Molecular Combing Research Articles

Localized micro- and nano-scale remodelling in the diabetic aorta

Diabetes is strongly associated with cardiovascular disease, but the mechanisms, structural and biomechanical consequences of aberrant blood vessel remodelling remain poorly defined. Using an experimental (streptozotocin, STZ) rat model of diabetes, we hypothesized that diabetes enhances extracellular protease activity in the aorta and induces morphological, compositional and localized micromechanical tissue remodelling. We found that the medial aortic layer underwent significant thickening in diabetic animals but without significant changes in collagen or elastin (abundance). Scanning acoustic microscopy demonstrated that such tissue remodelling was associated with a significant decrease in acoustic wave speed (an indicator of reduced material stiffness) in the inter-lamellar spaces of the vessel wall. This index of decreased stiffness was also linked to increased extracellular protease activity (assessed by semi-quantitative in situ gelatin zymography). Such a proteolytically active environment may affect the macromolecular structure of long-lived extracellular matrix molecules. To test this hypothesis, we also characterized the effects of diabetes on the ultrastructure of an important elastic fibre component: the fibrillin microfibril. Using size exclusion chromatography and atomic force microscopy, we isolated and imaged microfibrils from both healthy and diabetic aortas. Microfibrils derived from diabetic tissues were fragmented, morphologically disrupted and weakened (as assessed following molecular combing). These structural and functional abnormalities were not replicated by in vitro glycation. Our data suggest that proteolysis may be a key driver of localized mechanical change in the inter-lamellar space of diabetic rat aortas and that structural proteins (such as fibrillin microfbrils) may be biomarkers of diabetes induced damage.

Direct optical mapping of transcription factor binding sites on field-stretched λ-DNA in nanofluidic devices.

Mapping transcription factor (TF) binding sites along a DNA backbone is crucial in understanding the regulatory circuits that control cellular processes. Here, we deployed a method adopting bioconjugation, nanofluidic confinement and fluorescence single molecule imaging for direct mapping of TF (RNA polymerase) binding sites on field-stretched single DNA molecules. Using this method, we have mapped out five of the TF binding sites of E. coli RNA polymerase to bacteriophage λ-DNA, where two promoter sites and three pseudo-promoter sites are identified with the corresponding binding frequency of 45% and 30%, respectively. Our method is quick, robust and capable of resolving protein-binding locations with high accuracy (∼ 300 bp), making our system a complementary platform to the methods currently practiced. It is advantageous in parallel analysis and less prone to false positive results over other single molecule mapping techniques such as optical tweezers, atomic force microscopy and molecular combing, and could potentially be extended to general mapping of protein–DNA interaction sites.

Graphene Oxide Architectures Prepared by Molecular Combing on Hydrophilic‐Hydrophobic Micropatterns

A novel graphene oxide (GO) architecture is fabricated on hydrophilic-hydrophobic patterned alkanethiol self-assembled monolayers on Au by molecular combing of GO sheets. With hydrazine reduction, the reduced GO architecture-based device is demonstrated to detect NO2 gas. This simple method shows the potential to control the shape, orientation and position of GO sheets over large areas.

General and specific replication profiles are detected in normal human cells by genome-wide and single-locus molecular combing

Mammalian genomes are replicated under a flexible program, with random use of origins and variable fork rates, and many details of the process must be still unraveled. Molecular combing provides a set of direct data regarding the replication profile of eukaryotic cells: fork rates; organization of the replication clusters; proportion of unidirectional forks; and fork dynamics. In this study the replication profiles of different primary and immortalized non-cancer human cells (lymphocytes, lymphoblastoid cells, fibroblasts) were evaluated at the whole-genome level or within reference genomic regions harboring coding genes. It emerged that these different cell types are characterized by specific replication profiles. In primary fibroblasts, a remarkable fraction of the mammalian genome was found to be replicated by unidirectional forks, and interestingly, the proportion of unidirectional forks further increased in the replicating genome along the population divisions. A second difference concerned in the proportion of paused replication forks, again more frequent in primary fibroblasts than in PBL/lymphoblastoid cells. We concluded that these patterns, whose relevance could escape when genomic methods are applied, represent normal replication features. In single-locus analyses, unidirectional and paused replication forks were highly represented in all genomic regions considered with respect to the average estimates referring to the whole-genome. In addition, fork rates were significantly lower than whole-genome estimates. Instead, when considering the specificities of each genomic region investigated (early to late replication, normal or fragile site) no further differentiating features of replication profiles were detected. These data, representing the integration of genome-wide and single-locus analyses, highlight a large heterogeneity of replication profiles among cell types and within the genome, which should be considered for the correct use of replication datasets.

Abstract 578: Plk1 decreases DNA double-strand break (DSB) formation during replication-stalling genotoxic stress.

Abstract Polo-like kinase 1 (Plk1) is a key regulator of mitosis and its over-expression/ deregulation is linked to cellular transformation. Recent studies highlight its role in resumption of cell cycle progression after DNA damage. Plk1 has also been shown to enhance DNA replication under stress of stalled replication forks. We reported Plk1 activation-induced G2/M checkpoint bypass in normal human lung cells (HLFs), in the presence of acute genotoxic stress. The present aim was to determine the effect of Plk1 on DNA double strand breaks (DSBs) during genotoxic stress. In the present study, we measured DNA DSB induction in HLFs by Cr(VI), etoposide, and H2O2 after Plk1 activation using the comet assay and γH2AX immunofluorescence staining. We used equitoxic concentrations of the different genotoxins and found that DNA DSBs increased > 2 fold after exposure to 3 μM Cr(VI), 12.5 μM etoposide, and 150 μM H2O2, respectively, and persisted for at least 4h. Transfection of a Plk1 constitutively active (c/a) mutant (T210D) abrogated both Cr(VI)- and etoposide-induced DNA DSBs as early as 30 min for the duration of treatment, in comparison to the vector-transfected cells. In sharp contrast, Plk1 had no effect on H2O2-induced DNA DSBs. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Moreover, Plk1 enhanced S phase progression during Cr(VI) treatment, which was accompanied by increased replication fork velocity and longer inter-origin distances as determined by using a single-molecule approach based on molecular combing. Expression of a Plk1 “kinase-dead” mutant (K82R) also abrogated DNA DSBs during Cr(VI) treatment, suggesting that this effect may be independent of Plk1 kinase activity. In summary, our data underline the ability of Plk1 to override DNA DSB-induced checkpoints, and to decrease DSBs after replication-stalling genotoxin exposure. We postulate these effects to be due to the replication-enhancing ability of Plk1, which may occur at the expense of genomic stability. This work was supported in part by NIH grants CA107972 and ES017334 (S.C.); ES05304 (S.R.P.); and a PhRMA Foundation predoctoral fellowship (G.C.K.). Citation Format: Daniel Gebreselassie, Gina Chun-Kost, Elisabetta Leo, Yves Pommier, Erika Saito, Steven Patierno, Susan M. Ceryak. Plk1 decreases DNA double-strand break (DSB) formation during replication-stalling genotoxic stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 578. doi:10.1158/1538-7445.AM2013-578

Replication Dynamics: Biases and Robustness of DNA Fiber Analysis

The factors that govern replication programs are still poorly identified in metazoans, especially in mammalian cells. Thanks to molecular combing, the dynamics of DNA replication can be assessed at the genome-scale level from the cumulative analysis of single DNA fibers. This technique notably enables measurement of replication fork speed and fork asymmetry and that of distances separating either initiation or termination events. The results presented here aim to evaluate requirements critical to accurate measurement of replication parameters by molecular combing. We show that sample size, fiber length and DNA counterstaining are crucial to gain robust information concerning replication dynamics. Our results thus provide a methodological frame to investigate the DNA replication program through molecular combing analyses.

Alternate forms of molecular testing

This presentation will describe two groundbreaking techniques validated in our laboratory: Molecular Combing and SOMAmer Diagnosis. Molecular Combing, developed by Genomic Vision, allows the visualisation and size measurement of single DNA molecules for the purposes of molecular diagnostics. I will describe the technology and the validation of a molecular combing for the diagnosis of fascio-scapular-humeral dystrophy (FSHD). Potential future uses of this technology in the hereditary cancer syndromes will also be discussed. Developed by SomaLogic Inc., SOMAmers (slow off-rate modified aptamers) are a new generation of protein-binding aptamers. SOMAmer reagents are nucleic acid molecules of 40-80 bp that are capable of binding to single proteins. Using a proprietary method, high affinity SOMAmer reagents can be isolated for virtually any purified protein. By introducing modified bases, SOMAmer reagents can be selected that bind with high affinity and remain bound to proteins in solution long enough for a quantitative assay. I will discuss the development of the first ever clinical SOMAmer-based assay that measures unbound EGFR in serum. I will present the utility of a SOMAmer-based assay for the purposes of pro-teomics discovery and the translation into a clinical assay.

Molecular Combing of DNA: Methods and Applications

First proposed in 1994, molecular combing of DNA is a technique that allowsadsorption and alignment of DNA on the surface with no need for prior modi-fication of the molecule. Since then, many variations of the original methodhave been devised and used in a wide range of applications from genomicstudies to nanoelectronics. While molecular combing has been applied in avariety of DNA-related studies, no comprehensive review has been publishedon different combing methods proposed so far. In this review, the underly-ing mechanisms of molecular combing of DNA are described followed bydiscussion of the main methods in molecular combing as well as its majorapplications in nanotechnology.

On'mechanism'of'inactivation'of'bio-particles'by'the'plasma'exposure'and'evaluation'of'the'toxicity'using'single'DNA'molecules

NTP can also inactivate viruses. In the previous research, critical damages for the inactivation of λ phage were done on coat proteins and M13 phage on DNA. These results were obtained with wet phage sample [1]. We also tried to use dry sample. We used bacteriophage φX174 which is resistant to drying. When the bacteriophage is inactivated by NTP, the damage should exist either on viral nucleic acids or coat proteins or both. It is possible to extract the nucleic acids from the bacteriophage so that the damage to the DNA can be separately analyzed to exclude the effect of the proteins. The DNA extracted from the plasma-exposed phage can be assayed its plaque forming activity by transfection. The coat proteins were analysed by SDS-PAGE. Using these assays, the damages of viral DNA and coat proteins have been determined separately. The results for the dry φX174 phage inactivated by the exposure to DBD showed both DNA and coat proteins were damaged and the critical damage were done to the coat proteins. This is similar to the result of the wet sample. We also report a single-molecule-based analysis of strand breakages on large DNA molecules induced by the plasma exposure. Single-molecule observation of DNA that involved molecular combing was used to measure the length of individual DNA molecules. The measured DNA length showed that plasma exposure caused a marked change in length of DNA molecules. The rate of plasma-induced strand breakage on large random-coiled DNA molecules was determined using a simple mathematical model. The measured rate shows good relation with the plasma exposure time, and could be used for safety evaluation of the plasma treated water. References

Graphene Oxide Scrolls on Hydrophobic Substrates Fabricated by Molecular Combing and Their Application in Gas Sensing

Well-aligned graphene oxide (GO) scrolls are prepared through the controlled folding/scrolling of single-layer GO sheets using molecular combing on hydrophobic substrates, such as aged gold substrate, polydimethylsiloxane film, poly(L-lactic acid) film, and octadecyltrimethoxysilane-modified silicon dioxide. As a proof of concept, the gas sensor fabricated with a single reduced GO scroll is used to detect NO(2) gas with a concentration as low as 0.4 ppm.

Single DNA Condensation Induced by Hexammine Cobalt with Molecular Combing

We investigated the interaction between DNA and hexammine cobalt III [Co(NH3)6]3+ by a simple molecular combing method and dynamic light scattering. The average extension of λ-DNA-YOYO-1 complex is found to be 20.9 μm, about 30% longer than the contour length of the DNA in TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH=8.0), due to bis-intercalation of YOYO-1. A multivalent cation, hexammine cobalt, is used for DNA condensation. We find that the length of DNA-[Co(NH3)6]3+ complexes decrease from 20.9 μ to 5.9 μ as the concentration of the [Co(NH3)6]3+ vary from 0 to 3 μmol/L. This observation provides a direct visualization of single DNA condensation induced by hexammine cobalt. The results from the molecular combing studies are supported by dynamic light scattering investigation, where the average hydrodynamic radius of the DNA complex decreases from 203.8 nm to 39.26 nm under the same conditions. It shows that the molecular combing method is feasible for quantitative conformation characterization of single bio-macromolecules.

A diagnostic genetic test for the physical mapping of germline rearrangements in the susceptibility breast cancer genes BRCA1 and BRCA2

The BRCA1 and BRCA2 genes are involved in breast and ovarian cancer susceptibility. About 2 to 4% of breast cancer patients with positive family history, negative for point mutations, can be expected to carry large rearrangements in one of these two genes. We developed a novel diagnostic genetic test for the physical mapping of large rearrangements, based on molecular combing (MC), a FISH-based technique for direct visualization of single DNA molecules at high resolution. We designed specific Genomic Morse Codes (GMCs), covering the exons, the noncoding regions, and large genomic portions flanking both genes. We validated our approach by testing 10 index cases with positive family history of breast cancer and 50 negative controls. Large rearrangements, corresponding to deletions and duplications with sizes ranging from 3 to 40 kb, were detected and characterized on both genes, including four novel mutations. The nature of all the identified mutations was confirmed by high-resolution array comparative genomic hybridization (aCGH) and breakpoints characterized by sequencing. The developed GMCs allowed to localize several tandem repeat duplications on both genes. We propose the developed genetic test as a valuable tool to screen large rearrangements in BRCA1 and BRCA2 to be combined in clinical settings with an assay capable of detecting small mutations.

Potentiation of the novel topoisomerase I inhibitor indenoisoquinoline LMP-400 by the cell checkpoint and Chk1-Chk2 inhibitor AZD7762.

Novel topoisomerase I (Top1) inhibitors are in clinical development to circumvent the drawbacks of camptothecins (CPT). Here, we report molecular investigations into LMP-400, an indenoisoquinoline Top1 inhibitor in phase 1 clinical trial, by itself and in combination with the cell-cycle checkpoint inhibitor AZD7762. We examined drug effects on DNA replication and killing of cancer cells and found that LMP-400 showed synergistic antiproliferative activity when combined with AZD7762 in human colon carcinoma cells. Inhibition of S-phase progression and bromodeoxyuridine incorporation were similarly induced by LMP-400 and CPT and were abrogated by AZD7762. Replication studied by single DNA molecule analyses and immunofluorescence microscopy (molecular combing) showed rapid inhibition of fork progression in response to LMP-400 treatment with subsequent recapitulation after AZD7762 addition. AZD7762 inhibited both the activation/autophosphosphorylation of Chk1 and Chk2 at nanomolar concentrations in LMP-400-treated cells. This potent dual inhibition of Chk1 and Chk2 by AZD7762 was below the drug concentrations required to abrogate cell-cycle inhibition and produce synergism with LMP-400. Also, the synergism was independent of Chk2 both in Chk2-complemented cells and Chk2 knockout cells, suggesting additional mechanisms for cell-cycle abrogation by AZD7762. Together, our findings show a rationale for combining cell-cycle checkpoint inhibitors with the novel non-CPT indenoisoquinoline Top1 inhibitors.

Molecular combing in the analysis of developmentally regulated amplified segments of Bradysia hygida

Molecular combing technology is an important new tool for the functional and physical mapping of genome segments. It is designed to identify amplifications, microdeletions, and rearrangements in a DNA sequence and to study the process of DNA replication. This technique has recently been used to identify and analyze the dynamics of replication in amplified domains. In Bradysia hygida, multiple amplification initiation sites are predicted to exist upstream of the BhC4-1 gene. However, it has been impossible to identify them using the available standard techniques. The aim of this study was to optimize molecular combing technology to obtain DNA fibers from the polytene nuclei of the salivary glands of B. hygida to study the dynamics of DNA replication in this organism. Our results suggest that combing this DNA without prior purification of the polytene nuclei is possible. The density, integrity, and linearity of the DNA fibers were analyzed, fibers 50 to 300 kb in length were detected, and a 9-kb fragment within the amplified region was visualized using biotin detected by Alexa Fluor 488-conjugated streptavidin technique. The feasibility of physically mapping these fibers demonstrated in this study suggests that molecular combing may be used to identify the replication origin of the BhC4-1 amplicon.

Local orientational disorder in peptide fibrils probed by a combination of residue-specific 13C–18O labelling, polarised infrared spectroscopy and molecular combing

A novel combination of site-specific isotope labelling, polarised infrared spectroscopy and molecular combing reveals local orientational ordering in the fibril-forming peptide YTIAALLSPYSGGRADS. Use of (13)C-(18)O labelled alanine residues demonstrates that the N-terminal end of the peptide is incorporated into the cross-beta structure, while the C-terminal end shows orientational disorder.

Detection and Damage-analysis of Bio-Particles and for Safety-Evaluation of Plasma-treated water using DNA-manipulation

ABSTRACTEffect of non-thermal plasma (NTP) on bio-particles has been studied using Bacillus subtilis (B. subtilis), Escherichia coli (E. coli) and bacteriophages. NTP has been used, and states of different biological components were monitored during the course of the exposure. Analysis of green fluorescent protein (GFP), introduced into E.coli cells proved that NTP causes a prominent protein damages without cutting peptide bonds. We have developed a biological assay which evaluates in vivo DNA damage of the bacteriophages. Different doses of the plasma were applied to wet state of λ phages. From the plasma-exposed λ phages, DNA was purified and subjected to in vitro DNA packaging reactions. The re-packaged phages consist of the DNA from discharged phages and brand-new coat proteins. Survival curves of the re-packaged phages showed extremely large D value (D = 25 s) compared to the previous D value (D = 3 s) from the discharged phages. The results indicate that DNA damage hardly contributed to the inactivation, and the damage in coat proteins is responsible for inactivation of the phages. We also report a single-molecule-based analysis of strand breakages on large DNA molecules induced by the plasma exposure. Single-molecule observation of DNA that involved molecular combing was used to measure the length of individual DNA molecules. The measured DNA length showed that plasma exposure caused a marked change in length of DNA molecules. The rate of plasmainduced strand breakage on large random-coiled DNA molecules was determined using a simple mathematical model. The measured rate shows good relation with the plasma exposure time, and could be used for safety evaluation of the plasma treated water.

P2-13-10: Detection, Visualization and High Resolution Physical Mapping of Large Rearrangements by Molecular Combing in the Hereditary Breast Cancer Genes BRCA1 and BRCA2.

Abstract The BRCA1 and BRCA2 genes are involved, with high penetrance, in breast and ovarian cancer susceptibility. About 2% to 4% of breast cancer patients with a positive family history who are negative for BRCA1 and BRCA2 point mutations can be expected to carry large genomic alterations (deletion or duplication) in one of the two genes, and especially BRCA1. However, large rearrangements are missed by direct sequencing. Molecular Combing is a powerful FISH-based technique for direct visualization of single DNA molecules, allowing the entire genome to be examined at high resolution in a single analysis. We have developed a novel genetic test based on Molecular Combing. For that purpose, we designed specific BRCA1 and BRCA2 “Genomic Morse Codes” (GMC), also covering the non-coding regions and including large genomic portions flanking both genes. We developed a measurement strategy for the GMC signals, and validated our approach by blindly testing 10 breast cancer patients with a positive family history and 10 control patients. Large rearrangements, corresponding to deletions and duplications of one or several exons and with sizes ranging from 3 kb to 40 kb, were detected on both genes, including the characterization of 4 new mutations (for BRCA1: Del ex 3, Del ex 24 and Dup ex 3; for BRCA2: Dup ex 17–20). The identified mutations confirmed the results obtained with high-resolution zoom-in aCGH (11 k) in the same patients, with a resolution in the 1–2 kb range. Importantly, the developed GMC allowed to unambiguously localize several tandem repeat duplications on both genes, and to precisely map large rearrangements in the problematic Alu-rich 5'-region of BRCA1. We propose the developed Molecular Combing genetic test as a valuable tool for the screening of tandem repeat duplications, CNVs, and other complex rearrangements in BRCA1 and BRCA2, such as translocations and inversions, to be combined in clinical settings with an essay that allows the detection of point mutations. We see the main application of the developed molecular diagnostic tool as a predictive genetic test. However, we envisage to extended the application of the developed tool as a companion diagnostic test, for instance in the screening of BRCA-mutated cells in the context of the development of PARP inhibitors. Thus, the genetic test may be applied not only to clinical blood samples, but also to circulating cells and heterogeneous cell populations, such as tumor tissues. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-13-10.

Abstract C204: Potentiation of the novel topoisomerase I inhibitor indenoisoquinoline LMP-400 by the Chk1/2 inhibitor, AZD7762.

Abstract Novel topoisomerase I (Top1) inhibitors are in clinical development to circumvent the drawbacks of camptothecins (1–3). Here we report molecular investigations of the clinical indenoisoquinoline Top1 inhibitor, LMP-400 by itself and in combination with the cell cycle checkpoint inhibitor, AZD7762 (4). Drug effects on DNA replication and killing of cancer cells were examined. LMP-400 shows synergistic antiproliferative activity when combined with AZD7762 in human colon carcinoma HT-29 and HCT-15 cells. Inhibition of S-phase progression and bromodeoxyuridine incorporation was similarly induced by LMP-400 and CPT and was abrogated by AZD7762. Replication studied by single DNA molecule analyses and immunofluorescence microscopy (molecular combing) (5) showed rapid inhibition of fork progression in response to LMP-400 treatment with subsequent recapitulation after AZD7762 addition. Consistent with cell cycle checkpoint abrogation, AZD7762 inhibited both the activation of Chk1 and Chk2 autophosphorylations in LMP-400-treated cells. In spite of the potent dual inhibition of Chk1 and Chk2 at nanomolar concentrations of AZD7762, cell cycle inhibition and synergism with LMP-400 were only observed at higher concentrations and independently of Chk2 both in Chk2-complemented HT-15 and Chk2 knockout HCT-116 cells. Our study demonstrates the rationale for combining checkpoint kinase inhibitors and the novel non-camptothecin indenoisoquinoline inhibitors.

Single-molecule measurement of strand breaks on large DNA induced by atmospheric pressure plasma jet

We report a single-molecule-based analysis of strand breakages on large DNA molecules induced by an atmospheric pressure plasma jet. We exposed DNA solution to an argon plasma jet; single-molecule observation that involved molecular combing was then used to measure the length of individual DNA molecules. The measured DNA length showed that plasma exposure caused a marked change in length of DNA molecules. The rate of plasma-induced strand breakage on large random-coiled DNA molecules was determined using a simple mathematical model. For strand breaks on large DNA molecules the rate was estimated.

Molecular combing reveals allelic combinations in facioscapulohumeral dystrophy

The genetic variation underlying facioscapulohumeral muscular dystrophy (FSHD), 1 of the most common hereditary neuromuscular disorders, is complex, and associated with the contraction of a repeat array (D4Z4) at the subtelomeric end of chromosome 4q. Nonpathogenic variants of 4q and the presence of a homologous array on chromosome 10q make FSHD diagnosis extremely challenging, at least in individuals with nonstandard D4Z4 arrays. We aimed to improve FSHD molecular analysis by proposing an alternative technique to the Southern blot. We applied molecular combing (MC) to directly visualize allelic combinations associated with FSHD. MC enabled the accurate diagnosis of 32 FSHD patients. Unreported haplotypes and rearrangements, as well as somatic mosaicism, which is common in the 10 to 30% of cases that are sporadic, were detectable by MC. MC enables the detailed exploration of the FSHD locus and accurate diagnosis of FSHD, the first Mendelian disease to benefit from this technique. MC is also likely to be applicable to other copy number-variant or repeat expansion-associated human diseases.