Select recent publications
For the comprehensive list of publications, patents and scholarly works, visit PubMed, bioRxiv and Google Scholar.
2017
Kim, Soohong; Jonghe, Joachim De; Kulesa, Anthony B; Feldman, David; Vatanen, Tommi; Bhattacharyya, Roby P; Berdy, Brittany; Gomez, James; Nolan, Jill; Epstein, Slava; Blainey, Paul C
High-throughput automated microfluidic sample preparation for accurate microbial genomics Journal Article
In: Nat Commun, vol. 8, pp. 13919, 2017, ISSN: 2041-1723.
@article{pmid28128213,
title = {High-throughput automated microfluidic sample preparation for accurate microbial genomics},
author = {Soohong Kim and Joachim De Jonghe and Anthony B Kulesa and David Feldman and Tommi Vatanen and Roby P Bhattacharyya and Brittany Berdy and James Gomez and Jill Nolan and Slava Epstein and Paul C Blainey},
doi = {10.1038/ncomms13919},
issn = {2041-1723},
year = {2017},
date = {2017-01-01},
journal = {Nat Commun},
volume = {8},
pages = {13919},
abstract = {Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications.
2016
Xiong, Kan; Erwin, Graham S; Ansari, Aseem Z; Blainey, Paul C
Sliding on DNA: From Peptides to Small Molecules Journal Article
In: Angew Chem Int Ed Engl, vol. 55, no. 48, pp. 15110–15114, 2016, ISSN: 1521-3773.
@article{pmid27813331,
title = {Sliding on DNA: From Peptides to Small Molecules},
author = {Kan Xiong and Graham S Erwin and Aseem Z Ansari and Paul C Blainey},
doi = {10.1002/anie.201606768},
issn = {1521-3773},
year = {2016},
date = {2016-11-01},
journal = {Angew Chem Int Ed Engl},
volume = {55},
number = {48},
pages = {15110--15114},
abstract = {Many DNA binding proteins utilize one-dimensional (1D) diffusion along DNA to accelerate their DNA target recognition. Although 1D diffusion of proteins along DNA has been studied for decades, a quantitative understanding is only beginning to emerge and few chemical tools are available to apply 1D diffusion as a design principle. Recently, we discovered that peptides can bind and slide along DNA-even transporting cargo along DNA. Such molecules are known as molecular sleds. Here, to advance our understanding of structure-function relationships governing sequence nonspecific DNA interaction of natural molecular sleds and to explore the potential for controlling sliding activity, we test the DNA binding and sliding activities of chemically modified peptides and analogs, and show that synthetic small molecules can slide on DNA. We found new ways to control molecular sled activity, novel small-molecule synthetic sleds, and molecular sled activity in N-methylpyrrole/N-methylimidazole polyamides that helps explain how these molecules locate rare target sites.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Many DNA binding proteins utilize one-dimensional (1D) diffusion along DNA to accelerate their DNA target recognition. Although 1D diffusion of proteins along DNA has been studied for decades, a quantitative understanding is only beginning to emerge and few chemical tools are available to apply 1D diffusion as a design principle. Recently, we discovered that peptides can bind and slide along DNA-even transporting cargo along DNA. Such molecules are known as molecular sleds. Here, to advance our understanding of structure-function relationships governing sequence nonspecific DNA interaction of natural molecular sleds and to explore the potential for controlling sliding activity, we test the DNA binding and sliding activities of chemically modified peptides and analogs, and show that synthetic small molecules can slide on DNA. We found new ways to control molecular sled activity, novel small-molecule synthetic sleds, and molecular sled activity in N-methylpyrrole/N-methylimidazole polyamides that helps explain how these molecules locate rare target sites.
Xu, Liyi; Brito, Ilana L; Alm, Eric J; Blainey, Paul C
Virtual microfluidics for digital quantification and single-cell sequencing Journal Article
In: Nat Methods, vol. 13, no. 9, pp. 759–762, 2016, ISSN: 1548-7105.
@article{pmid27479330,
title = {Virtual microfluidics for digital quantification and single-cell sequencing},
author = {Liyi Xu and Ilana L Brito and Eric J Alm and Paul C Blainey},
doi = {10.1038/nmeth.3955},
issn = {1548-7105},
year = {2016},
date = {2016-09-01},
journal = {Nat Methods},
volume = {13},
number = {9},
pages = {759--762},
abstract = {We have developed hydrogel-based virtual microfluidics as a simple and robust alternative to complex engineered microfluidic systems for the compartmentalization of nucleic acid amplification reactions. We applied in-gel digital multiple displacement amplification (dMDA) to purified DNA templates, cultured bacterial cells and human microbiome samples in the virtual microfluidics system, and demonstrated whole-genome sequencing of single-cell MDA products with excellent coverage uniformity and markedly reduced chimerism compared with products of liquid MDA reactions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We have developed hydrogel-based virtual microfluidics as a simple and robust alternative to complex engineered microfluidic systems for the compartmentalization of nucleic acid amplification reactions. We applied in-gel digital multiple displacement amplification (dMDA) to purified DNA templates, cultured bacterial cells and human microbiome samples in the virtual microfluidics system, and demonstrated whole-genome sequencing of single-cell MDA products with excellent coverage uniformity and markedly reduced chimerism compared with products of liquid MDA reactions.
Blainey, Paul C
Funding: Interdisciplinary challenges Journal Article
In: Nat Microbiol, vol. 1, no. 8, pp. 16118, 2016, ISSN: 2058-5276.
@article{pmid27573117,
title = {Funding: Interdisciplinary challenges},
author = {Paul C Blainey},
doi = {10.1038/nmicrobiol.2016.118},
issn = {2058-5276},
year = {2016},
date = {2016-07-01},
journal = {Nat Microbiol},
volume = {1},
number = {8},
pages = {16118},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Brito, I L; Yilmaz, S; Huang, K; Xu, L; Jupiter, S D; Jenkins, A P; Naisilisili, W; Tamminen, M; Smillie, C S; Wortman, J R; Birren, B W; Xavier, R J; Blainey, P C; Singh, A K; Gevers, D; Alm, E J
Mobile genes in the human microbiome are structured from global to individual scales Journal Article
In: Nature, vol. 535, no. 7612, pp. 435–439, 2016, ISSN: 1476-4687.
@article{pmid27409808,
title = {Mobile genes in the human microbiome are structured from global to individual scales},
author = {I L Brito and S Yilmaz and K Huang and L Xu and S D Jupiter and A P Jenkins and W Naisilisili and M Tamminen and C S Smillie and J R Wortman and B W Birren and R J Xavier and P C Blainey and A K Singh and D Gevers and E J Alm},
doi = {10.1038/nature18927},
issn = {1476-4687},
year = {2016},
date = {2016-07-01},
journal = {Nature},
volume = {535},
number = {7612},
pages = {435--439},
abstract = {Recent work has underscored the importance of the microbiome in human health, and has largely attributed differences in phenotype to differences in the species present among individuals. However, mobile genes can confer profoundly different phenotypes on different strains of the same species. Little is known about the function and distribution of mobile genes in the human microbiome, and in particular whether the gene pool is globally homogenous or constrained by human population structure. Here, we investigate this question by comparing the mobile genes found in the microbiomes of 81 metropolitan North Americans with those of 172 agrarian Fiji islanders using a combination of single-cell genomics and metagenomics. We find large differences in mobile gene content between the Fijian and North American microbiomes, with functional variation that mirrors known dietary differences such as the excess of plant-based starch degradation genes found in Fijian individuals. Notably, we also observed differences between the mobile gene pools of neighbouring Fijian villages, even though microbiome composition across villages is similar. Finally, we observe high rates of recombination leading to individual-specific mobile elements, suggesting that the abundance of some genes may reflect environmental selection rather than dispersal limitation. Together, these data support the hypothesis that human activities and behaviours provide selective pressures that shape mobile gene pools, and that acquisition of mobile genes is important for colonizing specific human populations.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Recent work has underscored the importance of the microbiome in human health, and has largely attributed differences in phenotype to differences in the species present among individuals. However, mobile genes can confer profoundly different phenotypes on different strains of the same species. Little is known about the function and distribution of mobile genes in the human microbiome, and in particular whether the gene pool is globally homogenous or constrained by human population structure. Here, we investigate this question by comparing the mobile genes found in the microbiomes of 81 metropolitan North Americans with those of 172 agrarian Fiji islanders using a combination of single-cell genomics and metagenomics. We find large differences in mobile gene content between the Fijian and North American microbiomes, with functional variation that mirrors known dietary differences such as the excess of plant-based starch degradation genes found in Fijian individuals. Notably, we also observed differences between the mobile gene pools of neighbouring Fijian villages, even though microbiome composition across villages is similar. Finally, we observe high rates of recombination leading to individual-specific mobile elements, suggesting that the abundance of some genes may reflect environmental selection rather than dispersal limitation. Together, these data support the hypothesis that human activities and behaviours provide selective pressures that shape mobile gene pools, and that acquisition of mobile genes is important for colonizing specific human populations.
Xiong, Kan; Blainey, Paul C
Molecular sled sequences are common in mammalian proteins Journal Article
In: Nucleic Acids Res, vol. 44, no. 5, pp. 2266–2273, 2016, ISSN: 1362-4962.
@article{pmid26857546,
title = {Molecular sled sequences are common in mammalian proteins},
author = {Kan Xiong and Paul C Blainey},
doi = {10.1093/nar/gkw035},
issn = {1362-4962},
year = {2016},
date = {2016-03-01},
journal = {Nucleic Acids Res},
volume = {44},
number = {5},
pages = {2266--2273},
abstract = {Recent work revealed a new class of molecular machines called molecular sleds, which are small basic molecules that bind and slide along DNA with the ability to carry cargo along DNA. Here, we performed biochemical and single-molecule flow stretching assays to investigate the basis of sliding activity in molecular sleds. In particular, we identified the functional core of pVIc, the first molecular sled characterized; peptide functional groups that control sliding activity; and propose a model for the sliding activity of molecular sleds. We also observed widespread DNA binding and sliding activity among basic polypeptide sequences that implicate mammalian nuclear localization sequences and many cell penetrating peptides as molecular sleds. These basic protein motifs exhibit weak but physiologically relevant sequence-nonspecific DNA affinity. Our findings indicate that many mammalian proteins contain molecular sled sequences and suggest the possibility that substantial undiscovered sliding activity exists among nuclear mammalian proteins.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Recent work revealed a new class of molecular machines called molecular sleds, which are small basic molecules that bind and slide along DNA with the ability to carry cargo along DNA. Here, we performed biochemical and single-molecule flow stretching assays to investigate the basis of sliding activity in molecular sleds. In particular, we identified the functional core of pVIc, the first molecular sled characterized; peptide functional groups that control sliding activity; and propose a model for the sliding activity of molecular sleds. We also observed widespread DNA binding and sliding activity among basic polypeptide sequences that implicate mammalian nuclear localization sequences and many cell penetrating peptides as molecular sleds. These basic protein motifs exhibit weak but physiologically relevant sequence-nonspecific DNA affinity. Our findings indicate that many mammalian proteins contain molecular sled sequences and suggest the possibility that substantial undiscovered sliding activity exists among nuclear mammalian proteins.
Mangel, Walter F; McGrath, William J; Xiong, Kan; Graziano, Vito; Blainey, Paul C
Molecular sled is an eleven-amino acid vehicle facilitating biochemical interactions via sliding components along DNA Journal Article
In: Nat Commun, vol. 7, pp. 10202, 2016, ISSN: 2041-1723.
@article{pmid26831565,
title = {Molecular sled is an eleven-amino acid vehicle facilitating biochemical interactions via sliding components along DNA},
author = {Walter F Mangel and William J McGrath and Kan Xiong and Vito Graziano and Paul C Blainey},
doi = {10.1038/ncomms10202},
issn = {2041-1723},
year = {2016},
date = {2016-02-01},
journal = {Nat Commun},
volume = {7},
pages = {10202},
abstract = {Recently, we showed the adenovirus proteinase interacts productively with its protein substrates in vitro and in vivo in nascent virus particles via one-dimensional diffusion along the viral DNA. The mechanism by which this occurs has heretofore been unknown. We show sliding of these proteins along DNA occurs on a new vehicle in molecular biology, a 'molecular sled' named pVIc. This 11-amino acid viral peptide binds to DNA independent of sequence. pVIc slides on DNA, exhibiting the fastest one-dimensional diffusion constant, 26±1.8 × 10(6) (bp)(2) s(-1). pVIc is a 'molecular sled,' because it can slide heterologous cargos along DNA, for example, a streptavidin tetramer. Similar peptides, for example, from the C terminus of β-actin or NLSIII of the p53 protein, slide along DNA. Characteristics of the 'molecular sled' in its milieu (virion, nucleus) have implications for how proteins in the nucleus of cells interact and imply a new form of biochemistry, one-dimensional biochemistry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Recently, we showed the adenovirus proteinase interacts productively with its protein substrates in vitro and in vivo in nascent virus particles via one-dimensional diffusion along the viral DNA. The mechanism by which this occurs has heretofore been unknown. We show sliding of these proteins along DNA occurs on a new vehicle in molecular biology, a 'molecular sled' named pVIc. This 11-amino acid viral peptide binds to DNA independent of sequence. pVIc slides on DNA, exhibiting the fastest one-dimensional diffusion constant, 26±1.8 × 10(6) (bp)(2) s(-1). pVIc is a 'molecular sled,' because it can slide heterologous cargos along DNA, for example, a streptavidin tetramer. Similar peptides, for example, from the C terminus of β-actin or NLSIII of the p53 protein, slide along DNA. Characteristics of the 'molecular sled' in its milieu (virion, nucleus) have implications for how proteins in the nucleus of cells interact and imply a new form of biochemistry, one-dimensional biochemistry.
Biteen, Julie S; Blainey, Paul C; Cardon, Zoe G; Chun, Miyoung; Church, George M; Dorrestein, Pieter C; Fraser, Scott E; Gilbert, Jack A; Jansson, Janet K; Knight, Rob; Miller, Jeff F; Ozcan, Aydogan; Prather, Kimberly A; Quake, Stephen R; Ruby, Edward G; Silver, Pamela A; Taha, Sharif; van den Engh, Ger; Weiss, Paul S; Wong, Gerard C L; Wright, Aaron T; Young, Thomas D
Tools for the Microbiome: Nano and Beyond Journal Article
In: ACS Nano, vol. 10, no. 1, pp. 6–37, 2016, ISSN: 1936-086X.
@article{pmid26695070,
title = {Tools for the Microbiome: Nano and Beyond},
author = {Julie S Biteen and Paul C Blainey and Zoe G Cardon and Miyoung Chun and George M Church and Pieter C Dorrestein and Scott E Fraser and Jack A Gilbert and Janet K Jansson and Rob Knight and Jeff F Miller and Aydogan Ozcan and Kimberly A Prather and Stephen R Quake and Edward G Ruby and Pamela A Silver and Sharif Taha and Ger van den Engh and Paul S Weiss and Gerard C L Wong and Aaron T Wright and Thomas D Young},
doi = {10.1021/acsnano.5b07826},
issn = {1936-086X},
year = {2016},
date = {2016-01-01},
journal = {ACS Nano},
volume = {10},
number = {1},
pages = {6--37},
abstract = {The microbiome presents great opportunities for understanding and improving the world around us and elucidating the interactions that compose it. The microbiome also poses tremendous challenges for mapping and manipulating the entangled networks of interactions among myriad diverse organisms. Here, we describe the opportunities, technical needs, and potential approaches to address these challenges, based on recent and upcoming advances in measurement and control at the nanoscale and beyond. These technical needs will provide the basis for advancing the largely descriptive studies of the microbiome to the theoretical and mechanistic understandings that will underpin the discipline of microbiome engineering. We anticipate that the new tools and methods developed will also be more broadly useful in environmental monitoring, medicine, forensics, and other areas.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The microbiome presents great opportunities for understanding and improving the world around us and elucidating the interactions that compose it. The microbiome also poses tremendous challenges for mapping and manipulating the entangled networks of interactions among myriad diverse organisms. Here, we describe the opportunities, technical needs, and potential approaches to address these challenges, based on recent and upcoming advances in measurement and control at the nanoscale and beyond. These technical needs will provide the basis for advancing the largely descriptive studies of the microbiome to the theoretical and mechanistic understandings that will underpin the discipline of microbiome engineering. We anticipate that the new tools and methods developed will also be more broadly useful in environmental monitoring, medicine, forensics, and other areas.
Kimmerling, Robert J; Szeto, Gregory Lee; Li, Jennifer W; Genshaft, Alex S; Kazer, Samuel W; Payer, Kristofor R; de Riba Borrajo, Jacob; Blainey, Paul C; Irvine, Darrell J; Shalek, Alex K; Manalis, Scott R
A microfluidic platform enabling single-cell RNA-seq of multigenerational lineages Journal Article
In: Nat Commun, vol. 7, pp. 10220, 2016, ISSN: 2041-1723.
@article{pmid26732280,
title = {A microfluidic platform enabling single-cell RNA-seq of multigenerational lineages},
author = {Robert J Kimmerling and Gregory Lee Szeto and Jennifer W Li and Alex S Genshaft and Samuel W Kazer and Kristofor R Payer and Jacob de Riba Borrajo and Paul C Blainey and Darrell J Irvine and Alex K Shalek and Scott R Manalis},
doi = {10.1038/ncomms10220},
issn = {2041-1723},
year = {2016},
date = {2016-01-01},
journal = {Nat Commun},
volume = {7},
pages = {10220},
abstract = {We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function--including Granzyme B--are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function--including Granzyme B--are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology.
2014
Vestergaard, Christian L; Blainey, Paul C; Flyvbjerg, Henrik
Optimal estimation of diffusion coefficients from single-particle trajectories Journal Article
In: Phys Rev E Stat Nonlin Soft Matter Phys, vol. 89, no. 2, pp. 022726, 2014, ISSN: 1550-2376.
@article{pmid25353527,
title = {Optimal estimation of diffusion coefficients from single-particle trajectories},
author = {Christian L Vestergaard and Paul C Blainey and Henrik Flyvbjerg},
doi = {10.1103/PhysRevE.89.022726},
issn = {1550-2376},
year = {2014},
date = {2014-02-01},
journal = {Phys Rev E Stat Nonlin Soft Matter Phys},
volume = {89},
number = {2},
pages = {022726},
abstract = {How does one optimally determine the diffusion coefficient of a diffusing particle from a single-time-lapse recorded trajectory of the particle? We answer this question with an explicit, unbiased, and practically optimal covariance-based estimator (CVE). This estimator is regression-free and is far superior to commonly used methods based on measured mean squared displacements. In experimentally relevant parameter ranges, it also outperforms the analytically intractable and computationally more demanding maximum likelihood estimator (MLE). For the case of diffusion on a flexible and fluctuating substrate, the CVE is biased by substrate motion. However, given some long time series and a substrate under some tension, an extended MLE can separate particle diffusion on the substrate from substrate motion in the laboratory frame. This provides benchmarks that allow removal of bias caused by substrate fluctuations in CVE. The resulting unbiased CVE is optimal also for short time series on a fluctuating substrate. We have applied our estimators to human 8-oxoguanine DNA glycolase proteins diffusing on flow-stretched DNA, a fluctuating substrate, and found that diffusion coefficients are severely overestimated if substrate fluctuations are not accounted for.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
How does one optimally determine the diffusion coefficient of a diffusing particle from a single-time-lapse recorded trajectory of the particle? We answer this question with an explicit, unbiased, and practically optimal covariance-based estimator (CVE). This estimator is regression-free and is far superior to commonly used methods based on measured mean squared displacements. In experimentally relevant parameter ranges, it also outperforms the analytically intractable and computationally more demanding maximum likelihood estimator (MLE). For the case of diffusion on a flexible and fluctuating substrate, the CVE is biased by substrate motion. However, given some long time series and a substrate under some tension, an extended MLE can separate particle diffusion on the substrate from substrate motion in the laboratory frame. This provides benchmarks that allow removal of bias caused by substrate fluctuations in CVE. The resulting unbiased CVE is optimal also for short time series on a fluctuating substrate. We have applied our estimators to human 8-oxoguanine DNA glycolase proteins diffusing on flow-stretched DNA, a fluctuating substrate, and found that diffusion coefficients are severely overestimated if substrate fluctuations are not accounted for.
Bennett, Robert D; Ysasi, Alexandra B; Belle, Janeil M; Wagner, Willi L; Konerding, Moritz A; Blainey, Paul C; Pyne, Saumyadipta; Mentzer, Steven J
Laser microdissection of the alveolar duct enables single-cell genomic analysis Journal Article
In: Front Oncol, vol. 4, pp. 260, 2014, ISSN: 2234-943X.
@article{pmid25309876,
title = {Laser microdissection of the alveolar duct enables single-cell genomic analysis},
author = {Robert D Bennett and Alexandra B Ysasi and Janeil M Belle and Willi L Wagner and Moritz A Konerding and Paul C Blainey and Saumyadipta Pyne and Steven J Mentzer},
doi = {10.3389/fonc.2014.00260},
issn = {2234-943X},
year = {2014},
date = {2014-01-01},
journal = {Front Oncol},
volume = {4},
pages = {260},
abstract = {Complex tissues such as the lung are composed of structural hierarchies such as alveoli, alveolar ducts, and lobules. Some structural units, such as the alveolar duct, appear to participate in tissue repair as well as the development of bronchioalveolar carcinoma. Here, we demonstrate an approach to conduct laser microdissection of the lung alveolar duct for single-cell PCR analysis. Our approach involved three steps. (1) The initial preparation used mechanical sectioning of the lung tissue with sufficient thickness to encompass the structure of interest. In the case of the alveolar duct, the precision-cut lung slices were 200 μm thick; the slices were processed using near-physiologic conditions to preserve the state of viable cells. (2) The lung slices were examined by transmission light microscopy to target the alveolar duct. The air-filled lung was sufficiently accessible by light microscopy that counterstains or fluorescent labels were unnecessary to identify the alveolar duct. (3) The enzymatic and microfluidic isolation of single cells allowed for the harvest of as few as several thousand cells for PCR analysis. Microfluidics based arrays were used to measure the expression of selected marker genes in individual cells to characterize different cell populations. Preliminary work suggests the unique value of this approach to understand the intra- and intercellular interactions within the regenerating alveolar duct.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Complex tissues such as the lung are composed of structural hierarchies such as alveoli, alveolar ducts, and lobules. Some structural units, such as the alveolar duct, appear to participate in tissue repair as well as the development of bronchioalveolar carcinoma. Here, we demonstrate an approach to conduct laser microdissection of the lung alveolar duct for single-cell PCR analysis. Our approach involved three steps. (1) The initial preparation used mechanical sectioning of the lung tissue with sufficient thickness to encompass the structure of interest. In the case of the alveolar duct, the precision-cut lung slices were 200 μm thick; the slices were processed using near-physiologic conditions to preserve the state of viable cells. (2) The lung slices were examined by transmission light microscopy to target the alveolar duct. The air-filled lung was sufficiently accessible by light microscopy that counterstains or fluorescent labels were unnecessary to identify the alveolar duct. (3) The enzymatic and microfluidic isolation of single cells allowed for the harvest of as few as several thousand cells for PCR analysis. Microfluidics based arrays were used to measure the expression of selected marker genes in individual cells to characterize different cell populations. Preliminary work suggests the unique value of this approach to understand the intra- and intercellular interactions within the regenerating alveolar duct.
Blainey, Paul C; Quake, Stephen R
Dissecting genomic diversity, one cell at a time Journal Article
In: Nat Methods, vol. 11, no. 1, pp. 19–21, 2014, ISSN: 1548-7105.
@article{pmid24524132,
title = {Dissecting genomic diversity, one cell at a time},
author = {Paul C Blainey and Stephen R Quake},
doi = {10.1038/nmeth.2783},
issn = {1548-7105},
year = {2014},
date = {2014-01-01},
journal = {Nat Methods},
volume = {11},
number = {1},
pages = {19--21},
abstract = {Emerging technologies are bringing single-cell genome sequencing into the mainstream; this field has already yielded insights into the genetic architecture and variability between cells that highlight the dynamic nature of the genome.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Emerging technologies are bringing single-cell genome sequencing into the mainstream; this field has already yielded insights into the genetic architecture and variability between cells that highlight the dynamic nature of the genome.
2013
Blainey, Paul C
The future is now: single-cell genomics of bacteria and archaea Journal Article
In: FEMS Microbiol Rev, vol. 37, no. 3, pp. 407–427, 2013, ISSN: 1574-6976.
@article{pmid23298390,
title = {The future is now: single-cell genomics of bacteria and archaea},
author = {Paul C Blainey},
doi = {10.1111/1574-6976.12015},
issn = {1574-6976},
year = {2013},
date = {2013-05-01},
journal = {FEMS Microbiol Rev},
volume = {37},
number = {3},
pages = {407--427},
abstract = {Interest in the expanding catalog of uncultivated microorganisms, increasing recognition of heterogeneity among seemingly similar cells, and technological advances in whole-genome amplification and single-cell manipulation are driving considerable progress in single-cell genomics. Here, the spectrum of applications for single-cell genomics, key advances in the development of the field, and emerging methodology for single-cell genome sequencing are reviewed by example with attention to the diversity of approaches and their unique characteristics. Experimental strategies transcending specific methodologies are identified and organized as a road map for future studies in single-cell genomics of environmental microorganisms. Over the next decade, increasingly powerful tools for single-cell genome sequencing and analysis will play key roles in accessing the genomes of uncultivated organisms, determining the basis of microbial community functions, and fundamental aspects of microbial population biology.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Interest in the expanding catalog of uncultivated microorganisms, increasing recognition of heterogeneity among seemingly similar cells, and technological advances in whole-genome amplification and single-cell manipulation are driving considerable progress in single-cell genomics. Here, the spectrum of applications for single-cell genomics, key advances in the development of the field, and emerging methodology for single-cell genome sequencing are reviewed by example with attention to the diversity of approaches and their unique characteristics. Experimental strategies transcending specific methodologies are identified and organized as a road map for future studies in single-cell genomics of environmental microorganisms. Over the next decade, increasingly powerful tools for single-cell genome sequencing and analysis will play key roles in accessing the genomes of uncultivated organisms, determining the basis of microbial community functions, and fundamental aspects of microbial population biology.
Blainey, Paul C; Graziano, Vito; Pérez-Berná, Ana J; McGrath, William J; Flint, S Jane; Martín, Carmen San; Xie, X Sunney; Mangel, Walter F
In: J Biol Chem, vol. 288, no. 3, pp. 2092–2102, 2013, ISSN: 1083-351X.
@article{pmid23043138,
title = {Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: IV. viral proteinase slides along DNA to locate and process its substrates},
author = {Paul C Blainey and Vito Graziano and Ana J Pérez-Berná and William J McGrath and S Jane Flint and Carmen San Martín and X Sunney Xie and Walter F Mangel},
doi = {10.1074/jbc.M112.407460},
issn = {1083-351X},
year = {2013},
date = {2013-01-01},
journal = {J Biol Chem},
volume = {288},
number = {3},
pages = {2092--2102},
abstract = {Precursor proteins used in the assembly of adenovirus virions must be processed by the virally encoded adenovirus proteinase (AVP) before the virus particle becomes infectious. An activated adenovirus proteinase, the AVP-pVIc complex, was shown to slide along viral DNA with an extremely fast one-dimensional diffusion constant, 21.0 ± 1.9 × 10(6) bp(2)/s. In principle, one-dimensional diffusion can provide a means for DNA-bound proteinases to locate and process DNA-bound substrates. Here, we show that this is correct. In vitro, AVP-pVIc complexes processed a purified virion precursor protein in a DNA-dependent reaction; in a quasi in vivo environment, heat-disrupted ts-1 virions, AVP-pVIc complexes processed five different precursor proteins in DNA-dependent reactions. Sliding of AVP-pVIc complexes along DNA illustrates a new biochemical mechanism by which a proteinase can locate its substrates, represents a new paradigm for virion maturation, and reveals a new way of exploiting the surface of DNA.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Precursor proteins used in the assembly of adenovirus virions must be processed by the virally encoded adenovirus proteinase (AVP) before the virus particle becomes infectious. An activated adenovirus proteinase, the AVP-pVIc complex, was shown to slide along viral DNA with an extremely fast one-dimensional diffusion constant, 21.0 ± 1.9 × 10(6) bp(2)/s. In principle, one-dimensional diffusion can provide a means for DNA-bound proteinases to locate and process DNA-bound substrates. Here, we show that this is correct. In vitro, AVP-pVIc complexes processed a purified virion precursor protein in a DNA-dependent reaction; in a quasi in vivo environment, heat-disrupted ts-1 virions, AVP-pVIc complexes processed five different precursor proteins in DNA-dependent reactions. Sliding of AVP-pVIc complexes along DNA illustrates a new biochemical mechanism by which a proteinase can locate its substrates, represents a new paradigm for virion maturation, and reveals a new way of exploiting the surface of DNA.
Graziano, Vito; Luo, Guobin; Blainey, Paul C; Pérez-Berná, Ana J; McGrath, William J; Flint, S Jane; Martín, Carmen San; Xie, X Sunney; Mangel, Walter F
In: J Biol Chem, vol. 288, no. 3, pp. 2068–2080, 2013, ISSN: 1083-351X.
@article{pmid23043137,
title = {Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: II. adenovirus proteinase is activated in an unusual one-dimensional biochemical reaction},
author = {Vito Graziano and Guobin Luo and Paul C Blainey and Ana J Pérez-Berná and William J McGrath and S Jane Flint and Carmen San Martín and X Sunney Xie and Walter F Mangel},
doi = {10.1074/jbc.M112.407312},
issn = {1083-351X},
year = {2013},
date = {2013-01-01},
journal = {J Biol Chem},
volume = {288},
number = {3},
pages = {2068--2080},
abstract = {Late in an adenovirus infection, the viral proteinase (AVP) becomes activated to process virion precursor proteins used in virus assembly. AVP is activated by two cofactors, the viral DNA and pVIc, an 11-amino acid peptide originating from the C terminus of the precursor protein pVI. There is a conundrum in the activation of AVP in that AVP and pVI are sequence-independent DNA-binding proteins with nm equilibrium dissociation constants such that in the virus particle, they are predicted to be essentially irreversibly bound to the viral DNA. Here, we resolve that conundrum by showing that activation of AVP takes place on the one-dimensional contour of DNA. In vitro, pVI, a substrate, slides on DNA via one-dimensional diffusion, D(1) = 1.45 × 10(6) bp(2)/s, until it binds to AVP also on the same DNA molecule. AVP, partially activated by being bound to DNA, excises pVIc, which binds to the AVP molecule that cut it out. pVIc then forms a disulfide bond with AVP forming the fully active AVP-pVIc complex bound to DNA. In vivo, in heat-disrupted immature virus, AVP was also activated by pVI in DNA-dependent reactions. This activation mechanism illustrates a new paradigm for virion maturation and a new way, by sliding on DNA, for bimolecular complexes to form among proteins not involved in DNA metabolism.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Late in an adenovirus infection, the viral proteinase (AVP) becomes activated to process virion precursor proteins used in virus assembly. AVP is activated by two cofactors, the viral DNA and pVIc, an 11-amino acid peptide originating from the C terminus of the precursor protein pVI. There is a conundrum in the activation of AVP in that AVP and pVI are sequence-independent DNA-binding proteins with nm equilibrium dissociation constants such that in the virus particle, they are predicted to be essentially irreversibly bound to the viral DNA. Here, we resolve that conundrum by showing that activation of AVP takes place on the one-dimensional contour of DNA. In vitro, pVI, a substrate, slides on DNA via one-dimensional diffusion, D(1) = 1.45 × 10(6) bp(2)/s, until it binds to AVP also on the same DNA molecule. AVP, partially activated by being bound to DNA, excises pVIc, which binds to the AVP molecule that cut it out. pVIc then forms a disulfide bond with AVP forming the fully active AVP-pVIc complex bound to DNA. In vivo, in heat-disrupted immature virus, AVP was also activated by pVI in DNA-dependent reactions. This activation mechanism illustrates a new paradigm for virion maturation and a new way, by sliding on DNA, for bimolecular complexes to form among proteins not involved in DNA metabolism.
Graziano, Vito; McGrath, William J; Suomalainen, Maarit; Greber, Urs F; Freimuth, Paul; Blainey, Paul C; Luo, Guobin; Xie, X Sunney; Mangel, Walter F
In: J Biol Chem, vol. 288, no. 3, pp. 2059–2067, 2013, ISSN: 1083-351X.
@article{pmid23043136,
title = {Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: I. binding to DNA AND to hexon of the precursor to protein VI, pVI, of human adenovirus},
author = {Vito Graziano and William J McGrath and Maarit Suomalainen and Urs F Greber and Paul Freimuth and Paul C Blainey and Guobin Luo and X Sunney Xie and Walter F Mangel},
doi = {10.1074/jbc.M112.377150},
issn = {1083-351X},
year = {2013},
date = {2013-01-01},
journal = {J Biol Chem},
volume = {288},
number = {3},
pages = {2059--2067},
abstract = {The precursor to adenovirus protein VI, pVI, is a multifunctional protein with different roles early and late in virus infection. Here, we focus on two roles late in infection, binding of pVI to DNA and to the major capsid protein hexon. pVI bound to DNA as a monomer independent of DNA sequence with an apparent equilibrium dissociation constant, K(d)((app)), of 46 nm. Bound to double-stranded DNA, one molecule of pVI occluded 8 bp. Upon the binding of pVI to DNA, three sodium ions were displaced from the DNA. A ΔG(0)(0) of -4.54 kcal/mol for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy resulted from nonspecific interactions between pVI and DNA. The proteolytically processed, mature form of pVI, protein VI, also bound to DNA; its K(d)((app)) was much higher, 307 nm. The binding assays were performed in 1 mm MgCl(2) because in the absence of magnesium, the binding to pVI or protein VI to DNA was too tight to determine a K(d)((app)). Three molecules of pVI bound to one molecule of the hexon trimer with an equilibrium dissociation constant K(d)((app)) of 1.1 nm.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The precursor to adenovirus protein VI, pVI, is a multifunctional protein with different roles early and late in virus infection. Here, we focus on two roles late in infection, binding of pVI to DNA and to the major capsid protein hexon. pVI bound to DNA as a monomer independent of DNA sequence with an apparent equilibrium dissociation constant, K(d)((app)), of 46 nm. Bound to double-stranded DNA, one molecule of pVI occluded 8 bp. Upon the binding of pVI to DNA, three sodium ions were displaced from the DNA. A ΔG(0)(0) of -4.54 kcal/mol for the nonelectrostatic free energy of binding indicated that a substantial component of the binding free energy resulted from nonspecific interactions between pVI and DNA. The proteolytically processed, mature form of pVI, protein VI, also bound to DNA; its K(d)((app)) was much higher, 307 nm. The binding assays were performed in 1 mm MgCl(2) because in the absence of magnesium, the binding to pVI or protein VI to DNA was too tight to determine a K(d)((app)). Three molecules of pVI bound to one molecule of the hexon trimer with an equilibrium dissociation constant K(d)((app)) of 1.1 nm.
Landry, Zachary C; Giovanonni, Stephen J; Quake, Stephen R; Blainey, Paul C
Optofluidic cell selection from complex microbial communities for single-genome analysis Journal Article
In: Methods Enzymol, vol. 531, pp. 61–90, 2013, ISSN: 1557-7988.
@article{pmid24060116,
title = {Optofluidic cell selection from complex microbial communities for single-genome analysis},
author = {Zachary C Landry and Stephen J Giovanonni and Stephen R Quake and Paul C Blainey},
doi = {10.1016/B978-0-12-407863-5.00004-6},
issn = {1557-7988},
year = {2013},
date = {2013-01-01},
journal = {Methods Enzymol},
volume = {531},
pages = {61--90},
abstract = {Genetic analysis of single cells is emerging as a powerful approach for studies of heterogeneous cell populations. Indeed, the notion of homogeneous cell populations is receding as approaches to resolve genetic and phenotypic variation between single cells are applied throughout the life sciences. A key step in single-cell genomic analysis today is the physical isolation of individual cells from heterogeneous populations, particularly microbial populations, which often exhibit high diversity. Here, we detail the construction and use of instrumentation for optical trapping inside microfluidic devices to select individual cells for analysis by methods including nucleic acid sequencing. This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Genetic analysis of single cells is emerging as a powerful approach for studies of heterogeneous cell populations. Indeed, the notion of homogeneous cell populations is receding as approaches to resolve genetic and phenotypic variation between single cells are applied throughout the life sciences. A key step in single-cell genomic analysis today is the physical isolation of individual cells from heterogeneous populations, particularly microbial populations, which often exhibit high diversity. Here, we detail the construction and use of instrumentation for optical trapping inside microfluidic devices to select individual cells for analysis by methods including nucleic acid sequencing. This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy.
Dodsworth, Jeremy A; Blainey, Paul C; Murugapiran, Senthil K; Swingley, Wesley D; Ross, Christian A; Tringe, Susannah G; Chain, Patrick S G; Scholz, Matthew B; Lo, Chien-Chi; Raymond, Jason; Quake, Stephen R; Hedlund, Brian P
Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage Journal Article
In: Nat Commun, vol. 4, pp. 1854, 2013, ISSN: 2041-1723.
@article{pmid23673639,
title = {Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage},
author = {Jeremy A Dodsworth and Paul C Blainey and Senthil K Murugapiran and Wesley D Swingley and Christian A Ross and Susannah G Tringe and Patrick S G Chain and Matthew B Scholz and Chien-Chi Lo and Jason Raymond and Stephen R Quake and Brian P Hedlund},
doi = {10.1038/ncomms2884},
issn = {2041-1723},
year = {2013},
date = {2013-01-01},
journal = {Nat Commun},
volume = {4},
pages = {1854},
abstract = {OP9 is a yet-uncultivated bacterial lineage found in geothermal systems, petroleum reservoirs, anaerobic digesters and wastewater treatment facilities. Here we use single-cell and metagenome sequencing to obtain two distinct, nearly complete OP9 genomes, one constructed from single cells sorted from hot spring sediments and the other derived from binned metagenomic contigs from an in situ-enriched cellulolytic, thermophilic community. Phylogenomic analyses support the designation of OP9 as a candidate phylum for which we propose the name 'Atribacteria'. Although a plurality of predicted proteins is most similar to those from Firmicutes, the presence of key genes suggests a diderm cell envelope. Metabolic reconstruction from the core genome suggests an anaerobic lifestyle based on sugar fermentation by Embden-Meyerhof glycolysis with production of hydrogen, acetate and ethanol. Putative glycohydrolases and an endoglucanase may enable catabolism of (hemi)cellulose in thermal environments. This study lays a foundation for understanding the physiology and ecological role of the 'Atribacteria'.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
OP9 is a yet-uncultivated bacterial lineage found in geothermal systems, petroleum reservoirs, anaerobic digesters and wastewater treatment facilities. Here we use single-cell and metagenome sequencing to obtain two distinct, nearly complete OP9 genomes, one constructed from single cells sorted from hot spring sediments and the other derived from binned metagenomic contigs from an in situ-enriched cellulolytic, thermophilic community. Phylogenomic analyses support the designation of OP9 as a candidate phylum for which we propose the name 'Atribacteria'. Although a plurality of predicted proteins is most similar to those from Firmicutes, the presence of key genes suggests a diderm cell envelope. Metabolic reconstruction from the core genome suggests an anaerobic lifestyle based on sugar fermentation by Embden-Meyerhof glycolysis with production of hydrogen, acetate and ethanol. Putative glycohydrolases and an endoglucanase may enable catabolism of (hemi)cellulose in thermal environments. This study lays a foundation for understanding the physiology and ecological role of the 'Atribacteria'.
2012
Marshall, Ian P G; Blainey, Paul C; Spormann, Alfred M; Quake, Stephen R
A Single-cell genome for Thiovulum sp Journal Article
In: Appl Environ Microbiol, vol. 78, no. 24, pp. 8555–8563, 2012, ISSN: 1098-5336.
@article{pmid23023751,
title = {A Single-cell genome for Thiovulum sp},
author = {Ian P G Marshall and Paul C Blainey and Alfred M Spormann and Stephen R Quake},
doi = {10.1128/AEM.02314-12},
issn = {1098-5336},
year = {2012},
date = {2012-12-01},
journal = {Appl Environ Microbiol},
volume = {78},
number = {24},
pages = {8555--8563},
abstract = {We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 μm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 μm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.
Blainey, Paul C; Milla, Carlos E; Cornfield, David N; Quake, Stephen R
Quantitative analysis of the human airway microbial ecology reveals a pervasive signature for cystic fibrosis Journal Article
In: Sci Transl Med, vol. 4, no. 153, pp. 153ra130, 2012, ISSN: 1946-6242.
@article{pmid23019655,
title = {Quantitative analysis of the human airway microbial ecology reveals a pervasive signature for cystic fibrosis},
author = {Paul C Blainey and Carlos E Milla and David N Cornfield and Stephen R Quake},
doi = {10.1126/scitranslmed.3004458},
issn = {1946-6242},
year = {2012},
date = {2012-09-01},
journal = {Sci Transl Med},
volume = {4},
number = {153},
pages = {153ra130},
abstract = {Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the CF transmembrane conductance regulator. Disruption of electrolyte homeostasis at mucosal surfaces leads to severe lung, pancreatic, intestinal, hepatic, and reproductive abnormalities. Loss of lung function as a result of chronic lung disease is the primary cause of death from CF. Using high-throughput sequencing to survey microbes in the sputum of 16 CF patients and 9 control individuals, we identified diverse microbial communities in the healthy samples, contravening conventional wisdom that healthy airways are not significantly colonized. Comparing these communities with those from the CF patients revealed significant differences in microbial ecology, including differential representation of uncultivated phylotypes. Despite patient-specific differences, our analysis revealed a focal microbial profile characteristic of CF. The profile differentiated case and control groups even when classically recognized CF pathogens were excluded. As a control, lung explant tissues were also processed from a group of patients with pulmonary disease. The findings in lung tissue corroborated the presence of taxa identified in the sputum samples. Comparing the sequencing results with clinical data indicated that diminished microbial diversity is associated with severity of pulmonary inflammation within our adult CF cohort.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the CF transmembrane conductance regulator. Disruption of electrolyte homeostasis at mucosal surfaces leads to severe lung, pancreatic, intestinal, hepatic, and reproductive abnormalities. Loss of lung function as a result of chronic lung disease is the primary cause of death from CF. Using high-throughput sequencing to survey microbes in the sputum of 16 CF patients and 9 control individuals, we identified diverse microbial communities in the healthy samples, contravening conventional wisdom that healthy airways are not significantly colonized. Comparing these communities with those from the CF patients revealed significant differences in microbial ecology, including differential representation of uncultivated phylotypes. Despite patient-specific differences, our analysis revealed a focal microbial profile characteristic of CF. The profile differentiated case and control groups even when classically recognized CF pathogens were excluded. As a control, lung explant tissues were also processed from a group of patients with pulmonary disease. The findings in lung tissue corroborated the presence of taxa identified in the sputum samples. Comparing the sequencing results with clinical data indicated that diminished microbial diversity is associated with severity of pulmonary inflammation within our adult CF cohort.
Pamp, Sünje J; Harrington, Eoghan D; Quake, Stephen R; Relman, David A; Blainey, Paul C
Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB) Journal Article
In: Genome Res, vol. 22, no. 6, pp. 1107–1119, 2012, ISSN: 1549-5469.
@article{pmid22434425,
title = {Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB)},
author = {Sünje J Pamp and Eoghan D Harrington and Stephen R Quake and David A Relman and Paul C Blainey},
doi = {10.1101/gr.131482.111},
issn = {1549-5469},
year = {2012},
date = {2012-06-01},
journal = {Genome Res},
volume = {22},
number = {6},
pages = {1107--1119},
abstract = {Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated Candidatus Arthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which "holdfasts" and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences with each other, as well as to recently published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell cycle control and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated Candidatus Arthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which "holdfasts" and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences with each other, as well as to recently published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell cycle control and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract.
2011
Youssef, Noha H; Blainey, Paul C; Quake, Stephen R; Elshahed, Mostafa S
Partial genome assembly for a candidate division OP11 single cell from an anoxic spring (Zodletone Spring, Oklahoma) Journal Article
In: Appl Environ Microbiol, vol. 77, no. 21, pp. 7804–7814, 2011, ISSN: 1098-5336.
@article{pmid21908640,
title = {Partial genome assembly for a candidate division OP11 single cell from an anoxic spring (Zodletone Spring, Oklahoma)},
author = {Noha H Youssef and Paul C Blainey and Stephen R Quake and Mostafa S Elshahed},
doi = {10.1128/AEM.06059-11},
issn = {1098-5336},
year = {2011},
date = {2011-11-01},
journal = {Appl Environ Microbiol},
volume = {77},
number = {21},
pages = {7804--7814},
abstract = {Members of candidate division OP11 are widely distributed in terrestrial and marine ecosystems, yet little information regarding their metabolic capabilities and ecological role within such habitats is currently available. Here, we report on the microfluidic isolation, multiple-displacement-amplification, pyrosequencing, and genomic analysis of a single cell (ZG1) belonging to candidate division OP11. Genome analysis of the ∼270-kb partial genome assembly obtained showed that it had no particular similarity to a specific phylum. Four hundred twenty-three open reading frames were identified, 46% of which had no function prediction. In-depth analysis revealed a heterotrophic lifestyle, with genes encoding endoglucanase, amylopullulanase, and laccase enzymes, suggesting a capacity for utilization of cellulose, starch, and, potentially, lignin, respectively. Genes encoding several glycolysis enzymes as well as formate utilization were identified, but no evidence for an electron transport chain was found. The presence of genes encoding various components of lipopolysaccharide biosynthesis indicates a Gram-negative bacterial cell wall. The partial genome also provides evidence for antibiotic resistance (β-lactamase, aminoglycoside phosphotransferase), as well as antibiotic production (bacteriocin) and extracellular bactericidal peptidases. Multiple mechanisms for stress response were identified, as were elements of type I and type IV secretion systems. Finally, housekeeping genes identified within the partial genome were used to demonstrate the OP11 affiliation of multiple hitherto unclassified genomic fragments from multiple database-deposited metagenomic data sets. These results provide the first glimpse into the lifestyle of a member of a ubiquitous, yet poorly understood bacterial candidate division.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Members of candidate division OP11 are widely distributed in terrestrial and marine ecosystems, yet little information regarding their metabolic capabilities and ecological role within such habitats is currently available. Here, we report on the microfluidic isolation, multiple-displacement-amplification, pyrosequencing, and genomic analysis of a single cell (ZG1) belonging to candidate division OP11. Genome analysis of the ∼270-kb partial genome assembly obtained showed that it had no particular similarity to a specific phylum. Four hundred twenty-three open reading frames were identified, 46% of which had no function prediction. In-depth analysis revealed a heterotrophic lifestyle, with genes encoding endoglucanase, amylopullulanase, and laccase enzymes, suggesting a capacity for utilization of cellulose, starch, and, potentially, lignin, respectively. Genes encoding several glycolysis enzymes as well as formate utilization were identified, but no evidence for an electron transport chain was found. The presence of genes encoding various components of lipopolysaccharide biosynthesis indicates a Gram-negative bacterial cell wall. The partial genome also provides evidence for antibiotic resistance (β-lactamase, aminoglycoside phosphotransferase), as well as antibiotic production (bacteriocin) and extracellular bactericidal peptidases. Multiple mechanisms for stress response were identified, as were elements of type I and type IV secretion systems. Finally, housekeeping genes identified within the partial genome were used to demonstrate the OP11 affiliation of multiple hitherto unclassified genomic fragments from multiple database-deposited metagenomic data sets. These results provide the first glimpse into the lifestyle of a member of a ubiquitous, yet poorly understood bacterial candidate division.
Blainey, Paul C; Quake, Stephen R
Digital MDA for enumeration of total nucleic acid contamination Journal Article
In: Nucleic Acids Res, vol. 39, no. 4, pp. e19, 2011, ISSN: 1362-4962.
@article{pmid21071419,
title = {Digital MDA for enumeration of total nucleic acid contamination},
author = {Paul C Blainey and Stephen R Quake},
doi = {10.1093/nar/gkq1074},
issn = {1362-4962},
year = {2011},
date = {2011-03-01},
journal = {Nucleic Acids Res},
volume = {39},
number = {4},
pages = {e19},
abstract = {Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by φ29 DNA polymerase (DNAP). Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence. We use the new assay to show that our custom φ29 DNAP preparation is free of contamination at the limit of detection of the dMDA assay (1 contaminating molecule per assay microliter). Contamination in commercially available preparations is also investigated. The results of the dMDA assay provide strong evidence that the so-called 'template-independent' MDA background can be attributed to high-molecular weight contaminants and is not primer-derived in the commercial kits tested. dMDA is orders of magnitude more sensitive than PCR-based techniques for detection of microbial genomic DNA fragments and opens up new possibilities for the ultrasensitive quantification of DNA fragments in a wide variety of application areas using MDA chemistry and off-the-shelf hardware developed for digital PCR.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by φ29 DNA polymerase (DNAP). Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence. We use the new assay to show that our custom φ29 DNAP preparation is free of contamination at the limit of detection of the dMDA assay (1 contaminating molecule per assay microliter). Contamination in commercially available preparations is also investigated. The results of the dMDA assay provide strong evidence that the so-called 'template-independent' MDA background can be attributed to high-molecular weight contaminants and is not primer-derived in the commercial kits tested. dMDA is orders of magnitude more sensitive than PCR-based techniques for detection of microbial genomic DNA fragments and opens up new possibilities for the ultrasensitive quantification of DNA fragments in a wide variety of application areas using MDA chemistry and off-the-shelf hardware developed for digital PCR.
Blainey, Paul C; Mosier, Annika C; Potanina, Anastasia; Francis, Christopher A; Quake, Stephen R
Genome of a low-salinity ammonia-oxidizing archaeon determined by single-cell and metagenomic analysis Journal Article
In: PLoS One, vol. 6, no. 2, pp. e16626, 2011, ISSN: 1932-6203.
@article{pmid21364937,
title = {Genome of a low-salinity ammonia-oxidizing archaeon determined by single-cell and metagenomic analysis},
author = {Paul C Blainey and Annika C Mosier and Anastasia Potanina and Christopher A Francis and Stephen R Quake},
doi = {10.1371/journal.pone.0016626},
issn = {1932-6203},
year = {2011},
date = {2011-02-01},
journal = {PLoS One},
volume = {6},
number = {2},
pages = {e16626},
abstract = {Ammonia-oxidizing archaea (AOA) are thought to be among the most abundant microorganisms on Earth and may significantly impact the global nitrogen and carbon cycles. We sequenced the genome of AOA in an enrichment culture from low-salinity sediments in San Francisco Bay using single-cell and metagenomic genome sequence data. Five single cells were isolated inside an integrated microfluidic device using laser tweezers, the cells' genomic DNA was amplified by multiple displacement amplification (MDA) in 50 nL volumes and then sequenced by high-throughput DNA pyrosequencing. This microscopy-based approach to single-cell genomics minimizes contamination and allows correlation of high-resolution cell images with genomic sequences. Statistical properties of coverage across the five single cells, in combination with the contrasting properties of the metagenomic dataset allowed the assembly of a high-quality draft genome. The genome of this AOA, which we designate Candidatus Nitrosoarchaeum limnia SFB1, is ∼1.77 Mb with >2100 genes and a G+C content of 32%. Across the entire genome, the average nucleotide identity to Nitrosopumilus maritimus, the only AOA in pure culture, is ∼70%, suggesting this AOA represents a new genus of Crenarchaeota. Phylogenetically, the 16S rRNA and ammonia monooxygenase subunit A (amoA) genes of this AOA are most closely related to sequences reported from a wide variety of freshwater ecosystems. Like N. maritimus, the low-salinity AOA genome appears to have an ammonia oxidation pathway distinct from ammonia oxidizing bacteria (AOB). In contrast to other described AOA, these low-salinity AOA appear to be motile, based on the presence of numerous motility- and chemotaxis-associated genes in the genome. This genome data will be used to inform targeted physiological and metabolic studies of this novel group of AOA, which may ultimately advance our understanding of AOA metabolism and their impacts on the global carbon and nitrogen cycles.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Ammonia-oxidizing archaea (AOA) are thought to be among the most abundant microorganisms on Earth and may significantly impact the global nitrogen and carbon cycles. We sequenced the genome of AOA in an enrichment culture from low-salinity sediments in San Francisco Bay using single-cell and metagenomic genome sequence data. Five single cells were isolated inside an integrated microfluidic device using laser tweezers, the cells' genomic DNA was amplified by multiple displacement amplification (MDA) in 50 nL volumes and then sequenced by high-throughput DNA pyrosequencing. This microscopy-based approach to single-cell genomics minimizes contamination and allows correlation of high-resolution cell images with genomic sequences. Statistical properties of coverage across the five single cells, in combination with the contrasting properties of the metagenomic dataset allowed the assembly of a high-quality draft genome. The genome of this AOA, which we designate Candidatus Nitrosoarchaeum limnia SFB1, is ∼1.77 Mb with >2100 genes and a G+C content of 32%. Across the entire genome, the average nucleotide identity to Nitrosopumilus maritimus, the only AOA in pure culture, is ∼70%, suggesting this AOA represents a new genus of Crenarchaeota. Phylogenetically, the 16S rRNA and ammonia monooxygenase subunit A (amoA) genes of this AOA are most closely related to sequences reported from a wide variety of freshwater ecosystems. Like N. maritimus, the low-salinity AOA genome appears to have an ammonia oxidation pathway distinct from ammonia oxidizing bacteria (AOB). In contrast to other described AOA, these low-salinity AOA appear to be motile, based on the presence of numerous motility- and chemotaxis-associated genes in the genome. This genome data will be used to inform targeted physiological and metabolic studies of this novel group of AOA, which may ultimately advance our understanding of AOA metabolism and their impacts on the global carbon and nitrogen cycles.
2009
Blainey, Paul C; Luo, Guobin; Kou, S C; Mangel, Walter F; Verdine, Gregory L; Bagchi, Biman; Xie, X Sunney
Nonspecifically bound proteins spin while diffusing along DNA Journal Article
In: Nat Struct Mol Biol, vol. 16, no. 12, pp. 1224–1229, 2009, ISSN: 1545-9985.
@article{pmid19898474,
title = {Nonspecifically bound proteins spin while diffusing along DNA},
author = {Paul C Blainey and Guobin Luo and S C Kou and Walter F Mangel and Gregory L Verdine and Biman Bagchi and X Sunney Xie},
doi = {10.1038/nsmb.1716},
issn = {1545-9985},
year = {2009},
date = {2009-12-01},
journal = {Nat Struct Mol Biol},
volume = {16},
number = {12},
pages = {1224--1229},
abstract = {It is known that DNA-binding proteins can slide along the DNA helix while searching for specific binding sites, but their path of motion remains obscure. Do these proteins undergo simple one-dimensional (1D) translational diffusion, or do they rotate to maintain a specific orientation with respect to the DNA helix? We measured 1D diffusion constants as a function of protein size while maintaining the DNA-protein interface. Using bootstrap analysis of single-molecule diffusion data, we compared the results to theoretical predictions for pure translational motion and rotation-coupled sliding along the DNA. The data indicate that DNA-binding proteins undergo rotation-coupled sliding along the DNA helix and can be described by a model of diffusion along the DNA helix on a rugged free-energy landscape. A similar analysis including the 1D diffusion constants of eight proteins of varying size shows that rotation-coupled sliding is a general phenomenon. The average free-energy barrier for sliding along the DNA was 1.1 +/- 0.2 k(B)T. Such small barriers facilitate rapid search for binding sites.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
It is known that DNA-binding proteins can slide along the DNA helix while searching for specific binding sites, but their path of motion remains obscure. Do these proteins undergo simple one-dimensional (1D) translational diffusion, or do they rotate to maintain a specific orientation with respect to the DNA helix? We measured 1D diffusion constants as a function of protein size while maintaining the DNA-protein interface. Using bootstrap analysis of single-molecule diffusion data, we compared the results to theoretical predictions for pure translational motion and rotation-coupled sliding along the DNA. The data indicate that DNA-binding proteins undergo rotation-coupled sliding along the DNA helix and can be described by a model of diffusion along the DNA helix on a rugged free-energy landscape. A similar analysis including the 1D diffusion constants of eight proteins of varying size shows that rotation-coupled sliding is a general phenomenon. The average free-energy barrier for sliding along the DNA was 1.1 +/- 0.2 k(B)T. Such small barriers facilitate rapid search for binding sites.
White, Richard A; Blainey, Paul C; Fan, H Christina; Quake, Stephen R
Digital PCR provides sensitive and absolute calibration for high throughput sequencing Journal Article
In: BMC Genomics, vol. 10, pp. 116, 2009, ISSN: 1471-2164.
@article{pmid19298667,
title = {Digital PCR provides sensitive and absolute calibration for high throughput sequencing},
author = {Richard A White and Paul C Blainey and H Christina Fan and Stephen R Quake},
doi = {10.1186/1471-2164-10-116},
issn = {1471-2164},
year = {2009},
date = {2009-03-01},
journal = {BMC Genomics},
volume = {10},
pages = {116},
abstract = {BACKGROUND: Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing.
RESULTS: We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth.
CONCLUSION: The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing.
RESULTS: We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth.
CONCLUSION: The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.
RESULTS: We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth.
CONCLUSION: The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.
2008
Bagchi, Biman; Blainey, Paul C; Xie, X Sunney
Diffusion constant of a nonspecifically bound protein undergoing curvilinear motion along DNA Journal Article
In: J Phys Chem B, vol. 112, no. 19, pp. 6282–6284, 2008, ISSN: 1520-6106.
@article{pmid18321088,
title = {Diffusion constant of a nonspecifically bound protein undergoing curvilinear motion along DNA},
author = {Biman Bagchi and Paul C Blainey and X Sunney Xie},
doi = {10.1021/jp077568f},
issn = {1520-6106},
year = {2008},
date = {2008-05-01},
journal = {J Phys Chem B},
volume = {112},
number = {19},
pages = {6282--6284},
abstract = {The mechanism of a protein's diffusion along a DNA segment is a subject of much current interest because of the involvement of this diffusion in numerous biological processes, including the recognition of DNA sequences and chemical modifications of DNA. In this work we present a theoretical derivation of the diffusion coefficient of a nonspecifically bound protein, assuming that the protein follows a helical track along the DNA. It is shown that, for protein-sized molecules, the principal contribution to the total translational friction comes from the curvilinear motion along the helix, and this contribution is given by 6pietaRR(oc)(2) + 8pietaR(3), where R is the protein radius, ROC is the distance of separation between the center of mass of the protein and the helical axis of DNA, and eta is the viscosity of the medium. The translational diffusion of the protein along the helical track of DNA is thus predicted to have a nearly R(-3) size dependence, not the R(-1) dependence characterizing simple translational diffusion. It is shown that this expression gives rather good estimates of the translational diffusion coefficient measured in single molecule experiments.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The mechanism of a protein's diffusion along a DNA segment is a subject of much current interest because of the involvement of this diffusion in numerous biological processes, including the recognition of DNA sequences and chemical modifications of DNA. In this work we present a theoretical derivation of the diffusion coefficient of a nonspecifically bound protein, assuming that the protein follows a helical track along the DNA. It is shown that, for protein-sized molecules, the principal contribution to the total translational friction comes from the curvilinear motion along the helix, and this contribution is given by 6pietaRR(oc)(2) + 8pietaR(3), where R is the protein radius, ROC is the distance of separation between the center of mass of the protein and the helical axis of DNA, and eta is the viscosity of the medium. The translational diffusion of the protein along the helical track of DNA is thus predicted to have a nearly R(-3) size dependence, not the R(-1) dependence characterizing simple translational diffusion. It is shown that this expression gives rather good estimates of the translational diffusion coefficient measured in single molecule experiments.
2007
Kim, Sangjin; Blainey, Paul C; Schroeder, Charles M; Xie, X Sunney
Multiplexed single-molecule assay for enzymatic activity on flow-stretched DNA Journal Article
In: Nat Methods, vol. 4, no. 5, pp. 397–399, 2007, ISSN: 1548-7091.
@article{pmid17435763,
title = {Multiplexed single-molecule assay for enzymatic activity on flow-stretched DNA},
author = {Sangjin Kim and Paul C Blainey and Charles M Schroeder and X Sunney Xie},
doi = {10.1038/nmeth1037},
issn = {1548-7091},
year = {2007},
date = {2007-05-01},
journal = {Nat Methods},
volume = {4},
number = {5},
pages = {397--399},
abstract = {We report a single-molecule assay for nucleic-acid enzymes on flow-stretched DNA templates. To facilitate the detection of slow or intermittent enzymatic activities, we developed the assay with 15-nm spatial resolution at a frame rate of 1 Hz and approximately 10 nm mechanical stability over the timescale of hours. With multiplexed data collection, we applied the assay to phi29 DNA polymerase, HIV-1 reverse transcriptase, lambda exonuclease and Escherichia coli RNA polymerase.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We report a single-molecule assay for nucleic-acid enzymes on flow-stretched DNA templates. To facilitate the detection of slow or intermittent enzymatic activities, we developed the assay with 15-nm spatial resolution at a frame rate of 1 Hz and approximately 10 nm mechanical stability over the timescale of hours. With multiplexed data collection, we applied the assay to phi29 DNA polymerase, HIV-1 reverse transcriptase, lambda exonuclease and Escherichia coli RNA polymerase.
2006
Blainey, Paul C; van Oijen, Antoine M; Banerjee, Anirban; Verdine, Gregory L; Xie, X Sunney
A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA Journal Article
In: Proc Natl Acad Sci U S A, vol. 103, no. 15, pp. 5752–5757, 2006, ISSN: 0027-8424.
@article{pmid16585517,
title = {A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA},
author = {Paul C Blainey and Antoine M van Oijen and Anirban Banerjee and Gregory L Verdine and X Sunney Xie},
doi = {10.1073/pnas.0509723103},
issn = {0027-8424},
year = {2006},
date = {2006-04-01},
journal = {Proc Natl Acad Sci U S A},
volume = {103},
number = {15},
pages = {5752--5757},
abstract = {A central mystery in the function of site-specific DNA-binding proteins is the detailed mechanism for rapid location and binding of target sites in DNA. Human oxoguanine DNA glycosylase 1 (hOgg1), for example, must search out rare 8-oxoguanine lesions to prevent transversion mutations arising from oxidative stress. Here we report high-speed imaging of single hOgg1 enzyme molecules diffusing along DNA stretched by shear flow. Salt-concentration-dependent measurements reveal that such diffusion occurs as hOgg1 slides in persistent contact with DNA. At near-physiologic pH and salt concentration, hOgg1 has a subsecond DNA-binding time and slides with a diffusion constant as high as 5 x 10(6) bp(2)/s. Such a value approaches the theoretical upper limit for one-dimensional diffusion and indicates an activation barrier for sliding of only 0.5 kcal/mol (1 kcal = 4.2 kJ). This nearly barrierless Brownian sliding indicates that DNA glycosylases locate lesion bases by a massively redundant search in which the enzyme selectively binds 8-oxoguanine under kinetic control.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A central mystery in the function of site-specific DNA-binding proteins is the detailed mechanism for rapid location and binding of target sites in DNA. Human oxoguanine DNA glycosylase 1 (hOgg1), for example, must search out rare 8-oxoguanine lesions to prevent transversion mutations arising from oxidative stress. Here we report high-speed imaging of single hOgg1 enzyme molecules diffusing along DNA stretched by shear flow. Salt-concentration-dependent measurements reveal that such diffusion occurs as hOgg1 slides in persistent contact with DNA. At near-physiologic pH and salt concentration, hOgg1 has a subsecond DNA-binding time and slides with a diffusion constant as high as 5 x 10(6) bp(2)/s. Such a value approaches the theoretical upper limit for one-dimensional diffusion and indicates an activation barrier for sliding of only 0.5 kcal/mol (1 kcal = 4.2 kJ). This nearly barrierless Brownian sliding indicates that DNA glycosylases locate lesion bases by a massively redundant search in which the enzyme selectively binds 8-oxoguanine under kinetic control.
2003
van Oijen, Antoine M; Blainey, Paul C; Crampton, Donald J; Richardson, Charles C; Ellenberger, Tom; Xie, X Sunney
Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder Journal Article
In: Science, vol. 301, no. 5637, pp. 1235–1238, 2003, ISSN: 1095-9203.
@article{pmid12947199,
title = {Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder},
author = {Antoine M van Oijen and Paul C Blainey and Donald J Crampton and Charles C Richardson and Tom Ellenberger and X Sunney Xie},
doi = {10.1126/science.1084387},
issn = {1095-9203},
year = {2003},
date = {2003-08-01},
journal = {Science},
volume = {301},
number = {5637},
pages = {1235--1238},
abstract = {We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.
2001
Blainey, P C; Reid, P J
FTIR studies of intermolecular hydrogen bonding in halogenated ethanols Journal Article
In: Spectrochim Acta A Mol Biomol Spectrosc, vol. 57, no. 14, pp. 2763–2774, 2001, ISSN: 1386-1425.
@article{pmid11789877,
title = {FTIR studies of intermolecular hydrogen bonding in halogenated ethanols},
author = {P C Blainey and P J Reid},
doi = {10.1016/s1386-1425(01)00507-8},
issn = {1386-1425},
year = {2001},
date = {2001-12-01},
journal = {Spectrochim Acta A Mol Biomol Spectrosc},
volume = {57},
number = {14},
pages = {2763--2774},
abstract = {The effect of halogen substitution on intermolecular hydrogen-bonding in ethanol is studied. Specifically, Fourier-transform infrared (FTIR) spectra of ethanol, 2,2,2-trifluoroethanol (TFE), and 2,2,2-trichloroethanol dissolved in carbon tetrachloride are reported as a function of temperature and concentration. The spectral intensities corresponding to monomer, dimer, and multimer formation are used to determine the effect of halogen substitution on intermolecular hydrogen-bonding. The enthalpy for dimerization was found to evolve from -4.2+/-0.3 kcal/mol in ethanol to -6.8+/-1.0 kcal/mol in TFE. An opposite trend was observed for multimer formation with enthalpies of -3.7+/-0.5 in ethanol and -2.1+/-1.4 kcal/mol in TFE. The majority of this evolution is assigned to the ability of ethanols to form intramolecular hydrogen bonds involving the hydoxyl proton and the halogen substituents.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The effect of halogen substitution on intermolecular hydrogen-bonding in ethanol is studied. Specifically, Fourier-transform infrared (FTIR) spectra of ethanol, 2,2,2-trifluoroethanol (TFE), and 2,2,2-trichloroethanol dissolved in carbon tetrachloride are reported as a function of temperature and concentration. The spectral intensities corresponding to monomer, dimer, and multimer formation are used to determine the effect of halogen substitution on intermolecular hydrogen-bonding. The enthalpy for dimerization was found to evolve from -4.2+/-0.3 kcal/mol in ethanol to -6.8+/-1.0 kcal/mol in TFE. An opposite trend was observed for multimer formation with enthalpies of -3.7+/-0.5 in ethanol and -2.1+/-1.4 kcal/mol in TFE. The majority of this evolution is assigned to the ability of ethanols to form intramolecular hydrogen bonds involving the hydoxyl proton and the halogen substituents.