Structural basis for conformational switching and GTP loading of the large G protein atlastin.

Atlastin, a member of the dynamin superfamily, is known to catalyse homotypic membrane fusion in the smooth endoplasmic reticulum (ER). Recent studies of atlastin have elucidated key features about its structure and function; however, several mechanistic details, including the catalytic mechanism and GTP hydrolysis-driven conformational changes, are yet to be determined. Here, we present the crystal structures of atlastin-1 bound to GDP·AlF(4)(-) and GppNHp, uncovering an intramolecular arginine finger that stimulates GTP hydrolysis when correctly oriented through rearrangements within the G domain. Utilizing Förster Resonance Energy Transfer, we describe nucleotide binding and hydrolysis-driven conformational changes in atlastin and their sequence. Furthermore, we discovered a nucleotide exchange mechanism that is intrinsic to atlastin's N-terminal domains. Our results indicate that the cytoplasmic domain of atlastin acts as a tether and homotypic interactions are timed by GTP binding and hydrolysis. Perturbation of these mechanisms may be implicated in a group of atlastin-associated hereditary neurodegenerative diseases.

Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions.

The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.

Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells.

Proper placement of epigenetic marks on DNA and histones is fundamental to normal development, and perturbations contribute to a variety of disease states. Combinations of marks act together to control gene expression; therefore, detecting their colocalization is important, but because of technical challenges, such measurements are rarely reported. Instead, measurements of epigenetic marks are typically performed one at a time in a population of cells, and their colocalization is inferred by association. Here, we describe a single-molecule analytical approach that can perform direct detection of multiple epigenetic marks simultaneously and use it to identify mechanisms coordinating placement of three gene silencing marks, trimethylated histone H3 lysine 9, lysine 27 (H3K9me3, H3K27me3), and cytosine methylation (mC), in the normal and cancer genome. We show that H3K9me3 and mC are present together on individual chromatin fragments in mouse embryonic stem cells and that half of the H3K9me3 marks require mC for their placement. In contrast, mC and H3K27me3 coincidence is rare, and in fact, mC antagonizes H3K27me3 in both embryonic stem cells and primary mouse fibroblasts, indicating this antagonism is shared among primary cells. However, upon immortalization or tumorigenic transformation of mouse fibroblasts, mC is required for complete H3K27me3 placement. Importantly, in human promyelocytic cells, H3K27me3 is also dependent on mC. Because aberrant placement of gene silencing marks at tumor suppressor genes contributes to tumor progression, the improper dependency of H3K27me3 by mC in immortalized cells is likely to be fundamental to cancer. Our platform can enable other studies involving coordination of epigenetic marks and leverage efforts to discover disease biomarkers and epigenome-modifying drugs.

High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn device.

We describe a versatile 96-well microplate-based device that utilizes affinity microcolumn chromatography to complement downstream plate-based processing in aptamer selections. This device is reconfigurable and is able to operate in serial and/or parallel mode with up to 96 microcolumns. We demonstrate the utility of this device by simultaneously performing characterizations of target binding using five RNA aptamers and a random library. This was accomplished through 96 total selection tests. Three sets of selections tested the effects of target concentration on aptamer binding compared to the random RNA library using aptamers to the proteins green fluorescent protein (GFP), human heat shock factor 1 (hHSF1), and negative elongation factor E (NELF-E). For all three targets, we found significant effects consistent with steric hindrance with optimum enrichments at predictable target concentrations. In a fourth selection set, we tested the partitioning efficiency and binding specificity of our three proteins' aptamers, as well as two suspected background binding sequences, to eight targets running serially. The targets included an empty microcolumn, three affinity resins, three specific proteins, and a non-specific protein control. The aptamers showed significant enrichments only on their intended targets. Specifically, the hHSF1 and NELF-E aptamers enriched over 200-fold on their protein targets, and the GFP aptamer enriched 750-fold. By utilizing our device's plate-based format with other complementary plate-based systems for all downstream biochemical processes and analysis, high-throughput selections, characterizations, and optimization were performed to significantly reduce the time and cost for completing large-scale aptamer selections.

Multiplexed microcolumn-based process for efficient selection of RNA aptamers.

We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin-a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 10(8) with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.

Rapid prototyping of nanofluidic systems using size-reduced electrospun nanofibers for biomolecular analysis.

Biomolecular transport in nanofluidic confinement offers various means to investigate the behavior of biomolecules in their native aqueous environments, and to develop tools for diverse single-molecule manipulations. Recently, a number of simple nanofluidic fabrication techniques has been demonstrated that utilize electrospun nanofibers as a backbone structure. These techniques are limited by the arbitrary dimension of the resulting nanochannels due to the random nature of electrospinning. Here, a new method for fabricating nanofluidic systems from size-reduced electrospun nanofibers is reported and demonstrated. As it is demonstrated, this method uses the scanned electrospinning technique for generation of oriented sacrificial nanofibers and exposes these nanofibers to harsh, but isotropic etching/heating environments to reduce their cross-sectional dimension. The creation of various nanofluidic systems as small as 20 nm is demonstrated, and practical examples of single biomolecular handling, such as DNA elongation in nanochannels and fluorescence correlation spectroscopic analysis of biomolecules passing through nanochannels, are provided.

Electrophoretic stretching and imaging of single native chromatin fibers in nanoslits.

Stretching single chromosomal DNA fibers in nanofluidic devices has become a valuable tool for studying the genome and more recently the epigenome. Although nanofluidic technology has been extensively used in single molecular DNA analysis, compared to bare DNA, much less work has been done to elongate chromatin, and only a few studies utilize more biologically relevant samples such as native eukaryotic chromatin. Here, we provide a method for stretching and imaging individual chromatin fibers within a micro- and nanofluidic device. This device was used to electrophoretically stretch and image single native chromatin fibers extracted from human cancer cells (HeLa cells) by attaching the chromatin to microspheres held at the entrance of a nanoslit. To further demonstrate the potential of this device in epigenetics, histone modification H3k79me2 was optically detected by fluorescence microscopy.

Stretching of Tethered DNA in Nanoslits.

External forces and confinement are two fundamental and complementary approaches for biopolymer stretching. By employing micro- and nanofluidics, we study the force-extension dynamics by simultaneously applying external forces and confinement to single-DNA molecules. In particular, we apply external electric fields to stretch single DNA molecules that are attached to microspheres anchored at a nanoslit entrance. Using this method, we measure the force-extension relation of tethered DNA and describe this relation with modified wormlike chain models. This allowed experimental validations of several theoretical predictions, including the increase in the global persistence length of confined DNA with increasing degree of confinement and the "confined Pincus" regime in slit confinement.

RAPID-SELEX for RNA aptamers.

Aptamers are high-affinity ligands selected from DNA or RNA libraries via SELEX, a repetitive in vitro process of sequential selection and amplification steps. RNA SELEX is more complicated than DNA SELEX because of the additional transcription and reverse transcription steps. Here, we report a new selection scheme, RAPID-SELEX (RNA Aptamer Isolation via Dual-cycles SELEX), that simplifies this process by systematically skipping unnecessary amplification steps. Using affinity microcolumns, we were able to complete a multiplex selection for protein targets, CHK2 and UBLCP1, in a third of the time required for analogous selections using a conventional SELEX approach. High-throughput sequencing of the enriched pools from both RAPID and SELEX revealed many identical candidate aptamers from the starting pool of 5 × 10(15) sequences. For CHK2, the same sequence was preferentially enriched in both selections as the top candidate and was found to bind to its respective target. These results demonstrate the efficiency and, most importantly, the robustness of our selection scheme. RAPID provides a generalized approach that can be used with any selection technology to accelerate the rate of aptamer discovery, without compromising selection performance.

Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells.

We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for off-chip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level.