William Greenleaf is an Assistant Professor in the Genetics Department at Stanford. Efforts in his lab are split between building new tools to leverage the power of high-throughput sequencing and cutting-edge microscopies, and bringing these new technologies to bear against basic biological questions of genomic and epigenomic variation. Will earned his PhD in the Applied Physics Department at Stanford in the lab of Steve Block investigating the single molecule biophysics of transcription. As a postdoctoral fellow in the lab of Sunney Xie in the Chemistry and Chemical Biology Department at Harvard he was awarded a Damon Runyon Cancer Research Fellowship. He started his lab at Stanford in November 2011, and recently was named a Rita Allen Foundation Scholar.
Honors & Awards
Rita Allen Scholar, Rita Allen Foundation (2011-)
Damon Runyon Cancer Research Fellowship, Damon Runyon Foundation (2009-2011)
ARCS Fellowship, ARCS (2006)
Gates Cambridge Trust Scholar, Gates Foundation (2002-2003)
Graduate Fellowship, National Science Foundation (2003-2005)
Postdoctoral Fellow, Harvard University, Chemistry and Chemical Biology
PhD, Stanford University, Applied Physics (2008)
Dip Comp Sci, Trinity College, Cambridge University, UK, Computer Science (2003)
AB, Harvard University, Physics (2002)
Current Research and Scholarly Interests
Our lab focuses on developing methods to probe the genome and epigenome at the single-cell and single-molecule levels. Our efforts are split between building new tools to leverage the power of high-throughput sequencing and cutting-edge microscopies, and bringing these new technologies to bear against basic biological questions of genomic and epigenomic variation.
- Advanced Imaging Lab in Biophysics
APPPHYS 232, BIO 132, BIO 232, BIOPHYS 232, MCP 232 (Spr)
- Genetics and Developmental Biology Training Camp
GENE 200 (Aut)
- Next Generation Sequencing and Applications
BIOS 201 (Win)
Independent Studies (10)
- Biomedical Informatics Teaching Methods
BIOMEDIN 290 (Aut, Win, Spr)
- Directed Reading and Research
BIOMEDIN 299 (Aut, Win, Spr, Sum)
- Directed Reading in Biophysics
BIOPHYS 399 (Aut, Win, Spr)
- Directed Reading in Genetics
GENE 299 (Aut, Win, Spr, Sum)
- Graduate Research
BIOPHYS 300 (Aut, Win, Spr)
- Graduate Research
GENE 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
BIOMEDIN 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
GENE 370 (Aut, Win, Spr, Sum)
- Supervised Study
GENE 260 (Aut, Win, Spr, Sum)
- Undergraduate Research
GENE 199 (Aut, Win, Spr, Sum)
- Biomedical Informatics Teaching Methods
- Prior Year Courses
Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position
2013; 10 (12): 1213-?
We describe an assay for transposase-accessible chromatin using sequencing (ATAC-seq), based on direct in vitro transposition of sequencing adaptors into native chromatin, as a rapid and sensitive method for integrative epigenomic analysis. ATAC-seq captures open chromatin sites using a simple two-step protocol with 500-50,000 cells and reveals the interplay between genomic locations of open chromatin, DNA-binding proteins, individual nucleosomes and chromatin compaction at nucleotide resolution. We discovered classes of DNA-binding factors that strictly avoided, could tolerate or tended to overlap with nucleosomes. Using ATAC-seq maps of human CD4(+) T cells from a proband obtained on consecutive days, we demonstrated the feasibility of analyzing an individual's epigenome on a timescale compatible with clinical decision-making.
View details for DOI 10.1038/NMETH.2688
View details for Web of Science ID 000327698100025
View details for PubMedID 24097267
- Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries AMERICAN JOURNAL OF HUMAN GENETICS 2013; 93 (5): 852-864
Digital Polymerase Chain Reaction in an Array of Femtoliter Polydimethylsiloxane Microreactors
2012; 84 (10): 4262-4266
We developed a simple, compact microfluidic device to perform high dynamic-range digital polymerase chain reaction (dPCR) in an array of isolated 36-femtoliter microreactors. The density of the microreactors exceeded 20000/mm(2). This device, made from polydimethylsiloxane (PDMS), allows the samples to be loaded into all microreactors simultaneously. The microreactors are completely sealed through the deformation of a PDMS membrane. The small volume of the microreactors ensures a compact device with high reaction efficiency and low reagent and sample consumption. Future potential applications of this platform include multicolor dPCR and massively parallel dPCR for next generation sequencing library preparation.
View details for DOI 10.1021/ac300761n
View details for Web of Science ID 000303965500005
View details for PubMedID 22482776
Fluorogenic DNA sequencing in PDMS microreactors
2011; 8 (7): 575-U84
We developed a multiplex sequencing-by-synthesis method combining terminal phosphate-labeled fluorogenic nucleotides (TPLFNs) and resealable polydimethylsiloxane (PDMS) microreactors. In the presence of phosphatase, primer extension by DNA polymerase using nonfluorescent TPLFNs generates fluorophores, which are confined in the microreactors and detected. We immobilized primed DNA templates in the microreactors, then sequentially introduced one of the four identically labeled TPLFNs, sealed the microreactors and recorded a fluorescence image after template-directed primer extension. With cycle times of <10 min, we demonstrate 30 base reads with ?99% raw accuracy. Our 'fluorogenic pyrosequencing' offers benefits of pyrosequencing, such as rapid turnaround, one-color detection and generation of native DNA, along with high detection sensitivity and simplicity of parallelization because simultaneous real-time monitoring of all microreactors is not required.
View details for DOI 10.1038/NMETH.1629
View details for Web of Science ID 000292194500021
View details for PubMedID 21666670
AN OPTICAL APPARATUS FOR ROTATION AND TRAPPING
METHODS IN ENZYMOLOGY, VOL 475: SINGLE MOLECULE TOOLS, PT B
2010; 475: 377-404
We present details of the design, construction, and testing of a single-beam optical tweezers apparatus capable of measuring and exerting torque, as well as force, on microfabricated, optically anisotropic particles (an "optical torque wrench"). The control of angular orientation is achieved by rotating the linear polarization of a trapping laser with an electro-optic modulator (EOM), which affords improved performance over previous designs. The torque imparted to the trapped particle is assessed by measuring the difference between left- and right-circular components of the transmitted light, and constant torque is maintained by feeding this difference signal back into a custom-designed electronic servo loop. The limited angular range of the EOM (+/-180 degrees ) is extended by rapidly reversing the polarization once a threshold angle is reached, enabling the torque clamp to function over unlimited, continuous rotations at high bandwidth. In addition, we developed particles suitable for rotation in this apparatus using microfabrication techniques. Altogether, the system allows for the simultaneous application of forces (approximately 0.1-100 pN) and torques (approximately 1-10,000 pN nm) in the study of biomolecules. As a proof of principle, we demonstrate how our instrument can be used to study the supercoiling of single DNA molecules.
View details for DOI 10.1016/S0076-6879(10)75015-1
View details for Web of Science ID 000280733800015
View details for PubMedID 20627165
Applied force reveals mechanistic and energetic details of transcription termination
2008; 132 (6): 971-982
Transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a GC-rich RNA hairpin followed by a U-rich tract. We used single-molecule techniques to investigate the mechanism by which three representative terminators (his, t500, and tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias translocation did not affect termination efficiency (TE). However, the force-dependent kinetics of release and the force-dependent TE of a mutant imply a forward translocation mechanism for the t500 terminator. Tension on isolated U-tracts induced transcript release in a manner consistent with RNA:DNA hybrid shearing. We deduce that different mechanisms, involving hypertranslocation or shearing, operate at terminators with different U-tracts. Tension applied to RNA at terminators suggests that closure of the final 2-3 hairpin bases destabilizes the hybrid and that competing RNA structures modulate TE. We propose a quantitative, energetic model that predicts the behavior for these terminators and mutant variants.
View details for DOI 10.1016/j.cell.2008.01.027
View details for Web of Science ID 000254273600016
View details for PubMedID 18358810
Direct observation of hierarchical folding in single riboswitch aptamers
2008; 319 (5863): 630-633
Riboswitches regulate genes through structural changes in ligand-binding RNA aptamers. With the use of an optical-trapping assay based on in situ transcription by a molecule of RNA polymerase, single nascent RNAs containing pbuE adenine riboswitch aptamers were unfolded and refolded. Multiple folding states were characterized by means of both force-extension curves and folding trajectories under constant force by measuring the molecular contour length, kinetics, and energetics with and without adenine. Distinct folding steps correlated with the formation of key secondary or tertiary structures and with ligand binding. Adenine-induced stabilization of the weakest helix in the aptamer, the mechanical switch underlying regulatory action, was observed directly. These results provide an integrated view of hierarchical folding in an aptamer, demonstrating how complex folding can be resolved into constituent parts, and supply further insights into tertiary structure formation.
View details for DOI 10.1126/science.1151298
View details for Web of Science ID 000252772000044
View details for PubMedID 18174398
Single-molecule studies of RNA polymerase: Motoring along
ANNUAL REVIEW OF BIOCHEMISTRY
2008; 77: 149-176
Single-molecule techniques have advanced our understanding of transcription by RNA polymerase (RNAP). A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps (OTs) have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNAP identifies a promoter, initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement the knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNAP, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates.
View details for DOI 10.1146/annurev.biochem.77.073106.100741
View details for Web of Science ID 000257596800008
View details for PubMedID 18410247
- High-resolution, single-molecule optical trapping measurements of transcription with basepair accuracy: Instrumentation and methods OPTICAL TRAPPING AND OPTICAL MICROMANIPULATION IV 2007; 6644
High-resolution, single-molecule measurements of biomolecular motion
ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE
2007; 36: 171-190
Many biologically important macromolecules undergo motions that are essential to their function. Biophysical techniques can now resolve the motions of single molecules down to the nanometer scale or even below, providing new insights into the mechanisms that drive molecular movements. This review outlines the principal approaches that have been used for high-resolution measurements of single-molecule motion, including centroid tracking, fluorescence resonance energy transfer, magnetic tweezers, atomic force microscopy, and optical traps. For each technique, the principles of operation are outlined, the capabilities and typical applications are examined, and various practical issues for implementation are considered. Extensions to these methods are also discussed, with an eye toward future application to outstanding biological problems.
View details for DOI 10.1146/annurev.biophys.36.101106.101451
View details for Web of Science ID 000247773000009
View details for PubMedID 17328679
Single-molecule, motion-based DNA sequencing using RNA polymerase
2006; 313 (5788): 801-801
We present a method for sequencing DNA that relies on the motion of single RNA polymerase molecules. When a given nucleotide species limits the rate of transcription, polymerase molecules pause at positions corresponding to the rare base. An ultrastable optical trapping apparatus capable of base pair resolution was used to monitor transcription under limiting amounts of each of the four nucleotide species. From the aligned patterns of pauses recorded from as few as four molecules, we determined the DNA sequence. This proof of principle demonstrates that the motion of a processive nucleic acid enzyme may be used to extract sequence information directly from DNA.
View details for DOI 10.1126/science.1130105
View details for Web of Science ID 000239671300049
View details for PubMedID 16902131
Direct observation of base-pair stepping by RNA polymerase
2005; 438 (7067): 460-465
During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 +/- 0.6 A, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force-velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site.
View details for DOI 10.1038/nature04268
View details for Web of Science ID 000233458200041
View details for PubMedID 16284617
Passive all-optical force clamp for high-resolution laser trapping
PHYSICAL REVIEW LETTERS
2005; 95 (20)
Optical traps are useful for studying the effects of forces on single molecules. Feedback-based force clamps are often used to maintain a constant load, but the response time of the feedback limits bandwidth and can introduce instability. We developed a novel force clamp that operates without feedback, taking advantage of the anharmonic region of the trapping potential where the differential stiffness vanishes. We demonstrate the utility of such a force clamp by measuring the unfolding of DNA hairpins and the effect of trap stiffness on opening distance and transition rates.
View details for DOI 10.1103/PhysRevLett.95.208102
View details for Web of Science ID 000233243500069
View details for PubMedID 16384102
Molecule by molecule, the physics and chemistry of life: SMB 2007.
Interdisciplinary work in the life sciences at the boundaries of biology, chemistry and physics is making enormous strides. This progress was showcased at the recent Single Molecule Biophysics conference.
View details for PubMedID 17372599