Xi-Miao Hou

Cisplatin induces loop structures and condensation of single DNA molecules

Structural properties of single λ DNA treated with anti-cancer drug cisplatin were studied with magnetic tweezers and AFM. Under the effect of low-concentration cisplatin, the DNA became more flexible, with the persistence length decreased significantly from ∼52 to 15 nm. At a high drug concentration, a DNA condensation phenomenon was observed. Based on experimental results from both single-molecule and AFM studies, we propose a model to explain this kind of DNA condensation by cisplatin: first, di-adducts induce local distortions of DNA. Next, micro-loops of ∼20 nm appear through distant crosslinks. Then, large aggregates are formed through further crosslinks. Finally, DNA is condensed into a compact globule. Experiments with Pt(dach)Cl2 indicate that oxaliplatin may modify the DNA structures in the same way as cisplatin. The observed loop structure formation of DNA may be an important feature of the effect of platinum anti-cancer drugs that are analogous to cisplatin in structure.

Direct Measurement of Sequential Folding Pathway and Energy Landscape of Human Telomeric G-quadruplex Structures

Single-stranded guanine-rich sequences fold into compact G-quadruplexes. Although G-triplexes have been proposed and demonstrated as intermediates in the folding of G-quadruplexes, there is still a debate on their folding pathways. In this work, we employed magnetic tweezers to investigate the folding kinetics of single human telomeric G-quadruplexes in 100 mM Na(+) buffer. The results are consistent with a model in which the G-triplex is an in-pathway intermediate in the folding of the G-quadruplex. By finely tuning the force exerted on the G-quadruplex, we observed reversible transitions from the G-quadruplex to the G-triplex as well as from the G-triplex to the unfolded coil when the force was increased from 26 to 39 pN. The energy landscape derived from the probability distribution shows clearly that the G-quadruplex goes through an intermediate when it is unfolded, and vice versa.

Optical trapping of individual human immunodeficiency viruses in culture fluid reveals heterogeneity with single-molecule resolution

Optical tweezers use the momentum of photons to trap and manipulate microscopic objects contact-free in three dimensions. Although this technique has been widely used in biology and nanotechnology to study molecular motors, biopolymers and nanostructures, its application in viruses has been very limited largely due to the small size of these nanoparticles. Using optical tweezers that can simultaneously resolve two-photon fluorescence at single-molecule level, here we show that individual HIV-1 can be optically trapped and manipulated, which allows multi-parameter analysis of single virions in culture fluid under native conditions. We show that individual HIV-1 differs in the numbers of envelope glycoproteins by more than one order of magnitude, which implies substantial heterogeneity of these virions in transmission and infection at single-particle level. Analogous to flow cytometry for cells, this fluid-based technique may allow ultrasensitive detection, multi-parameter analysis and sorting of viruses and other nanoparticles in biological fluid with single-molecule resolution.

Comparative Dispersion Studies of Single-Walled Carbon Nanotubes in Aqueous Solution

Single-walled carbon nanotubes (SWCNT) produced by various methods are commercially available but systematic characterization of their dispersion behavior in aqueous solution is rare. Here, we compare the properties of various SWCNT after their dispersion in aqueous solution assisted by DNA oligo. UV-vis-NIR absorbance measurement and atomic force microscopy (AFM) imaging showed marked differences among SWCNT produced from arc-discharge (AD) method, chemical vapor deposition (CVD), and high-pressure carbon monoxide process (HiPCO). To our surprise, the SWCNT produced from AD method showed the highest nanotube purity and the cleanest AFM image, better than HiPCO SWCNT that has been used extensively for biological applications. We also report our systematic studies on optimizing dispersing conditions to maximize SWCNT solubility and remove insoluble materials. We recommend a low power and short time of sonication to disperse SWCNT to preserve their average lengths. These results altogether serve as a future guide for the usage of commercial SWCNT in water-based applications.

BLM unfolds G-quadruplexes in different structural environments through different mechanisms

Mutations in the RecQ DNA helicase gene BLM give rise to Blooms syndrome, which is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition. BLM helicase is highly active in binding and unwinding G-quadruplexes (G4s), which are physiological targets for BLM, as revealed by genome-wide characterizations of gene expression of cells from BS patients. With smFRET assays, we studied the molecular mechanism of BLM-catalyzed G4 unfolding and showed that ATP is required for G4 unfolding. Surprisingly, depending on the molecular environments of G4, BLM unfolds G4 through different mechanisms: unfolding G4 harboring a 3′-ssDNA tail in three discrete steps with unidirectional translocation, and unfolding G4 connected to dsDNA by ssDNA in a repetitive manner in which BLM remains anchored at the ss/dsDNA junction, and G4 was unfolded by reeling in ssDNA. This indicates that one BLM molecule may unfold G4s in different molecular environments through different mechanisms.

Use of tapered amplifier diode laser for biological-friendly high-resolution optical trapping

A 1064 nm laser is commonly used for biological optical trapping. However, it has the problem of generating reactive oxygen species in the presence of a sensitizer, which leads to photo damage in biological samples. Here we constructed optical tweezers using a tapered amplifier diode laser that operates at 830 nm. Compared to a 1064 nm laser, this laser is friendly to live cells, eliminates photo damage associated with reactive oxygen species, and allows simultaneous two-photon fluorescence imaging of green fluorescent proteins in live mammalian cells. All these advantages could significantly benefit future application of this single molecule technique in biological studies.

Single-molecule detection using continuous wave excitation of two-photon fluorescence

Two-photon fluorescence (TPF) is one of the most important discoveries for biological imaging. Although a cw laser is known to excite TPF, its application in TPF imaging has been very limited due to the perceived low efficiency of excitation. Here we directly excited fluorophores with an IR cw laser used for optical trapping and achieved single-molecule fluorescence sensitivity: discrete stepwise photobleaching of enhanced green fluorescent proteins was observed. The single-molecule fluorescence intensity analysis and on-time distribution strongly indicate that a cw laser can generate TPF detectable at the single-molecule level, and thus opens the door to single-molecule TPF imaging using cw lasers.

Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding

Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase’s DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method. We showed that WRN exhibits repetitive DNA unwinding and translocation behaviors on different DNA structures, including forked, overhanging and G-quadruplex-containing DNAs with an apparently limited unwinding processivity. It was further revealed that the repetitive behaviors were caused by reciprocating of WRN along the same ssDNA, rather than by complete dissociation from and rebinding to substrates or by strand switching. The present study sheds new light on the mechanism for WRN functioning.

Involvement of G-triplex and G-hairpin in the multi-pathway folding of human telomeric G-quadruplex

Abstract G-quadruplex (G4) can be formed by G-rich DNA sequences that are widely distributed throughout the human genome. Although G-triplex and G-hairpin have been proposed as G4 folding intermediates, their formation still requires further investigation by experiments. Here, we employed single-molecule FRET to characterize the folding dynamics of G4 from human telomeric sequence. First, we observed four states during G4 folding initially assigned to be anti-parallel G4, G-triplex, G-hairpin and unfolded ssDNA. Then we constructed putative intra-strand G-triplex, G-hairpin structures and confirmed their existences in both NaCl and KCl. Further studies revealed those structures are going through dynamic transitions between different states and show relatively weak dependence on cations, unlike G4. Based on those results and molecular dynamics simulations, we proposed a multi-pathway folding mechanism for human telomeric G4. The present work may shed new light on our current understanding about the existence and stability of G4 intermediate states.

Detection of single fluorescent proteins inside eukaryotic cells using two-photon fluorescence.

Imaging single fluorescent proteins in a live cell is a challenging task because of the strong cellular autofluorescence. Autofluorescence can be minimized by reducing fluorescence excitation volume. Total internal reflection fluorescence (TIRF) microscopy has been routinely used to reduce excitation volume and detect single protein molecules in or close to cell membrane. However, the limited penetration depth of evanescent field excludes imaging of single fluorescent proteins that reside deep inside a eukaryotic cell. Here we report detection of single fluorescent proteins inside eukaryotic cells by two-photon fluorescence (TPF) microscopy. TPF has an excitation volume less than 0.1 femtoliter (fL). Cell autofluorescence under TPF is low and thus enables us to detect single enhanced green fluorescent proteins (EGFP) and single monomeric teal fluorescent proteins (mTFP1.0) that reside several microns deep inside the cell. Discrete stepwise photobleaching of TPF was observed for both proteins inside the cell. Quantitative analysis of single-molecule fluorescence trajectories show that mTFP1.0 is about twofold brighter than EGFP, while its fluorescence on-time before bleaching is about 10 fold shorter. These findings demonstrate the sensitivity of TPF for imaging of eukaryotic cells at single-molecule level and will be useful for measurement of protein stoichiometry inside the cell.