted Cryptophane-A Derivatives

Co-reporter:Olena Taratula, Michael P. Kim, Yubin Bai, John P. Philbin, Brittany A. Riggle, Danniebelle N. Haase, and Ivan J. Dmochowski

Organic Letters 2012 Volume 14(Issue 14) pp:3580-3583

Publication Date(Web):July 11, 2012

DOI:10.1021/ol300943w

The efficient synthesis of enantiopure, trisubstituted cryptophane-A derivatives, organic host molecules with unusually high xenon affinity, is reported. Synthesis and chromatographic separation of (±) tri-Mosher’s acid substituted cryptophane diastereomers gave ready access to the enantiopure cryptophanes, which are critical components in the design of enantiomerically pure 129Xe biosensors. Hyperpolarized 129Xe NMR spectroscopy identified single resonances for both trisubstituted cryptophane diastereomers that were separated by 9.5 ppm. This highlights opportunities for using enantiopure xenon biosensors in the simultaneous detection of 129Xe in different biochemical environments.

Utilizing a Water-Soluble Cryptophane with Fast Xenon Exchange Rates for Picomolar Sensitivity NMR Measurements

Co-reporter:Yubin Bai, P. Aru Hill, and Ivan J. Dmochowski

Analytical Chemistry 2012 Volume 84(Issue 22) pp:9935

Publication Date(Web):October 29, 2012

DOI:10.1021/ac302347y

Hyperpolarized 129Xe chemical exchange saturation transfer (129Xe Hyper-CEST) NMR is a powerful technique for the ultrasensitive, indirect detection of Xe host molecules (e.g., cryptophane-A). Irradiation at the appropriate Xe-cryptophane resonant radio frequency results in relaxation of the bound hyperpolarized 129Xe and rapid accumulation of depolarized 129Xe in bulk solution. The cryptophane effectively “catalyzes” this process by providing a unique molecular environment for spin depolarization to occur, while allowing xenon exchange with the bulk solution during the hyperpolarized lifetime (T1 ≈ 1 min). Following this scheme, a triacetic acid cryptophane-A derivative (TAAC) was indirectly detected at 1.4 picomolar concentration at 320 K in aqueous solution, which is the record for a single-unit xenon host. To investigate this sensitivity enhancement, the xenon binding kinetics of TAAC in water was studied by NMR exchange lifetime measurement. At 297 K, kon ≈ 1.5 × 106 M–1s–1 and koff = 45 s–1, which represent the fastest Xe association and dissociation rates measured for a high-affinity, water-soluble xenon host molecule near rt. NMR line width measurements provided similar exchange rates at rt, which we assign to solvent-Xe exchange in TAAC. At 320 K, koff was estimated to be 1.1 × 103 s–1. In Hyper-CEST NMR experiments, the rate of 129Xe depolarization achieved by 14 pM TAAC in the presence of radio frequency (RF) pulses was calculated to be 0.17 μM·s–1. On a per cryptophane basis, this equates to 1.2 × 104129Xe atoms s–1 (or 4.6 × 104 Xe atoms s–1, all Xe isotopes), which is more than an order of magnitude faster than koff, the directly measurable Xe-TAAC exchange rate. This compels us to consider multiple Xe exchange processes for cryptophane-mediated bulk 129Xe depolarization, which provide at least 107-fold sensitivity enhancements over directly detected hyperpolarized 129Xe NMR signals.

Mismatch discrimination and efficient photomodulation with split 10–23 DNAzymes

Co-reporter:Brittani K. Ruble, Julia L. Richards, Jasmina C. Cheung-Lau, Ivan J. Dmochowski

Inorganica Chimica Acta 2012 380() pp: 386-391

Publication Date(Web):

DOI:10.1016/j.ica.2011.10.068

Cell-compatible, integrin-targeted cryptophane-129XeNMR biosensors

Co-reporter:Garry K. Seward, Yubin Bai, Najat S. Khan and Ivan J. Dmochowski

Chemical Science 2011 vol. 2(Issue 6) pp:1103-1110

Publication Date(Web):23 Mar 2011

DOI:10.1039/C1SC00041A

Peptide-modified cryptophane enables sensitive detection ofproteinanalytes using hyperpolarized 129Xe NMR spectroscopy. Here we report improved targeting and delivery of cryptophane to cells expressing αvβ3 integrin receptor, which is overexpressed in many human cancers. Cryptophane was functionalized with cyclic RGDyKpeptide and Alexa Fluor 488dye, and cellular internalization was monitored by confocal laser scanning microscopy. Competitive blocking assays confirmed cryptophaneendocytosis through an αvβ3 integrin receptor-mediated pathway. The peptide–cryptophane conjugate was determined to be nontoxic in normal human lung fibroblasts by MTT assay at the micromolar cryptophane concentrations typically used for hyperpolarized 129Xe NMR biosensing experiments. Flow cytometry revealed 4-fold higher cellular internalization in cancer cells overexpressing the integrin receptor compared to normal cells. Nanomolar inhibitory concentrations (IC50 = 20–30 nM) were measured for cryptophane biosensors against vitronectin binding to αvβ3 integrin and fibrinogen binding to αIIbβ3 integrin. Functionalization of the conjugate with two propionic acidgroups improved water solubility for hyperpolarized 129Xe NMR spectroscopic studies, which revealed a single resonance at 67 ppm for the 129Xe-cryptophane–cyclic RGDyK biosensor. Introduction of αIIbβ3 integrin receptor in detergent solution generated a new “ bound” 129Xe biosensor peak that was shifted 4 ppm downfield from the “free” 129Xe biosensor.

Shorter Synthesis of Trifunctionalized Cryptophane-A Derivatives

Co-reporter:Olena Taratula, P. Aru Hill, Yubin Bai, Najat S. Khan, and Ivan J. Dmochowski

Organic Letters 2011 Volume 13(Issue 6) pp:1414-1417

Publication Date(Web):February 18, 2011

DOI:10.1021/ol200088f

Efficient syntheses of trisubstituted cryptophane-A derivatives that are versatile host molecules for many applications are reported. Trihydroxy cryptophane was synthesized in six or seven steps with yields as high as 9.5%. By a different route, trihydroxy cryptophane modified with three propargyl, allyl, or benzyl protecting groups was synthesized with yields of 4.1−5.8% in just six steps. Hyperpolarized 129Xe NMR chemical shifts of 57−65 ppm were measured for these trisubstituted cryptophanes.

Protein Assembly at the Air–Water Interface Studied by Fluorescence Microscopy

Co-reporter:Zhengzheng Liao, Joshua W. Lampe, Portonovo S. Ayyaswamy, David M. Eckmann, and Ivan J. Dmochowski

Langmuir 2011 Volume 27(Issue 21) pp:12775-12781

Publication Date(Web):September 26, 2011

DOI:10.1021/la203053g

Protein assembly at the air–water interface (AWI) occurs naturally in many biological processes and provides a method for creating biomaterials. However, the factors that control protein self-assembly at the AWI and the dynamic processes that occur during adsorption are still underexplored. Using fluorescence microscopy, we investigated assembly at the AWI of a model protein, human serum albumin minimally labeled with Texas Red fluorophore. Static and dynamic information was obtained under low subphase concentrations. By varying the solution protein concentration, ionic strength, and redox state, we changed the microstructure of protein assembly at the AWI accordingly. The addition of pluronic surfactant caused phase segregation to occur at the AWI, with fluid surfactant domains and more rigid protein domains revealed by fluorescence recovery after photobleaching experiments. Protein domains were observed to coalesce during this competitive adsorption process.

Multiple Hindered Rotators in a Gyroscope-Inspired Tribenzylamine Hemicryptophane

Co-reporter:Najat S. Khan, Jose Manuel Perez-Aguilar, Tara Kaufmann, P. Aru Hill, Olena Taratula, One-Sun Lee, Patrick J. Carroll, Jeffery G. Saven, and Ivan J. Dmochowski

The Journal of Organic Chemistry 2011 Volume 76(Issue 5) pp:1418-1424

Publication Date(Web):January 27, 2011

DOI:10.1021/jo102480s

A gyroscope-inspired tribenzylamine hemicryptophane provides a vehicle for exploring the structure and properties of multiple p-phenylene rotators within one molecule. The hemicryptophane was synthesized in three steps in good overall yield using mild conditions. Three rotator-forming linkers were cyclized to form a rigid cyclotriveratrylene (CTV) stator framework, which was then closed with an amine. The gyroscope-like molecule was characterized by 1H NMR and 13C NMR spectroscopy, and the structure was solved by X-ray crystallography. The rigidity of the two-component CTV-trismethylamine stator was investigated by 1H variable-temperature (VT) NMR experiments and molecular dynamics simulations. These techniques identified gyration of the three p-phenylene rotators on the millisecond time scale at −93 °C, with more dynamic but still hindered motion at room temperature (27 °C). The activation energy for the p-phenylene rotation was determined to be ∼10 kcal mol−1. Due to the propeller arrangement of the p-phenylenes, their rotation is hindered but not strongly correlated. The compact size, simple synthetic route, and molecular motions of this gyroscope-inspired tribenzylamine hemicryptophane make it an attractive starting point for controlling the direction and coupling of rotators within molecular systems.

Measurement of radon and xenon binding to a cryptophane molecular host

Co-reporter:David R. Jacobson;Najat S. Khan;Ronald Collé;Ryan Fitzgerald;Lizbeth Laureano-Pérez;Yubin Bai;

Proceedings of the National Academy of Sciences 2011 108(27) pp:10969-10973

Publication Date(Web):June 20, 2011

DOI:10.1073/pnas.1105227108

Xenon and radon have many similar properties, a difference being that all 35 isotopes of radon (¹⁹⁵Rn–²²⁹Rn) are radioactive. Radon is a pervasive indoor air pollutant believed to cause significant incidence of lung cancer in many geographic regions, yet radon affinity for a discrete molecular species has never been determined. By comparison, the chemistry of xenon has been widely studied and applied in science and technology. Here, both noble gases were found to bind with exceptional affinity to tris-(triazole ethylamine) cryptophane, a previously unsynthesized water-soluble organic host molecule. The cryptophane–xenon association constant, K_a = 42,000 ± 2,000 M^-1 at 293 K, was determined by isothermal titration calorimetry. This value represents the highest measured xenon affinity for a host molecule. The partitioning of radon between air and aqueous cryptophane solutions of varying concentration was determined radiometrically to give the cryptophane–radon association constant K_a = 49,000 ± 12,000 M^-1 at 293 K.

Sensing membrane stress with near IR-emissive porphyrins

Co-reporter:Neha P. Kamat;Zhengzheng Liao;Laurel E. Moses;Jeff Rawson;Michael J. Therien;Daniel A. Hammer

PNAS 2011 108 (34 ) pp:

Publication Date(Web):2011-08-23

DOI:10.1073/pnas.1102125108

Probes embedded within a structure can enable prediction of material behavior or failure. Carefully assembled composites that respond intelligently to physical changes within a material could be useful as intrinsic sensors. Molecular rotors are one such tool that can respond optically to physical environmental changes. Here, we propose to use molecular rotors within a polymersome membrane to report membrane stress. Using supermolecular porphyrin-based fluorophores as rotors, we characterize changes in the optical emission of these near-infrared (NIR) emissive probes embedded within the hydrophobic core of the polymersome membrane. The configuration of entrapped fluorophore depends on the available space within the membrane; in response to increased volume, emission is blue shifted. We used this feature to study how shifts in fluorescence correlate to membrane integrity, imparted by membrane stress. We monitored changes in emission of these porphyrin-based fluorophores resulting from membrane stress produced through a range of physical and chemical perturbations, including surfactant-induced lysis, hydrolytic lysis, thermal degradation, and applied stress by micropipette aspiration. This paper comprehensively illustrates the potential for supermolecular porphyrin-based fluorophores to detect intrinsic physical changes in a wide variety of environments, and suggests how molecular rotors may be used in soft materials science and biology as sensors.

Turning the 1023 DNAzyme On and Off with Light

Co-reporter:Julia L. Richards;Garry K. Seward;Yu-Hsiu Wang

ChemBioChem 2010 Volume 11( Issue 3) pp:320-324

Publication Date(Web):

DOI:10.1002/cbic.200900702

Identification of a fluorescent general anesthetic, 1-aminoanthracene

Co-reporter:Christopher A. Butts;Jin Xi;Grace Brannigan;Abdalla A. Saad;Srinivasan P. Venkatachalan;Robert A. Pearce;Michael L. Klein;Roderic G. Eckenhoff

PNAS 2009 Volume 106 (Issue 16 ) pp:6501-6506

Publication Date(Web):2009-04-21

DOI:10.1073/pnas.0810590106

We identified a fluorophore, 1-aminoanthracene (1-AMA), that is anesthetic, potentiates GABAergic transmission, and gives an appropriate dissociation constant, Kd ≈ 0.1 mM, for binding to the general anesthetic site in horse spleen apoferritin (HSAF). 1-AMA fluorescence is enhanced when bound to HSAF. Thus, displacement of 1-AMA from HSAF by other anesthetics attenuates the fluorescence signal and allows determination of Kd, as validated by isothermal titration calorimetry. This provides a unique fluorescence assay for compound screening and anesthetic discovery. Additional electrophysiology experiments in isolated cells indicate that 1-AMA potentiates chloride currents elicited by GABA, similar to many general anesthetics. Furthermore, 1-AMA reversibly immobilizes stage 45–50 Xenopus laevis tadpoles (EC50 = 16 μM) and fluorescence micrographs show 1-AMA localized to brain and olfactory regions. Thus, 1-AMA provides an unprecedented opportunity for studying general anesthetic distribution in vivo at the cellular and subcellular levels.

Efficient Self-Assembly of Archaeoglobus fulgidus Ferritin around Metallic Cores

Co-reporter:Joe Swift, Christopher A. Butts, Jasmina Cheung-Lau, Vijay Yerubandi and Ivan J. Dmochowski

Langmuir 2009 Volume 25(Issue 9) pp:5219-5225

Publication Date(Web):March 4, 2009

DOI:10.1021/la8040743

Interfacing biological systems with inorganic nanoparticles is of great interest, as it offers means of particle stabilization and spatial control in electronic or biomedical applications. We report on the particle-directed assembly of hyperthermophile Archaeoglobus fulgidus ferritin subunits around negatively charged colloidal gold. An annealing process allows rapid assembly of the protein in near-native stoichiometry. Transmission electron microscopy suggests that greater than 95% of nanoparticles are encapsulated while the self-assembly process ensures that almost 100% of the assembled ferritin cavities are occupied.

Peptide-Mediated Cellular Uptake of Cryptophane

Co-reporter:Garry K. Seward, Qian Wei and Ivan J. Dmochowski

Bioconjugate Chemistry 2008 Volume 19(Issue 11) pp:2129

Publication Date(Web):October 16, 2008

DOI:10.1021/bc8002265

Cryptophane-A has generated considerable interest based on its high affinity for xenon and potential for creating biosensors for 129Xe nuclear magnetic resonance (NMR) spectroscopy. Here, we report the cellular delivery of three peptide-functionalized cryptophane biosensors. Cryptophanes were delivered using two cationic cell penetrating peptides into several human cancer and normal cell lines. An RGD peptide targeting αvβ3 integrin receptor was shown to increase specificity of cryptophane cell uptake. Labeling the peptides with Cy3 made it possible to monitor cellular delivery using confocal laser scanning microscopy. The peptido-cryptophanes were determined to be relatively nontoxic by MTT assay at the micromolar cryptophane concentrations that are required for 129Xe NMR biosensing experiments.

Directing Noble Metal Ion Chemistry within a Designed Ferritin Protein†,‡

Co-reporter:Christopher A. Butts, Joe Swift, Seung-gu Kang, Luigi Di Costanzo, David W. Christianson, Jeffery G. Saven and Ivan J. Dmochowski

Biochemistry 2008 Volume 47(Issue 48) pp:12729-12739

Publication Date(Web):November 7, 2008

DOI:10.1021/bi8016735

Human H ferritin (HuHF) assembles from 24 four-helix bundles to form an ∼500 kDa protein with an 8 nm internal cavity. HuHF provides a useful model for studying the transport of metal ions in solution to buried reaction sites in proteins. In this study, HuHF was redesigned to facilitate noble metal ion (Au3+, Ag+) binding, reduction, and nanoparticle formation within the cavity. Computationally determined amino acid substitutions were targeted at four external and four internal surface sites. A variant with a total of 96 cysteines and histidines removed from the exterior surface and 96 non-native cysteines added to the interior surface retained wild-type stability and structure, as confirmed by X-ray crystallography, and promoted the formation of silver or gold nanoparticles within the protein cavity. Crystallographic studies with HuHF variants provide insight into how ferritins control access of metal ions to interior residues that perform chemistry.

Regulating gene expression with light-activated oligonucleotides

Co-reporter:XinJing Tang and Ivan J. Dmochowski

Molecular BioSystems 2007 vol. 3(Issue 2) pp:100-110

Publication Date(Web):20 Nov 2006

DOI:10.1039/B614349K

Since the development of light-responsive amino acids, the activity of numerous biomolecules has been photomodulated in biochemical, biophysical, and cellular assays. Biological problems of even greater complexity motivate the development of quantitative methods for controlling gene activity with high spatial and temporal resolution, using light as an external trigger. Photoresponsive DNA and RNA oligonucleotides would optimally serve this purpose, but have proven difficult to expand from proofs-of-concept to in vivo experiments. Until recently, the development of this technology was limited by the synthesis of oligonucleotides whose function could be significantly modulated with near-UV light. New synthetic protocols and strategies for both up- and down-regulating gene activity finally make it possible to address biological considerations. In the near future, we can expect photoresponsive DNA and RNA molecules that are relatively non-toxic, nuclease-resistant, and maintain their specificity and activity in vivo. Quantitative, laser-initiated methods for controlling DNA and RNA function will illuminate new areas in cell and developmental biology.

Structure and activity of apoferritin-stabilized gold nanoparticles

Co-reporter:Lei Zhang, Joe Swift, Christopher A. Butts, Vijay Yerubandi, Ivan J. Dmochowski

Journal of Inorganic Biochemistry 2007 Volume 101(11–12) pp:1719-1729

Publication Date(Web):November 2007

DOI:10.1016/j.jinorgbio.2007.07.023

A simple method for synthesizing gold nanoparticles stabilized by horse spleen apoferritin (HSAF) is reported using NaBH4 or 3-(N -morpholino)propanesulfonic acid (MOPS) as the reducing agent. AuCl4- reduction by NaBH4 was complete within a few seconds, whereas reduction by MOPS was much slower; in all cases, protein was required during reduction to keep the gold particles in aqueous solution. Transmission electron microscopy (TEM) showed that the gold nanoparticles were associated with the outer surface of the protein. The average particle diameters were 3.6 and 15.4 nm for NaBH4-reduced and MOPS-reduced Au-HSAF, respectively. A 5-nm difference in the UV–Vis absorption maximum was observed for NaBH4-reduced (530 nm) and MOPS-reduced Au-HSAF (535 nm), which was attributed to the greater size and aggregation of the MOPS-reduced gold sample. NaBH4-reduced Au-HSAF was much more effective than MOPS-reduced Au-HSAF in catalyzing the reduction of 4-nitrophenol by NaBH4, based on the greater accessibility of the NaBH4-reduced gold particle to the substrate. Rapid reduction of AuCl4- by NaBH4 was determined to result in less surface passivation by the protein. Methods for studying ferritin–gold nanoparticle assemblies may be readily applied to other protein–metal colloid systems.

Controlling RNA Digestion by RNase H with a Light-Activated DNA Hairpin

Co-reporter:XinJing Tang

Angewandte Chemie 2006 Volume 118(Issue 21) pp:

Publication Date(Web):24 APR 2006

DOI:10.1002/ange.200600954

UV-empfindlich: Eine DNA-Haarnadel wurde aus einem 20-meren Antisense-Oligodesoxynucleotid (asODN) und einem Sense-ODN mithilfe eines difunktionellen photospaltbaren Linkers hergestellt. Das Konjugat wurde bei UV-Photoaktivierung erheblich destabilisiert, was das Binden des asODN an komplementäre RNA erleichterte. Auf diese Art ließ sich die RNase-H-Aktivität gegenüber einer Vielzahl von RNA-Zielen photomodulieren.

Controlling RNA Digestion by RNase H with a Light-Activated DNA Hairpin

Co-reporter:XinJing Tang,Ivan J. Dmochowski

Angewandte Chemie International Edition 2006 45(21) pp:3523-3526

Publication Date(Web):

DOI:10.1002/anie.200600954

Taxane modulation of anesthetic sensitivity in surgery for nonmetastatic breast cancer

Co-reporter:Regina E. Linganna, Warren J. Levy, Ivan J. Dmochowski, Roderic G. Eckenhoff, Rebecca M. Speck

Journal of Clinical Anesthesia (September 2015) Volume 27(Issue 6) pp:481-485

Publication Date(Web):1 September 2015

DOI:10.1016/j.jclinane.2015.05.001

•Destabilization of microtubules is a possible mechanism of general anesthetics.•Breast cancer patients treated with taxanes have hyperstabilized microtubules.•Taxane-treated patients will be resistant to anesthetics that destabilize microtubules.•Hemodynamic changes in response to incision were a surrogate for anesthetic depth.•We report a greater change in blood pressure for taxane treated patients.Study objective and designThe mechanism of action of commonly used general anesthetics is largely unknown. One hypothesized mechanism is through modulation of microtubule stability. Taxanes, a subset of chemotherapeutic drugs known to alter microtubule stability and commonly used to treat breast cancer, offer a natural experiment to test our hypothesis that patients exposed to taxanes prior to surgery, as compared to after surgery, would have a partial resistance to general anesthetics.Setting, patients, and measurementsThe anesthetic record of adult women with nonmetastatic breast cancer was used to obtain changes in heart rate and blood pressure surrounding incision, and the amount of inhaled anesthetic agent, induction, and rescue drugs administered.Main resultsChange in blood pressure in response to incision was significantly higher in the neoadjuvant group (P = .03), whereas change in heart rate was not (P = .53). A greater amount of morphine was administered in the neoadjuvant group (26.3 vs 15.5 mg, P = .02), although not a higher concentration of inhaled anesthetics (P = .15).ConclusionThese results suggest that the alteration of microtubule stability is one of a number of mechanisms of inhaled anesthetics.

Bacterial spore detection and analysis using hyperpolarized 129Xe chemical exchange saturation transfer (Hyper-CEST) NMR

Co-reporter:Yubin Bai, Yanfei Wang, Mark Goulian, Adam Driks and Ivan J. Dmochowski

Chemical Science (2010-Present) 2014 - vol. 5(Issue 8) pp:NaN3203-3203

Publication Date(Web):2014/05/23

DOI:10.1039/C4SC01190B

Previously, we reported hyperpolarized 129Xe chemical exchange saturation transfer (Hyper-CEST) NMR techniques for the ultrasensitive (i.e., 1 picomolar) detection of xenon host molecules known as cryptophane. Here, we demonstrate a more general role for Hyper-CEST NMR as a spectroscopic method for probing nanoporous structures, without the requirement for cryptophane or engineered xenon-binding sites. Hyper-CEST 129Xe NMR spectroscopy was employed to detect Bacillus anthracis and Bacillus subtilis spores in solution, and interrogate the layers that comprise their structures. 129Xe–spore samples were selectively irradiated with radiofrequency pulses; the depolarized 129Xe returned to aqueous solution and depleted the 129Xe-water signal, providing measurable contrast. Removal of the outermost spore layers in B. anthracis and B. subtilis (the exosporium and coat, respectively) enhanced 129Xe exchange with the spore interior. Notably, the spores were invisible to hyperpolarized 129Xe NMR direct detection methods, highlighting the lack of high-affinity xenon-binding sites, and the potential for extending Hyper-CEST NMR structural analysis to other biological and synthetic nanoporous structures.

Cell-compatible, integrin-targeted cryptophane-129XeNMR biosensors

Co-reporter:Garry K. Seward, Yubin Bai, Najat S. Khan and Ivan J. Dmochowski

Chemical Science (2010-Present) 2011 - vol. 2(Issue 6) pp:NaN1110-1110

Publication Date(Web):2011/03/23

DOI:10.1039/C1SC00041A

Peptide-modified cryptophane enables sensitive detection ofproteinanalytes using hyperpolarized 129Xe NMR spectroscopy. Here we report improved targeting and delivery of cryptophane to cells expressing αvβ3 integrin receptor, which is overexpressed in many human cancers. Cryptophane was functionalized with cyclic RGDyKpeptide and Alexa Fluor 488dye, and cellular internalization was monitored by confocal laser scanning microscopy. Competitive blocking assays confirmed cryptophaneendocytosis through an αvβ3 integrin receptor-mediated pathway. The peptide–cryptophane conjugate was determined to be nontoxic in normal human lung fibroblasts by MTT assay at the micromolar cryptophane concentrations typically used for hyperpolarized 129Xe NMR biosensing experiments. Flow cytometry revealed 4-fold higher cellular internalization in cancer cells overexpressing the integrin receptor compared to normal cells. Nanomolar inhibitory concentrations (IC50 = 20–30 nM) were measured for cryptophane biosensors against vitronectin binding to αvβ3 integrin and fibrinogen binding to αIIbβ3 integrin. Functionalization of the conjugate with two propionic acidgroups improved water solubility for hyperpolarized 129Xe NMR spectroscopic studies, which revealed a single resonance at 67 ppm for the 129Xe-cryptophane–cyclic RGDyK biosensor. Introduction of αIIbβ3 integrin receptor in detergent solution generated a new “ bound” 129Xe biosensor peak that was shifted 4 ppm downfield from the “free” 129Xe biosensor.

Ruthenium-caged antisense morpholinos for regulating gene expression in zebrafish embryos

Co-reporter:Julianne C. Griepenburg, Teresa L. Rapp, Patrick J. Carroll, James Eberwine and Ivan J. Dmochowski

Chemical Science (2010-Present) 2015 - vol. 6(Issue 4) pp:NaN2346-2346

Publication Date(Web):2015/01/29

DOI:10.1039/C4SC03990D

Photochemical approaches afford high spatiotemporal control over molecular structure and function, for broad applications in materials and biological science. Here, we present the first example of a visible light responsive ruthenium-based photolinker, Ru(bipyridine)2(3-ethynylpyridine)2 (RuBEP), which was reacted stoichiometrically with a 25mer DNA or morpholino (MO) oligonucleotide functionalized with 3′ and 5′ terminal azides, via Cu(I)-mediated [3+2] Huisgen cycloaddition reactions. RuBEP-caged circular morpholinos (Ru-MOs) targeting two early developmental zebrafish genes, chordin and notail, were synthesized and tested in vivo. One-cell-stage zebrafish embryos microinjected with Ru-MO and incubated in the dark for 24 h developed normally, consistent with caging, whereas irradiation at 450 nm dissociated one 3-ethynylpyridine ligand (Φ = 0.33) and uncaged the MO to achieve gene knockdown. As demonstrated, Ru photolinkers provide a versatile method for controlling structure and function of biopolymers.

Cucurbit[6]uril is an ultrasensitive 129Xe NMR contrast agent

Co-reporter:Yanfei Wang and Ivan J. Dmochowski

Chemical Communications 2015 - vol. 51(Issue 43) pp:NaN8985-8985

Publication Date(Web):2015/04/21

DOI:10.1039/C5CC01826A

A lack of molecular contrast agents has slowed the application of ultrasensitive hyperpolarized 129Xe NMR methods. Here, we report that commercially available cucurbit[6]uril (CB[6]) undergoes rapid xenon exchange kinetics at 300 K, and is detectable by Hyper-CEST NMR at 1.8 pM in PBS and at 1 μM in human plasma where many molecules, including polyamines, can compete with xenon for CB[6] binding.