Seok Cheol Hong

Two-Photon Fluorescent Turn-On Probe for Lipid Rafts in Live Cell and Tissue

We report a new two-photon fluorescence turn-on probe 6-[(E)-3-oxo-1-dodecenyl]-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (CL2) that is designed specifically for visualizing lipid rafts in living cells and tissues. This probe emits much brighter two-photon excited fluorescence in lipid rafts than in non-raft domains and allows direct visualization of the lipid rafts in the live cells and pyramidal neuron layer of the CA1 region at a depth of 100-250 mum in live tissues using two-photon microscopy.

Two-photon fluorescent probes for acidic vesicles in live cells and tissue.

enzymes and secretory proteins exhibiting a variety of functions. To determine their functions, a variety of membrane-permeable fluorescent pH and lysosomal probes have been developed, some of which are commercially available. However, use of these probes with one-photon microscopy (OPM) requires excitation with short-wavelength light (ca. 350–550 nm) that limits their application in deeptissue imaging, owing to the shallow penetration depth (less than 80 mm) as well as to photobleaching, photodamage, and cellular autofluorescence. To overcome these problems, it is crucial to use two-photon microscopy (TPM). TPM employs two near-infrared photons for excitation and offers a number of advantages over OPM, including increased penetration depth (greater than 500 mm), localized excitation, and prolonged observation time. The extra penetration depth that TPM affords is of particular interest in tissue imaging, because surface preparation artifacts such as damaged cells extend over 70 mm into the tissue interior. However, most of the OP fluorescent probes presently used for TPM have small TP action cross sections (Fd) that limit their usage. Although a TP pH probe with appreciable Fd (ca. 42 GM) has been reported, the utility of this probe in TPM imaging has not been verified. Therefore, there is a need to develop an efficient TP probe that can visualize acidic vesicles deep inside tissue for a long period of time. To design an efficient TP probe for acidic vesicles, we chose acedan as the TP fluorophore, because acedan-derived TP probes for Mg (AMg1) and Ca (ACa1) exhibited high photostability as well as significant TP action cross sections for the bright TPM image at low probe concentration, thus allowing the detection of the metal ions deep inside live tissues for over 1100 s. We have introduced an aniline, o-methoxy aniline (pKa(BH ) 4), or tertiary amine (pKa(BH ) 10) substituent as the proton-binding site through an amide linkage to the fluorophore. It is expected that AH1 and AH2 would emit TP-excited fluorescence (TPEF) upon protonation at pH< 4, whereas AL1 would emit TPEF in the acidic vesicles, where it should accumulate as the protonated form. Herein, we report that these probes are capable of imaging the acidic vesicles in live cells and living tissues at greater than 100 mm depth without mistargeting and photobleaching problems. Moreover, AL1 can visualize the transportation of the acidic vesicles in the hippocampal cornu ammonis CA3 region for a long period of time with the use of TPM. AH1, AH2, and AL1 were prepared in 47–77% yields by reactions of 6-acyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene and a p-phenylenediamine derivative or N,Ndimethylethylenediamine (see the Supporting Information). The solubilities of AH1, AH2, and AL1 in water are in the range of 5.0–9.0 mm, which are sufficient to stain the cells (Figure S2 in the Supporting Information). The fluorescence spectra of AH1, AH2, and AL1 show gradual bathochromic shifts with solvent polarity (ET) in the order 1,4-dioxane< DMF<EtOH<H2O (Figure S1 and Table S1 in the Supporting Information). The large bathochromic shifts with increasing solvent polarity indicate the utility of these molecules as polarity probes. TP action cross section was determined by investigating the TPEFof the probes using fluorescein as the reference (see the Supporting Information). The TP action spectra of AH1, Scheme 1. The structures of AH1, AH2, and AL1.

Minute negative superhelicity is sufficient to induce the B-Z transition in the presence of low tension

Left-handed Z-DNA has fascinated biological scientists for decades by its extraordinary structure and potential involvement in biological phenomena. Despite its instability relative to B-DNA, Z-DNA is stabilized in vivo by negative supercoiling. A detailed understanding of Z-DNA formation is, however, still lacking. In this study, we have examined the B-Z transition in a short guanine/cytosine (GC) repeat in the presence of controlled tension and superhelicity via a hybrid technique of single-molecule FRET and magnetic tweezers. The hybrid scheme enabled us to identify the states of the specific GC region under mechanical control and trace conformational changes synchronously at local and global scales. Intriguingly, minute negative superhelicity can facilitate the B-Z transition at low tension, indicating that tension, as well as torsion, plays a pivotal role in the transition. Dynamic interconversions between the states at elevated temperatures yielded thermodynamic and kinetic constants of the transition. Our single-molecule studies shed light on the understanding of Z-DNA formation by highlighting the highly cooperative and dynamic nature of the B-Z transition.

Orientations of phenyl sidegroups and liquid crystal molecules on a rubbed polystyrene surface

Surface–specific sum–frequency vibrational spectroscopy and second-harmonic generation have been used to study the structure of a rubbed polystyrene (PS) surface and the orientation of 4′-n-pentyl-4-cyanobiphenyl (5CB) liquid crystal molecules on it. The results show that the phenyl sidegroups are well aligned by rubbing in the direction perpendicular to rubbing but tilt from the surface normal with a broad distribution. Although the PS surface is nonpolar, the 5CB molecules appear to adsorb on PS preferentially with the terminal cyano group facing the PS surface.

Kinetics of the Triplex-Duplex Transition in DNA

The kinetics of triplex folding/unfolding is investigated by the single-molecule fluorescence resonance energy transfer (FRET) technique. In neutral pH conditions, the average dwell times in both high-FRET (folded) and low-FRET (unfolded) states are comparable, meaning that the triplex is marginally stable. The dwell-time distributions are qualitatively different: while the dwell-time distribution of the high-FRET state should be fit with at least a double-exponential function, the dwell-time distribution of the low-FRET state can be fit with a single-exponential function. We propose a model where the folding can be trapped in metastable states, which is consistent with the FRET data. Our model also accounts for the fact that the relevant timescales of triplex folding/unfolding are macroscopic.

Artifacts identification in apertureless near-field optical microscopy

The aim of this paper is to provide criteria for optical artifacts recognition in reflection-mode apertureless scanning near-field optical microscopy, implementing demodulation techniques at higher harmonics. We show that optical images acquired at different harmonics, although totally uncorrelated from the topography, can be entirely due to far-field artifacts. Such observations are interpreted by developing the dipole-dipole model for the detection scheme at higher harmonics. The model, confirmed by the experiment, predicts a lack of correlation between the topography and optical images even for structures a few tens of nanometers high, due to the rectification effect introduced by the lock-in amplifier used for signal demodulation. Analytical formulas deduced for the far-field background permit to simulate and identify all the different fictitious patterns to be expected from metallic nanowires or nanoparticles of a given shape. In particular, the background dependence on the tip-oscillation amplitude is put forward as the cause of the error-signal artifacts, suggesting, at the same time, specific fine-tuning configurations for background-free imaging. Finally a careful analysis of the phase signal is carried out. In particular, our model correctly interprets the steplike dependence observed experimentally of the background phase signal versus the tip-sample distance, and suggests to look for smooth variations of the phase signal for unambiguous near-field imaging assessment.

Rubbing-induced polar ordering in nylon-11

Sum-frequency vibrational spectroscopy was used to show that mechanical rubbing could induce domains of ferroelectric ordering in films of odd-numbered nylon. In each domain, the dipole groups of NH and CO were aligned perpendicular to the rubbing direction and parallel to the surface.

Destabilization of i-Motif by Submolar Concentrations of a Monovalent Cation

Counterions are crucial for self-assembly of nucleic acids. Submolar monovalent cations are generally deemed to stabilize various types of base pairs in nucleic acids such as Watson-Crick and Hoogsteen base pairs via screening of electrostatic repulsion. Besides monovalent cations, acidic pH is required for i-motif formation because protons facilitate pairing between cytosines. Here we report that Li(+) ions destabilize i-motif, whereas other monovalent cations, Na(+) and K(+), have the usual stabilizing effect. The thermodynamics data alone, however, cannot reveal which mechanism, enhanced unfolding or suppressed folding or both, is responsible for the Li(+)-induced destabilization. To gain further insight, we examined the kinetics of i-motif. To deal with slow kinetics of i-motif, we developed a method dubbed HaRP to construct a long FRET time trace to observe a sufficient number of transitions. Our kinetics analysis shows clearly that Li(+) ions promote unfolding of i-motif but do not hinder its folding, lending strong support for our hypothesis on the origin of this unusual effect of Li(+). Although the subangstrom size of Li(+) ions allows them to infiltrate the space between cytosines in competition with protons, they cannot adequately fulfill the role of protons in mediating the hydrogen bonding of cytosine pairs.

In situ analysis of cisplatin binding to DNA: the effects of physiological ionic conditions

Platinum-based anti-cancer drugs form a major family of cancer chemotherapeutic agents. Cisplatin, the first member of the family, remains a potent anti-cancer drug and exhibits its clinical effect by inducing local DNA kinks and subsequently interfering with DNA metabolism. Although its mechanism is reasonably well understood, effects of intracellular ions on cisplatin activity are left to be elucidated because cisplatin binding to DNA, thus its drug efficacy, is modified by various ions. One such issue is the effect of carbonate ions: cisplatin binding to DNA is suppressed under physiological carbonate conditions. Here, we examined the role of common cellular ions (carbonate and chloride) by measuring cisplatin binding in relevant physiological buffers via a DNA micromanipulation technique. Using two orthogonal single-molecule methods, we succeeded in detecting hidden monofunctional adducts (kink-free, presumably clinically inactive form) and clearly showed that the major effect of carbonates was to form such adducts and to prevent them from converting to bifunctional adducts (kinked, clinically active). The chloride-rich environment also led to the formation of monofunctional adducts. Our approach is widely applicable to the study of the transient behaviours of various drugs and proteins that bind to DNA in different modes depending on various physical and chemical factors such as tension, torsion, ligands, and ions.

Deciphering Kinetic Information from Single-Molecule FRET Data That Show Slow Transitions.

Single-molecule FRET is one of the most powerful and widely used biophysical techniques in biological sciences. It, however, often suffers from limitations such as weak signal and limited measurement time intrinsic to single-molecule fluorescence measurements. Despite several ameliorative measures taken to increase measurement time, it is nearly impossible to acquire meaningful kinetic information on a molecule if conformational transitions of the molecule are ultraslow such that transition times (⟨τ⟩orig) are comparable to or longer than measurement times (δt) limited by the finite lifetime of fluorescent dye. Here, to extract a reliable and accurate mean transition time from a series of short time traces with ultraslow kinetics, we suggest a scheme called sHaRPer (serialized Handshaking Repeated Permutation with end removal) that concatenates multiple time traces. Because data acquisition frequency f and measurement time (δt) affect the estimation of mean transition time (⟨τ⟩), we provide mathematical criteria that f, δt, and ⟨τ⟩ should satisfy to make ⟨τ⟩ close enough to ⟨τ⟩orig. Although application of the sHaRPer method has a potential risk of distorting the time constants of individual kinetic phases if the data are described with kinetic partitioning, we also provide criteria to avoid such distortion. Our sHaRPer method is a useful way to handle single-molecule data with slow transition kinetics. This study provides a practical guide to use sHaRPer.