The massive parallelization of biological assays and realization of single-molecule resolution have yielded profound advances in the ways that biological systems are characterized and monitored and the way in which biological disorders are treated. Assays are able to interrogate thousands of individual molecules simultaneously, often in real time. In particular, the combination of solid state electronics technologies to biological research applications has provided a number of important advances including, e.g., molecular array technology, i.e., DNA arrays (see, e.g., U.S. Pat. No. 6,261,776), microfluidic chip technologies (see e.g., U.S. Pat. No. 5,976,336), chemically sensitive field effect transistors (ChemFETs), and other valuable sensor technologies.
These biochemical and medical assays often rely on the accurate and precise positioning of individual assay components on a molecular scale. Thousands of nanoscale assays are often patterned on a substrate for macro-manipulation, analysis, and data recording. Accordingly, new tools are needed to arrange and construct assay components with accuracy and precision at a molecular resolution.
Components of Molecular Motors
One of the fundamental processes occurring in biological cells is active transport of individual molecules (e.g., a macromolecule such as a protein or a DNA) on a sub-micrometer scale. The simplest eukaryotic cell contains thousands of components that must be processed, packaged, sorted, and delivered to specific sites at specific times within the cell. These essential transport processes are carried out by motor proteins (e.g., kinesins and dyneins) that travel along microtubules reaching into every corner of the cell. Motor proteins can be conceptualized as biological machines that transduce chemical energy into mechanical forces and motion.
Microtubules, cytoskeletal fibers with a diameter of about 24 nm, have multiple roles in the cell. Bundles of microtubules form cilia and flagella, which are whip-like extensions of the cell membrane that are necessary for sweeping materials across an epithelium and for swimming of sperm, respectively. Marginal bands of microtubules in red blood cells and platelets are important for these cells' pliability. Organelles, membrane vesicles, and proteins are transported in the cell along tracks of microtubules. For example, microtubules run through nerve cell axons, allowing bi-directional transport of materials and membrane vesicles between the cell body and the nerve terminal. Failure to supply the nerve terminal with these vesicles blocks the transmission of neural signals. Microtubules are also critical to chromosomal movement during cell division. Both stable and short-lived populations of microtubules exist in the cell.
Microtubules are polymers of GTP-binding tubulin protein subunits. Each subunit is a heterodimer of alpha-and beta-tubulin, multiple isoforms of which exist. The hydrolysis of GTP is linked to the addition of tubulin subunits at the end of a microtubule. The subunits interact head-to-tail to form protofilaments; the protofilaments interact side-to-side to form a microtubule. A microtubule is polarized, one end ringed with alpha-tubulin (e.g., the “−end”) and the other with beta-tubulin (e.g., the “+end”), and the two ends differ in their rates of assembly. Generally, each microtubule is composed of 13 protofilaments although 11 or 15 protofilament-microtubules are sometimes found. Cilia and flagella contain doublet microtubules.
Methods have been developed for manipulation of microtubules. Microtubules can be routinely reassembled in vitro from tubulin purified from bovine brains. For example, the nucleation, assembly, and disassembly reactions of microtubules have been well characterized in, e.g., L. U. Cassimeris et al., Dynamic Instability of Microtubules, 7 Bioessays 149 (1988). More recently, considerable progress has been made toward producing recombinant tubulin in yeast. See A. Davis et al., Purification and Biochemical Characterization of Tubulin from the Budding Yeast Saccharomyces cerevisiae, 32 Biochemistry 8823 (1993).
The motor protein, kinesin, was discovered in 1985 in squid axoplasm. R. D. Vale et al., Identification of a Novel Force-generating Protein, Kinesin, Involved in Microtubule based Motility, 42 Cell 39-50 (1985). It has been discovered that kinesin is just one member of a very large family of motor proteins. E.g., S. A. Endow, The Emerging Kinesin Family of Microtubule Motor Proteins, 16 Trends Biochem. Sci. 221 (1991); L. S. B. Goldstein, The Kinesin Superfamily: Tails of Functional Redundancy, 1 Trends Cell Biol. 93 (1991); R. J. Stewart et al., Identification and Partial Characterization of Six Members of the Kinesin Superfamily in Drosophila. 88 Proc. Nat'l Acad. Sci. USA 8470 (1991). Other motor proteins include dynein, e.g., M.-G. Li et al., Drosophila Cytoplasmic Dynein, a Microtubule Motor that is Asymmetrically Localized in the Oocyte, 126 J. Cell Biol. 1475-93 (1994), and myosin, e.g., T. Q. P. Uyeda et al, 214 J. Molec. Biol. 699-710 (1990). Kinesin, dynein, and related proteins move along microtubules, whereas myosin moves along actin filaments.
Kinesins are motor proteins that act on microtubules and that typically move toward the +end of the microtubule. The prototypical kinesin molecule is involved in the transport of membrane-bound vesicles and organelles. This function is particularly important for axonal transport in neurons. Kinesin is also important in all cell types for the transport of vesicles from the Golgi complex to the endoplasmic reticulum. This role is critical for maintaining the identity and functionality of these secretory organelles.
Kinesins define a ubiquitous, conserved family of over 50 proteins that can be classified into at least 8 subfamilies based on primary amino acid sequence, domain structure, velocity of movement, and cellular function. (Reviewed in Moore, J. D. and S. A. Endow (1996) Bioessays 18:207-219; and Hoyt, A. M. (1994) Curr. Opin. Cell Biol. 6:63-68.) The prototypical kinesin molecule is a heterotetramer composed of two heavy polypeptide chains (KHCs) and two light polypeptide chains (KLCs). The KHC subunits are typically referred to as “kinesin.” KHC is about 1000 amino acids in length (having a mass of about 120 kDa) and KLC is about 550 amino acids in length (having a mass of about 60 kDa). Two KHCs dimerize to form a rod-shaped molecule with three distinct regions of secondary structure. At one end of the molecule is a globular motor domain that functions in ATP hydrolysis and microtubule binding. Kinesin motor domains are highly conserved and share over 70% identity. Beyond the motor domain is an alpha-helical coiled-coil region that mediates dimerization. At the other end of the molecule is a fan-shaped tail that associates with molecular cargo. The tail is formed by the interaction of the KHC C-termini with the two KLCs.
The kinesin heavy chains comprise three structural domains: (a) an amino-terminal head domain, which contains the sites for ATP and microtubule binding and for motor activity; (b) a middle or stalk domain, which may form an alpha-helical coiled coil that entwines two heavy chains to form a dimer; and (c) a carboxyl-terminal domain, which probably forms a globular tail that interacts with the light chains and possibly with vesicles and organelles. Kinesin and kinesin-like proteins are all related by sequence similarity within an approximately 340-amino acid region of the head domain, but outside of this conserved region they show no sequence similarity.
The motility activity of purified kinesin on microtubules has been demonstrated in vitro. R. D. Vale et al., Identification of a Novel Force-generating Protein, Kinesin, Involved in Microtubule-based Motility, 42 Cell 39-50 (1985). Further, full-length kinesin heavy chain and several types of truncated kinesin heavy chain molecules produced in E. coli are also capable of generating in vitro microtubule motility. J. T. Yang et al., Evidence that the Head of Kinesin is Sufficient for Force Generation and Motility In Vitro, 249 Science 42 (1990); R. J. Stewart et al, Direction of Microtubule Movement is an Intrisic Property of the Motor Domains of Kinesin Heavy Chain and Drosophila NCD Protein, 90 Proc. Nat'l Acad. Sci. USA 5209-13 (1993). The kinesin motor domain has also been shown to retain motor activity in vitro after genetic fusion to several other proteins including spectrin, J. T. Yang et al., The Head of Kinesin is Sufficient for Force Generation and Motility In Vitro, 249 Science 42 (1990), glutathione S-transferase, R. J. Stewart et al., Direction of Microtubule Movement is an Intrinsic Property of the NCD and Kinesin Heavy Chain Motor Domains, 90 Proc. Nat'l Acad. Sci. USA 5209 (1993), and biotin carboxyl carrier protein, E. Berliner, Microtubule Movement by a Biotinated Kinesin Bound to a Streptavidincoated Surface, 269 J Biol. Chem. 8610 (1994).
In addition to kinesins, dyneins are also motor proteins that bind to and act on microtubules and typically move toward the −end of the microtubule. Two classes of dyneins, cytosolic and axonemal, have been identified. Cytosolic dyneins are responsible for translocation of materials along cytoplasmic microtubules, for example, for transport from the nerve terminal to the cell body and transport of endocytic vesicles to lysosomes. As well, viruses often take advantage of cytoplasmic dyneins to be transported to the nucleus and establish a successful infection. Sodeik, B. et al. 136 J. Cell Biol. 1007-21 (1997). Virion proteins of herpes simplex virus 1, for example, interact with the cytoplasmic dynein intermediate chain. Ye, G. J. et al. 74 J. Virol. 1355-63 (2000). Cytoplasmic dyneins are also reported to play a role in mitosis. Axonemal dyneins are responsible for the beating of flagella and cilia. Dynein on one microtubule doublet walks along the adjacent microtubule doublet. This sliding force produces bending that causes the flagellum or cilium to beat. Dyneins have a native mass between 1000 and 2000 kDa and contain either two or three force-producing heads driven by the hydrolysis of ATP. The heads are linked via stalks to a basal domain which is composed of a highly variable number of accessory intermediate and light chains. Cytoplasmic dynein is the largest and most complex of the motor proteins.
Myosins are actin-activated ATPases, found in eukaryotic cells, that couple hydrolysis of ATP with motion. Myosin provides the motor function for muscle contraction and intracellular movements such as phagocytosis and rearrangement of cell contents during mitotic cell division (cytokinesis). The contractile unit of skeletal muscle, termed the sarcomere, consists of highly ordered arrays of thin actin-containing filaments and thick myosin-containing filaments. Crossbridges form between the thick and thin filaments, and the ATP-dependent movement of myosin heads within the thick filaments pulls the thin filaments, shortening the sarcomere and thus the muscle fiber. Myosins are composed of one or two heavy chains and associated light chains. Myosin heavy chains contain an amino-terminal motor or head domain, a neck that is the site of light-chain binding, and a carboxy-terminal tail domain. The tail domains may associate to form an alpha-helical coiled coil. Conventional myosins, such as those found in muscle tissue, are composed of two myosin heavy-chain subunits, each associated with two light-chain subunits that bind at the neck region and play a regulatory role. Unconventional myosins, believed to function in intracellular motion, may contain either one or two heavy chains and associated light chains. There is evidence for about 25 myosin heavy chain genes in vertebrates, more than half of them unconventional. Actin is the most abundant intracellular protein in the eukaryotic cell.
Actin filaments interact with myosin in muscles and provide a framework to support the plasma membrane and determine cell shape. In muscle cells, thin filaments containing actin slide past thick filaments containing the motor protein myosin during contraction. Microfilaments are the polymerized form of actin and are vital to cell locomotion, cell shape, cell adhesion, cell division, and muscle contraction. Assembly and disassembly of the microfilaments allow cells to change their morphology. Human cells contain six isoforms of actin. The three alpha-actins are found in different kinds of muscle, nonmuscle beta-actin, and nonmuscle gamma-actin are found in nonmuscle cells, and another gamma-actin is found in intestinal smooth muscle cells. G-actin, the monomeric form of actin, polymerizes into polarized, helical F-actin filaments, accompanied by the hydrolysis of ATP to ADP. A family of actin-related proteins exist that are not part of the actin cytoskeleton, but rather associate with microtubules and dynein.
Zero Mode Waveguides
In some assays, molecules are confined in a series, array, or other arrangement of small holes, pores, or wells, for example, a zero mode waveguide (ZMW). ZMW arrays have been applied to a range of biochemical analyses and have found particular usefulness for genetic analysis. ZMWs typically comprise a nanoscale core, well, or opening disposed in an opaque cladding layer that is disposed upon a transparent substrate, e.g., a circular hole in an aluminum cladding film deposited on a clear silica substrate. J. Korlach et al., Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures. 105 PNAS 1176-81 (2008). A typical ZMW hole is ˜70 nm in diameter and ˜100 nm in depth. ZMW technology allows the sensitive analysis of single molecules because, as light travels through a small aperture, the optical field decays exponentially inside the chamber. That is, due to the narrow dimensions of the well, electromagnetic radiation that is of a frequency above a particular cut-off frequency will be prevented from propagating all the way through the core. Notwithstanding the foregoing, the radiation will penetrate a limited distance into the core, providing a very small illuminated volume within the core. By illuminating a very small volume, one can potentially interrogate very small quantities of reagents, including, e.g., single molecule reactions. The observation volume within an illuminated ZMW is ˜20 zeptoliters (20×10−21 liters). Within this volume, the activity of DNA polymerase incorporating a single nucleotide can be readily detected.
By monitoring reactions at the single molecule level, one can precisely identify and/or monitor a given reaction. The technology is not limited in the types of single molecule interactions that can be observed (e.g., a non-limiting list is protein-protein, protein-DNA, DNA-DNA, DNA-RNA, RNA-RNA, protein-RNA, lipid-lipid, protein-lipid, enzyme-substrate, enzyme-intermediate, enzyme-product, enzyme-metabolite, enzyme-cofactor, enzyme-inhibitor, etc.). In particular, the technology is the basis for a particularly promising field of single molecule DNA sequencing technology that monitors the molecule-by-molecule (e.g., nucleotide-by-nucleotide) synthesis of a DNA strand in a template-dependent fashion by a single polymerase enzyme (e.g., Single Molecule Real Time (SMRT) DNA Sequencing as performed, e.g., by a Pacific Biosciences RS Sequencer (Pacific Biosciences, Menlo Park, Calif.)). See, e.g., U.S. Pat. Nos. 7,476,503; 7,486,865; 7,907,800; and 7,170,050; and U.S. patent application Ser. Nos. 12/553,478, 12/767,673; 12/814,075; 12/413,258; and 12/413,466, each incorporated herein by reference in its entirety for all purposes. See also, Eid, J. et al. 2009. “Real-time DNA sequencing from single polymerase molecules”, 323 Science: 133-38 (2009); Korlach, J. et al. “Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides”, 27 Nucleosides, Nucleotides & Nucleic Acids: 1072-82 (2008); Lundquist, P. M. et al., “Parallel confocal detection of single molecules in real time”, 33 Optics Letters: 1026-28 (2008); Korlach, J. et al., “Selective aluminum passivation for targeted immobilization of single dna polymerase molecules in zero-mode waveguide nanostructures”, 105 Proc Natl Acad Sci USA: 1176-81 (2008); Foquet, M. et al., “Improved fabrication of zero-mode waveguides for single-molecule detection”, 103 Journal of Applied Physics (2008); and Levene, M. J. et al. “Zero-mode waveguides for single-molecule analysis at high concentrations”, 299 Science: 682-86 (2003), each incorporated herein by reference in its entirety for all purposes.
In conventional use, placing components in the wells of the ZMW relies on simple diffusion to deliver components (e.g., macromolecules such as DNA polymerase and/or DNA and/or DNA/DNA polymerase complexes) to the desired site (e.g., the bottom of the ZMW well) in the zero mode waveguides. As a result, a significant amount of the macromolecule (e.g., the DNA polymerase/DNA complex) needs to be added to the ZMWs to achieve a critical mass sufficient enough to drive the diffusion of the complexes into the bottom of the wells. This process is not efficient: e.g., only a fraction of the complexes reaches the desired sites in the wells and incubation times are required to position the assay components in the proper sites. Moreover, extensive incubation times (e.g., 4 or more hours) are required to form the complexes to be delivered to the ZMWs.