Active microtubule-based separations by kinesins

A method and system for separating a selected molecule from a heterogeneous mixture of molecules in aqueous solution are described. The method comprises (a) providing a separation device comprising a loading reservoir and a receiving reservoir coupled by a channel bearing immobilized microtubules aligned parallel to the longitudinal axis thereof the channel; (b) placing an aqueous solution containing the heterogeneous mixture of molecules in the loading reservoir; (c) adding a motor-ligand composition and ATP to the aqueous solution, wherein the motor-ligand composition comprises a motor protein for attaching to microtubules and moving therealong in the presence of ATP and the ligand is capable of binding the selected molecule, such that the ligand binds the selected molecule to form a complex and the complex moves along the immobilized microtubules to the receiving reservoir; and (d) removing the selected molecule from the receiving chamber.

BACKGROUND OF THE INVENTION 
This invention relates to a method and system for separating a selected 
molecule from a heterogeneous mixture of molecules. More particularly, the 
invention relates to separating a selected molecule from a heterogeneous 
mixture of molecules by reversibly coupling the selected molecule to a 
motor protein such that the motor protein can transport the selected 
molecule away from the heterogeneous mixture by moving on microtubules 
immobilized in a separation device. 
One of the fundamental processes occurring in biological cells is active 
transport on a sub-micrometer scale. The simplest eukaryotic cell contains 
thousands of components that must be processed, packaged, sorted, and 
delivered to specific locations at specific times within the cell. These 
essential transport processes are carried out by motor proteins 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. 
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). In the last 
few years, 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. 1475-1493 (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. It has now become apparent that eukaryotic cells use motor 
proteins to mediate numerous transport requirements. In addition to its 
motor activity, kinesin is also a microtubule-activated adenosine 
triphosphatase (ATPase). 
Kinesin is composed of two heavy chains (each about 120 kDa) and two light 
chains (each about 60 kDa). 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, fulllength 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 42-47 (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 5209-5213 (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 Streptavidin-coated Surface, 269 J. Biol. 
Chem. 8610 (1994). 
Similarly, methods have been developed for manipulation of microtubules. 
Microtubules can be routinely reassembled in vitro from tubulin purified 
from bovine brains. The nucleation, assembly, and disassembly reactions of 
microtubules have been well characterized. 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. A. Davis et al., Purification and Biochemical Characterization 
of Tubulin from the Budding Yeast Saccharomyces cerevisiae, 32 
Biochemistry 8823 (1993). 
Separation of selected molecules from complex mixtures of molecules is of 
great importance in chemical, pharmaceutical, biotechnological, 
health-related and medical, and many other industries. Great amounts of 
time and money are spent on performing such separations. There is also an 
interest in instrument miniaturization driven by potential for 
substantially decreased analysis time, decreased reagent volumes and cost, 
decreased analyte volumes, integration of analytical techniques in a 
single device, and the economy of batch fabrication of complex devices. 
In view of the foregoing, it will be appreciated that providing a method of 
separating a selected molecule from a heterogeneous mixture of molecules 
by reversibly coupling the selected molecule to a motor protein for 
transport on microtubules immobilized in a separation device would be a 
significant advancement in the art. 
BRIEF SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a method for separating 
a selected molecule from a heterogeneous mixture of molecules. 
It is also an object of the invention to provide a method for separating a 
selected molecule from a heterogeneous mixture of molecules by reversibly 
coupling the selected molecule to a motor protein, which transports the 
selected molecule on immobilized microtubules. 
It is another object of the invention to provide a microfabricated device 
comprising immobilized microtubules for performing separations using a 
motor protein reversibly coupled to a selected molecule to be isolated. 
It is still another object of the invention to provide a separation system 
that recognizes, separates, and detects selected molecules on a single 
micromachined chip. 
These and other objects are accomplished by providing a method for 
separating a selected molecule from a heterogeneous mixture of molecules 
comprising: 
(a) providing a separation device comprising a loading reservoir and a 
receiving reservoir coupled by a channel having immobilized to a surface 
thereof a plurality of microtubules aligned substantially parallel to a 
longitudinal axis of the channel; 
(b) placing an aqueous solution comprising the heterogeneous mixture of 
molecules in the loading reservoir; 
(c) adding a motor-ligand composition and an effective amount of ATP to the 
aqueous solution, wherein the motor-ligand composition comprises 
(i) a motor protein capable of attaching to the immobilized microtubules 
and moving therealong in the presence of ATP as a source of chemical 
energy, and 
(ii) a ligand coupled to the motor protein, wherein the ligand is capable 
of selectively binding the selected molecule, 
such that the ligand selectively binds the selected molecule and the motor 
protein attaches to the immobilized microtubules and transports the bound 
selected molecule therealong to the receiving reservoir; and 
(d) removing the selected molecule from the receiving reservoir. 
Preferably, the motor protein comprises the N-terminal 410 amino acid 
residues of kinesin. In one illustrative embodiment, the ligand comprises 
an oligonucleotide having a nucleotide sequence capable of hybridizing to 
a target site on the selected molecule. A preferred oligonucleotide has a 
nucleotide sequence capable of hybridizing to a phage .lambda. cos site, 
wherein the target site comprises a phage .lambda. cos site. In another 
preferred embodiment, the ligand comprises an oligonucleotide and the 
method further comprises providing an adaptor oligonucleotide comprising a 
first hybridization site and a second hybridization site, wherein the 
ligand is capable of hybridizing to the first hybridization site and the 
second hybridization site is capable of hybridizing to a target site on 
the selected molecule. 
In another preferred embodiment, the ligand comprises a peptide, such as 
streptavidin, protein A, or an immunoglobulin such as a single chain 
antibody. With a streptavidin, the ligand will bind any biotinylated 
molecule, or a biotinylated bead can be used to simultaneously bind a 
plurality of motor-ligand compositions and a plurality of selected 
molecules conjugated to streptavidin. 
In another preferred embodiment, the method further comprises, prior to 
removing the selected molecule from the receiving reservoir, detecting the 
presence of the selected molecule in the receiving reservoir. For example, 
detection of a nucleic acid, protein, or other selected molecule can be 
with an appropriate fluorescent dye. 
The invention also comprises aligning the microtubules in the channel of 
the separation device. Preferred methods of aligning the microtubules 
include flow alignment, nucleating with centrosomes or axoneme fragments, 
and fletching. 
A preferred separation device is a micrometer-scale device wherein the 
loading reservoir, receiving reservoir, and channel are micromachined into 
a substrate. 
Another aspect of the invention is a system for separating a selected 
molecule from a heterogeneous mixture of molecules in aqueous solution 
comprising: 
(a) a separation device comprising a loading reservoir and a receiving 
reservoir coupled by a channel having immobilized to a surface thereof a 
plurality of microtubules aligned substantially parallel to a longitudinal 
axis of the channel; 
(b) a motor-ligand composition comprising 
(i) a motor protein capable of attaching to the immobilized microtubules 
and moving therealong in the presence of ATP as a source of chemical 
energy, and 
(ii) a ligand coupled to the motor protein, wherein the ligand is capable 
of selectively binding the selected molecule; 
(c) an effective amount of ATP for providing chemical energy to the motor 
protein for supporting movement thereof along the immobilized microtubules 
.

DETAILED DESCRIPTION 
Before the present method and system for separating a selected molecule 
from a heterogeneous mixture of molecules are disclosed and described, it 
is to be understood that this invention is not limited to the particular 
configurations, process steps, and materials disclosed herein as such 
configurations, process steps, and materials may vary somewhat. It is also 
to be understood that the terminology employed herein is used for the 
purpose of describing particular embodiments only and is not intended to 
be limiting since the scope of the present invention will be limited only 
by the appended claims and equivalents thereof. 
It must be noted that, as used in this specification and the appended 
claims, the singular forms "a," "an," and "the" include plural referents 
unless the context clearly dictates otherwise. Thus, for example, 
reference to a separation system containing "a microtubule" includes a 
system containing two or more of such microtubules, reference to "a 
motor-ligand composition" includes reference to two or more of such 
motor-ligand compositions, and reference to a separation system containing 
"a channel" includes reference to two or more of such channels. 
In describing and claiming the present invention, the following terminology 
will be used in accordance with the definitions set out below. 
As used herein, "micromachining," "micromachined," and similar terms refer 
to the processes used to create micrometer-sized structures with primarily 
mechanical functions on a glass, silicon, silica, or photoreactive 
polymer-coated chip or other suitable substrate. The processes of 
micromachining are based on techniques developed in the microelectronics 
industry to create layered structures in integrated circuits, e.g. 
photolithography and film deposition procedures. In a preferred embodiment 
of the present invention, the dimensions of a microchannel connecting a 
loading reservoir and a receiving reservoir are about 125 .mu.m in length 
by about 25 .mu.m in width by about 10 .mu.m in depth, but the dimensions 
of such microchannels are limited only by functionality. The dimensions of 
the loading and receiving reservoirs are not considered to be critical and 
are also limited only by functionality. The microchannel is constructed of 
sufficient length such that the motor-ligand composition can transport a 
selected molecule from the loading reservoir to the receiving reservoir 
before contaminating molecules reach the receiving reservoir by diffusion. 
Kinesins move at a rate of about 60 .mu.m/min. Diffusion of undesirable 
molecules can be retarded by application of an electrical field and/or 
increasing the viscosity of the liquid medium, and the like. 
As used herein, "hybridization," "hybridizing," and similar terms refers to 
forming double-stranded nucleic acid molecules by hydrogen bonding of 
complementary base pairs, as is well known in the art. A person skilled in 
the art will recognize that a certain amount of mismatching is permitted 
under certain circumstances such that hybridization will still occur. 
Further, the conditions of hybridization can be manipulated by varying the 
lengths and GC ratios of complementary sequences that are to be 
hybridized, the amount of mismatching, the monovalent salt concentration, 
the presence of certain solvents such as formamide, and the temperature, 
according to principles well known in the art, such as are described in J. 
Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., 1989); T. 
Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); F. Ausubel 
et al., Current Protocols in Molecular Biology (1987), hereby incorporated 
by reference. 
As used herein, "motor protein" means a protein that transduces chemical 
energy into mechanical forces and motion. Preferred motor proteins for the 
present invention are kinesin and related proteins, such as ncd, S. A. 
Endow et al., Mediation of Meiotic and Early Mitotic Chromosome 
Segregation in Drosophila by a Protein Related to Kinesin, 345 Nature 
81-83 (1990), hereby 11 incorporated by reference, that are highly 
processive, i.e. do not readily detach from the microtubule tracks to 
which they are coupled. Once such highly processive motor proteins attach 
to a microtubule, there is a relatively high likelihood that they will 
move for many micrometers along the microtubule before becoming detached. 
Motor proteins such as myosin and dynein are considered unsuitable for use 
in the present invention because they lack high processivity. Preferred 
motor proteins are "double-headed," that is they are heavy chain dimers, 
which in part explains their processivity. Kinesin moves toward the 
plus-end of microtubules, whereas ncd moves toward the minus-end thereof. 
As used herein, "ligand" refers to a moiety that reversibly binds a 
selected molecule. In coordination compound chemistry, a ligand is a 
molecule or anion that donates a pair of electrons to a central metal atom 
to form a coordinate covalent bond between the ligand and the metal atom; 
thus, the ligand binds the metal atom. "Ligand" is used more broadly 
herein to refer to any moiety that reversibly binds a selected molecule 
that is to be separated from a mixture of molecules. For example, a ligand 
can be a single-stranded nucleic acid molecule that is adapted for and is 
capable of hybridizing to a selected complementary nucleic acid molecule. 
In another illustrative example, a ligand can be an antibody, Fab, 
F(ab').sub.2, F(ab'), single chain antibody, or the like that is capable 
of binding a selected antigen. In another illustrative embodiment, a 
ligand can be a protein A molecule, which is capable of binding IgG 
molecules. In another illustrative example, a ligand can be avidin or 
streptavidin, which is capable of binding biotin or a biotinylated 
molecule of interest. 
As used herein, "motor-ligand composition" refers to a motor protein 
coupled to a ligand molecule. The motor protein portion of the 
motor-ligand composition is preferably derived from kinesin, ncd, or 
another highly processive kinesin-related motor protein. The motor protein 
portion should be double-headed, therefore it will contain at least about 
the N-terminal 410 amino acid residues of the heavy chain protein. Amino 
acid residues in addition to the N-terminal 410 amino acid residues can 
also be present, and in this respect the length of the motor protein 
molecules is limited only by functionality, but preferably the motor 
protein chain contains no more than about 900 amino acid residues. Several 
illustrative constructions are exemplified herein. Recombinant motor 
proteins are also considered within the scope of the present invention. A 
few illustrative motor-ligand compositions are described herein, but it 
should be recognized that a person of skill in the art could easily 
construct additional motor-ligand compositions by recombinant DNA 
technology. 
The ligand portion of the motor-ligand composition can be any ligand that 
will selectively bind to a selected molecule to be separated from a 
mixture of molecules, provided that the ligand can be coupled to the motor 
protein without destroying the ability of the ligand to bind the selected 
molecule or the ability of the motor protein to move on the microtubules. 
For example, nucleic acids and certain proteins are preferred ligands. 
Selected oligonucleotides can be coupled to a motor protein, as will be 
discussed in more detail momentarily, such that the oligonucleotide is 
capable of hybridizing to a selected molecule, i.e. a nucleic acid 
molecule, that is to be separated in the separation process. The variety 
of molecules that can be subject to such separations is extremely wide, as 
will be appreciated. By way of further example, proteins such as 
streptavidin, protein A, and single chain antibodies can be coupled to a 
motor protein for binding a wide variety of molecules. Streptavidin is 
known to bind biotin, thus any molecule that can be biotinylated, such as 
DNA and proteins, can be separated with such a ligand. Protein A is known 
to bind to IgG molecules. Single chain antibodies can be produced that 
will bind to virtually any immunogen. 
Coupling of an oligonucleotide ligand to a motor protein can be by any 
method known in the art such that the motility of the motor protein 
portion and ability of the oligonucleotide to hybridize are preserved. An 
illustrative method of coupling an oligonucleotide to a motor protein will 
be exemplified below. Coupling of a motor protein to a protein or 
polypeptide ligand can also be carried out by known methods, such as 
chemical coupling or, preferably, expression of a fusion protein by 
recombinant DNA technology. Such recombinant DNA methods are described in 
the Sambrook et al., Maniatis et al., and Ausubel references. Briefly, a 
gene encoding a motor protein is spliced to a gene encoding a selected 
ligand polypeptide to form a gene fusion, and then the gene fusion is 
expressed in a suitable expression system such as E. coli or yeast to 
produce the motor-ligand composition, which is then purified and used in 
the separation system. 
As used herein, "peptide" means peptides of any length and includes 
proteins. The terms "polypeptide" and "oligopeptide" are used herein 
without any particular intended size limitation, unless a particular size 
is otherwise stated. 
As used herein, "effective amount" means an amount of a source of chemical 
energy, such as ATP, sufficient to permit a selected motor protein to 
generate mechanical force and thus move along a microtubule track. An 
effective amount can easily be determined by a person skilled in the art 
without undue experimentation. 
As used herein, "ATP" means adenosine triphosphate, a mononucleotide that 
stores chemical energy that is used by motor proteins, such as kinesin, 
for producing movement. 
Eukaryotic cells contain thousands of components that are sorted and 
distributed through specific bio-recognition and directed active 
transport. Numerous cellular components are synthesized, processed, and 
utilized in distinct cellular locations, often undergoing additional 
processing during transit. Families of motor proteins, which transduce 
chemical energy released by ATP hydrolysis into mechanical force and 
motion, haul these cellular components along tracks of actin or 
microtubule filaments to specific locations. Individual motor proteins are 
hitched to their specific cargo through unique recognition domains, which 
specify their cellular function. The specific function of kinesin is to 
recognize and transport a subset of neuronal vesicles from the cell body 
to axonal synapses. W. M. Saxton et al., Kinesin Heavy Chain Is Essential 
for Viability and Neuromuscular Functions in Drosophila, but Mutants Show 
No Defects in Mitosis, 64 Cell 1093 (1991). The present invention mimics 
the separation functions of kinesin in nerve cells, as will become clear 
from the following description. 
FIG. 1 shows a diagrammatic representation of a nerve cell 4 or neuron 
comprising a cell body 8, containing a nucleus 12 and neuronal vesicles 
16, 20, and 24; an axon 28, containing microtubules 32; and a synapse 34. 
Kinesin molecules 36 bind to a subset of neuronal vesicles 40 (FIG. 2) and 
transport them on microtubules through the axon. Vesicle transport can 
occur over distances up to a meter. 
FIGS. 3-5 depict an illustrative micromachined separation device according 
to the present invention. FIG. 3 shows a schematic diagram of a 
micro-fabricated device that exploits a motor protein, such as kinesin, 
and immobilized microtubules for recognizing, separating, and detecting a 
selected molecule on a single silicon chip. The device 44 comprises a 
loading reservoir 48 joined to a receiving reservoir 52 by a channel 56 
containing immobilized microtubules. Advantageously, access ports or holes 
60 are provided in the loading reservoir and receiving reservoir to permit 
loading of the loading reservoir and removal of separated molecules from 
the receiving reservoir. Microtubules are aligned and immobilized in the 
channel such that the long axes of the microtubules are substantially 
parallel to the long axis of the channel. 
FIG. 4 shows an illustrative embodiment of such a micro-fabricated device. 
The device 64 comprises a substrate 68 into which are micromachined a 
loading reservoir, a channel, and a receiving reservoir (as best shown in 
FIG. 3). A coverslip or cover plate 70 is bonded to the substrate 68 to 
enclose the loading reservoir, channel and receiving reservoir, as will be 
explained in more detail momentarily. Pipet tips 72 are preferably coupled 
to access holes (illustrated in FIG. 3) formed in the cover plate 70 to 
permit access to the loading reservoir and receiving reservoir. It will be 
appreciated that access ports could be provide in other designs, such as 
through micromachining in the substrate. FIG. 5 shows a cross section 
through a channel 76 formed in a substrate 80 and covered or enclosed by a 
cover plate 84. By "enclosed" is meant that the cover plate is placed over 
the loading reservoir, channel, and receiving reservoir, and is preferably 
bonded to the substrate such that liquid placed in the loading reservoir, 
channel, or receiving reservoir does not leak out and such that the 
coverslip does not move with respect to the substrate and thus disturb the 
contents of the device. Thus, it is intended that the loading reservoir, 
channel, and receiving reservoir are in liquid communication, but that the 
liquid does not leak from the loading reservoir or channel into the 
receiving reservoir, or vice versa. The access holes permit loading and 
removal of solutions in the device. 
Suitable materials for the substrate and cover plate include glass, 
silicon, silica, and the like. Any other material that would be functional 
for undergoing the micromachining process and would be compatible with 
immobilizing microtubules, the motor-ligand composition, ATP, the selected 
molecules to be separated, and a detection system that may be employed 
would also be suitable. 
In another embodiment of the present invention, a detection system is 
coupled to the separation system previously described for monitoring the 
progress of separating a selected molecule from a mixture of molecules. 
FIG. 8 shows a schematic representation of such a detection system 100. 
There is shown a micromachined device 104 to which is coupled a standard 
epifluorescence microscope. An argon ion laser 108 emits a laser beam 112 
(488 nm) that is reflected by a dichroic beam splitter 116 such that the 
beam passes through an objective lens 120 (Zeiss 63+, 1.4 NA) onto the 
microchannel of the separation device 104. A fluorescent intercalating 
dye, such as YOYO-1 (Molecular Probes, Eugene, Oreg.), with an excitation 
maximum of 491 nm and an emission maximum of 509 nm is suitable for 
detecting separation of DNA. The focused laser beam contacts the dye to 
excite fluorescence from transported DNA molecules. The fluorescence 124 
is collected by the objective lens 120 and focused, and then passed 
through a bandpass filter 128, and onto a slit in the front of a 
photomultiplier tube 132. The photomultiplier tube produces a signal that 
is transmitted to a PC-based data acquisition system 136 (Labview, 
National Instruments, Inc.) for processing, quantitation, a storage. 
One of the characteristics of kinesin that makes it particularly 
well-suited for application in a separation device is that it remains 
associated with the microtubule surface through thousands of ATP 
hydrolysis and motility cycles. J. Howard et al., Movement of Microtubules 
by Single Kinesin Molecules, 342 Nature 154 (1989). This means that a 
single kinesin molecule will move many micrometers, often completely to 
the end of a microtubule, without dissociating from its microtubule track. 
This property is likely due to cooperativity between the two motor domains 
of kinesin heavy chain dimers that results in one or the other of the 
motor domains being tightly bound at all times. D. D. Hackney, Evidence 
for Alternating Head Catalysis by Kinesin During Microtubule-stimulated 
ATP Hydrolysis, 91 Proc. Nat'l Acad. Sci. USA 6865 (1994). Myosin and 
dynein do not exhibit this property, but dissociate from their tracks 
between cycles. Microtubules are polar filaments because they are 
assembled from asymmetric tubulin subunits. The asymmetry is recognized by 
kinesin, which moves only toward what is referred to as the plus-end of 
the microtubules. Another member of the kinesin family, ncd, moves toward 
the minus-end of the microtubules. 
As reviewed briefly above, the kinesin heavy chain can be divided into 
three domains: the motor domain (amino acid residues 1-340), the 
coiled-coil stalk (amino acid residues 341-800), and the tail domain 
(amino acid residues 801-975). The motor domain of Drosophila kinesin 
contains 5 cysteine residues. Apparently, these cysteine residues are not 
critical to kinesin activity since kinesin motility is not sensitive to 
treatment with N-ethyl maleimide. Therefore, it is possible to chemically 
couple a probe, such as an oligonucleotide, to cysteine residues in the 
kinesin stalk without disrupting kinesin motor domain function. The 
relevant region (amino acid residues 340-595) of the Drosphila kinesin 
stalk contains only one cysteine residue, at position 441. In initial 
examples of the operation of the present invention, oligonucleotide 
ligands are coupled to this cysteine residue. In other embodiments, a 
modified kinesin molecule has been constructed wherein the stalk is 
truncated at residue 410 and a cysteine residue is coupled thereto. In 
practice, the length of the kinesin molecule is limited only by 
functionality. Generally, however, it is advantageous to limit the size of 
the kinesin molecule to about 410-900 amino acid residues per chain 
because expression and manipulation of proteins is generally easier with 
smaller proteins as opposed to larger proteins. SEQ ID NO:2 contains the 
nucleotide sequence of the Drosophila kinesin gene from kinesin cDNA 
including the 5' untranslated region, the complete coding region up, and 
the 3' untranslated region. This sequence of the entire gene is set forth 
in J. T. Yang et al., A Three-domain Structure of Kinesin Heavy Chain 
Revealed by DNA Sequence and Microtubule Binding Analyses, 56 Cell 879-89 
(1989), hereby incorporated by reference. 
Methods have been developed for manipulation of the microtubule component 
of the active separation device. Microtubules can be routinely reassembled 
in vitro from tubulin purified from bovine brains. The nucleation, 
assembly, and disassembly reactions of microtubules have been well 
characterized over the last 20 years. L. U. Cassimeris et al., Dynamic 
instability of microtubules, 7 Bioessays 149 (1988). 
EXAMPLE 1 
In this example, standard cross-linking chemistry is used to covalently 
attach an oligonucleotide to the carboxy-terminus of a genetically 
truncated kinesin protein. Oligonucleotides can be synthesized with 
modified nucleotides that contain either a thiol or an amino group for 
crosslinking to the truncated kinesin protein. Oligonucleotides are 
synthesized according to methods well known in the art, such as S. A. 
Narang et al., 68 Meth. Enzymol. 90 (1979); E. L. Brown et al., 68 Meth. 
Enzymol. 109 (1979); U.S. Pat. Nos. 4,356,270; 4,458,066; 4,416,988; 
4,293,652, which are hereby incorporated by reference. 
The kinesin motor protein used in this example is a 441 amino acid residue 
genetically truncated version with an additional 6 histidine residues 
coupled to the C-terminal Cys residue to aid in purification (SEQ ID 
NO:3). This kinesin protein is expressed in E. coli according to methods 
well known in the art. This kinesin motor protein can be made by digesting 
pET-K447, described in J. G. Yang et al., Evidence That the Head of 
Kinesin Is Sufficient for Force Generation and Motility in Vitro, 249 
Science 42-47 (1990), with PvuII, and then digesting with exonuclease, 
polishing the ends, and religating to obtain a plasmid that encodes the 
441 amino acid residue kinesin. Expression of the protein is obtained by 
transforming E. coli strain BL21 (DE3), A. H. Rosenberg et al., 56 Gene 
125 (1987), growing overnight cultures of the transformed bacteria, 
diluting the overnight culture 1:100 in LB medium, J. Miller, Experiments 
in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y. (1972), supplemented with ampicillin and shaking at 37.degree. C. for 
2 hours. The culture is then made 0.1 mM with IPTG, and shaken at 
22.degree. C. for 10 hours. The cells are then lysed for protein 
preparation as follows. The cells are harvested by centrifugation (SORVALL 
GSA rotor; 8000 rpm, 5 minutes, 4.degree. C.). The pellet is resuspended 
in lysis buffer (0.1 M PIPES, pH 6.9, 1 mM MgCl.sub.2, 1 mM EGTA, 2 mM 
dithiothreitol) and centrifuged in a weighed tube (5 minutes, 8000 rpm, 
SORVALL SS34 rotor, 4.degree. C.). The pellet is weighed and resuspended 
in lysis buffer supplemented with the protease inhibitor 
phenylmethylsulfonyl fluoride (PMSF) at 1 mM. Each gram of cells is 
resuspended in 4 ml of buffer. The resuspended cells are lysed by 
sonication. The lysed cells are released into a tube sitting on ice, and 
centrifuged (SS34 rotor, 10,000 rpm, 30 minutes, 4.degree. C.). The 
supernatant is referred to as the cell extract. 
In one illustrative method, the kinesin heavy chain protein is enriched by 
microtubule affinity as follows. The cell extract is mixed with 
microtubules, prepared according to the procedure of Example 4, incubated 
at room temperature for 15 minutes, and centrifuged through a 2-ml sucrose 
cushion (15% sucrose, 20 .mu.M taxol, 1 mM GTP in lysis buffer with 1 mM 
PMSF) in a swinging bucket rotor (54,000 g, 35 minutes, 22.degree. C.). 
The pellet is resuspended in lysis buffer supplemented with protease 
inhibitor, taxol, and GTP, and centrifuged at 100,000 g. The kinesin heavy 
chain protein is released from microtubules by resuspending the pellet 
(from 1 ml of cell extract) in 100 .mu.l of lysis buffer containing 10 mM 
ATP, 10 mM MgSO.sub.4, and 0.1 M KCl, incubating at room temperature for 
15 minutes, and centrifuging at 100,000 g for 30 minutes at 22.degree. C. 
The supernatant containing enriched kinesin protein is divided into 
portions, frozen with liquid nitrogen, and stored at -70.degree. C. 
An alternative illustrative method of enrichment is by ammonium sulfate 
precipitation. The kinesin heavy chain protein is precipitated in a 
saturated ammonium sulfate solution (supplemented with 10 mM EDTA, 
adjusted to pH 8.2 with NH.sub.4 OH, and stored at 4.degree. C.), which is 
added dropwise with constant stirring until the final concentration of 
ammonium sulfate is 35%. This concentration gives the best enrichment of 
kinesin heavy chain protein relative to other bacterial proteins. The 
mixture is stirred in the cold for 30 minutes, and centrifuged (SS34 
rotor) at 10,000 rpm for 15 minutes. The pellet is resuspended in lysis 
buffer with protease inhibitors (200 .mu.l of buffer for 10 ml of cell 
extract), and dialyzed in 1 liter of lysis buffer for 6 hours with one 
change. The dialyzed sample is clarified by centrifugation at 150,000 g 
for 30 minutes at 4.degree. C. 
Residue 441 of the kinesin protein is a Cys residue to which the SEQ ID 
NO:1 oligonucleotide is coupled. This cysteine residue of kinesin is 
crosslinked to a 3' amino group of the oligonucleotide using the 
heterobifunctional crosslinker, succinimidyl 
4-N!maleiminomethyl!cyclohexane-1-carboxylate (SMCC, Pierce Chemical Co., 
Milwaukee, Wis.), according to the procedures outlined by the supplier, 
hereby incorporated by reference. In this example, the oligonucleotide 
(AGGTCGCCGC CCAT-amino (SEQ ID NO:1)) is complementary to the 
single-stranded cos site of lambda DNA. 
EXAMPLE 2 
In this Example, the procedure of Example 1 is followed with the exception 
that the kinesin motor protein comprises the N-terminal 410 amino acid 
residues of the kinesin protein to which is added a C-terminal Cys residue 
(SEQ ID NO:4). 
EXAMPLE 3 
In this example, a kinesin motor protein is coupled to a streptavidin 
ligand according to the construction set forth in FIG. 9 and as follows. 
The plasmid pMON-kinesin was constructed by ligating an XbaI-EcoRI 
fragment containing the kinesin gene from the pET-kin plasmid into the 
pMON plasmid (R. J. Duronio et al., 87 Proc. Nat'l Acad. Sci. USA 1506 
(1990)) that was digested with XbaI and EcoRI. The construction of pET-kin 
is described in J. T. Yang et al., Evidence That the Head of Kinesin Is 
Sufficient for Force Generation and Motility in Vitro, 249 Science 42-47 
(1990), hereby incorporated by reference. The pET-kin plasmid contains the 
entire protein coding region of the kinesin heavy chain cDNA. Proteins 
produced from pET-kin have an alanine residue inserted after the first 
methionine residue of the kinesin heavy chain and an alteration of 
Ala.sup.3 to Arg. The PMON-kin plasmid is an expression plasmid for 
expression of kinesin in E. coli under the control of a T7 promoter. This 
plasmid is digested with the restriction endonuclease AflII, the 3' 
recessed ends are filled in with the Klenow fragment of E. coli DNA 
polymerase I, and then the resulting blunt-ended plasmid is digested with 
EcoRI. Streptomyces avidinii DNA was purified from an S. avidinii culture 
(ATCC no. 27419) according to methods well known in the art, e.g. Sambrook 
et al., supra; Maniatis et al., supra; and Ausubel, supra. A portion of S. 
avidinii DNA containing the streptaviding gene was amplified by polymerase 
chain reaction (PCR) with the primers described below according to methods 
well known in the art, for example, U.S. Pat. No. 4,683,195; U.S. Pat. No. 
4,683,202; U.S. Pat. No. 4,800,159; U.S. Pat. No. 4,965,188; PCR 
Technology: Principles and Applications for DNA Amplification (H. Erlich 
ed., Stockton Press, New York, 1989); PCR Protocols: A Guide to Methods 
and Applications (Innis et al. eds, Academic Press, San Diego, Calif., 
1990), hereby incorporated by reference. The coding primer is identified 
as SEQ ID NO:5, having a sequence of GAAGGCCTTG ACCCCTCCAA GGACTC. The 
non-coding primer is identified herein as SEQ ID NO:6, having a sequence 
of GGAATTCAAT GATGATGATG ATGATGCTGA ACGGCGTCGA. This non-coding primer 
introduces six consecutive histidine codons, a stop codon, and an EcoRI 
site at 3' end of the streptaviding gene. The amplified DNA was then 
digested with restriction endonucleases StuI and EcoRI, and the resulting 
fragment was ligated into the digested pMON-kinesin plasmid with T4 
ligase. Restriction endonuclease digestions and ligase reactions were 
performed according to procedures well known in the art, e.g., J. Sambrook 
et al., Molecular Cloning: A Laboratory Manual (2d ed., 1989); T. Maniatis 
et al., Molecular Cloning: A Laboratory Manual (1982); F. Ausubel et al., 
Current Protocols in Molecular Biology (1987), hereby incorporated by 
reference. The resulting plasmid is designated pMON-kin-sav and comprises 
a gene fusion of kinesin, truncated at amino acid 595, with streptavidin. 
Expression of this plasmid in E. coli results in a kinesin-streptavidin 
fusion protein. 
This fusion protein is purified from bacterial cells according to the 
method of Example 1 except the cell extract is purified by a one-step 
immobilized metal affinity chromatography step instead of by microtubule 
affinity or ammonium sulfate enrichment. The six consecutive histidine 
residues at the C-terminus of the kinesin/streptavidin fusion protein have 
an affinity for a nickel-charged resin (such as "PROBOND," Invitrogen, San 
Diego, Calif.). Thus, the cell extract is caused to pass through the 
nickel-charged resin such that the kinesin/streptavidin fusion protein 
binds to the metal atoms through the 6 His residues. The column is then 
washed with lysis buffer to remove other bacterial proteins, and the 
kinesin/streptavidin fusion protein is eluted in lysis buffer containing 
250 mM imidizole. The eluted protein is then desalted with SEPHADEX G-25 
into PEM 80 buffer (80 mM PIPES, pH 6.9, 1 mM EGTA, 4 mM MgSO.sub.4). 
Streptavidin has a high affinity for biotin. Thus, this 
kinesin-streptavidin fusion protein is useful for separations wherein the 
molecule of interest, e.g. nucleic acid, protein, or other, is 
biotinylated. The avidin/streptavidin reaction with biotin is described in 
P. Langer et al., 78 Proc. Nat'l Acad. Sci USA 6633-37 (1981); A. Forster 
et al., 13 Nucleic Acids Res. 745-61 (1985); L. Riley et al., 5 DNA 333-37 
(1986), hereby incorporated by reference. 
EXAMPLE 4 
In this example, there is described an illustrative method of purifying 
tubulin from bovine brain. Brain tissue was homogenized at 4.degree. C. 
for 45 seconds at low speed in a Waring blender in 0.5 ml of 
polymerization buffer per gram of wet weight. The polymerization buffer 
(PM) is 50 mM (piperzine-N,N'-bis 2-ethanesulfonic acid)-KOH (PIPES-KOH) 
pH 6.9, 0.5 mM MgSO.sub.4, 1 mM EGTA, 0.5 mM GTP. The resulting homogenate 
was centrifuged at 130,000 g for 75 minutes at 4.degree. C. The 
supernatant solution was then diluted 1:1 with PM containing 8 M glycerol 
and incubated at 37.degree. C. for 30 minutes to assemble microtubules. 
The microtubules were then sedimented at 130,000 g for 75 minutes at 
25.degree. C., and the pellet was resuspended in cold PM (about 0.2-0.25 
the volume of the crude supernatant solution) using a Dounce homogenizer 
and incubated at 4.degree. C. for 30 minutes. The solution was then 
centrifuged at 130,000 g for 30 minutes at 4.degree. C. to sediment any 
remaining microtubules and aggregates not dissociated by cold treatment. 
The supernatant solution from this centrifugation was made 8 M in glycerol 
and stored for up to 3-4 days at 20.degree. C. before use. 
Typically, an appropriate aliquot of the stored tubulin solution was 
diluted 1:1 with PM containing 2 mm GTP and incubated at 37.degree. C. for 
39 minutes to assemble microtubules. The microtubules were collected as 
described above, resuspended, and depolymerized in column buffer (CB; 50 
mM PIPES-KOH pH 6.9, 0.5 mM MgSO.sub.4, 1 mM EGTA, 0.1 mM GTP), and 
incubated at 4.degree. C. for 30 minutes to assemble microtubules. The 
solution was then clarified by centrifuging at 130,000 g for 30 minutes at 
4.degree. C. The supernatant from this centrifugation was designated as 
2X-microtubule protein. 
Phosphocellulose (Whatman P11) was precycled by suspending it in 0.5 N KOH 
for 30 minutes (1 g phosphocellulose/15 ml); the exchanger was allowed to 
settle, the supernatant decanted, and the phosphocellulose washed with 
distilled water until the effluent was pH 8. The exchanger was then 
suspended in 0.5 N HCl for 30 minutes (1 g/15 ml), the supernatant 
decanted, and this step repeated. The phosphocellulose was then washed 
with distilled water until the effluent was near neutrality, suspended in 
50 mM PIPES-KOH (pH 6.9), and stored at 4.degree. C. until use. 
Phosphocellulose columns, 25.times.1.5 cm, were equilibrated by washing 
with Cs; 2X-microtubule protein (3 mg protein/ml bed volume) was run into 
this column. The tubulin flows through the phosphocellulose column without 
binding, thus the flow-through solution containing tubulin was collected, 
GTP was added to 0.1 mM, and then the tubilin was frozen with liquid 
nitrogen and stored at -80.degree. C. If desired, protein was quantitated 
by the method of M. Bradford, A Rapid and Sensitive Method for the 
Quantitation of Microgram Quantities of Protein Utilizing the Principle of 
Protein Dye Binding, 72 Anal. Biochem. 248-54 (1976). 
Microtubules were assembled as described above using 
phosphocellulose-purified tubulin with 2 mM GTP and at least about 40 
.mu.M taxol. 
EXAMPLE 5 
In this example, there is described an illustrative method of 
micromachining a device for separating selected molecules according to the 
present invention. Micromachining is carried out according to procedures 
well known in the art, i.e. photolithographical processes, for etching 
micrometer-scale channels into glass, particularly procedures used for 
microchannel electrophoresis applications, such as are described in A. T. 
Wooley & R. A. Mathies, Ultra-high-speed DNA Fragment Separations Using 
Microfabricated Capillary Array Electrophoresis Chips, 91 Biophysics 
11348-52 (1994); C. S. Effenhauser et al., High-speed Separation of 
Antisense Oligonucleotides on a Micromachined Capillary Electrophoresis 
Device, 66 Anal. Chem. 2949 (1994); C. Effenhauser et al., 65 Anal. Chem. 
2637 (1993); Z. H. Fan & D. J. Harrison, Micromachining of Capillary 
Electrophoresis Injectors and Separators on Class Chips and Evaluation of 
Flow at Capillary Intersections, 66 Anal. Chem. 177-84 (1994); W. H. Ko et 
al., in Sensors: A Comprehensive Survey, T. Grandke, W. H. Ko, eds., VCH 
Press: Weinhein, Germany, Vol. 1, pp. 107-68 (1989); K. E. Petersen, 70 
Proc. IEEE 420-57 (1982), which are hereby incorporated by reference. 
Micromachining is done at the University of Utah Hedco Microelectronics 
Research Laboratory. 
Glass plates are cleaned ultrasonically in detergent (5% SKLEEN, Fisher 
Scientific), methanol (reagent grade), acetone (semiconductor grade, Olin 
Hunt, N.J.), and deionized water in an ultrasonic bath in a class 100 
clean room environment. A photomask is created with the patterns of the 
reservoirs and channels of the separation device. The metal mask, 
nominally consisting of 200-.ANG. Cr and 1000-.ANG. Au, is evaporatively 
deposited under vacuum (&lt;10.sup.-6 Torr), and trace organics are then 
removed in H.sub.2 SO.sub.4 --H.sub.2 O.sub.2. A 1.4-.mu.m-thick positive 
photoresist (Waycoat HPR 504, Olin Hunt) is spin-coated on the metal with 
a Solitec photoresist coater-developer (3500 rpm) and then soft-baked at 
110.degree. C. for 5 min. 
Photomask layout is performed on a Princess CAD system, SUN 3/160 
workstation, and the master mask is manufactured by Precision Photomask 
(Montreal, Canada). A Quintel contact mask aligner is used to expose the 
photoresist, and Microposit 354 (Shipley, Newton, Mass.) is used as a 
developer to obtain a selected line width for channel definition. 
Following a hard bake at 120.degree. C. (5 min), the metal layer is etched 
away with aqua regia and a commercial Cr etch (KTI Chemicals, Sunnyside, 
Calif.). 
The photoresist is not removed from the remaining metal layer, so as to 
reduce the impact of pinholes in the metal. The exposed glass is etched in 
a slowly stirred mixture of concentrated HF--HNO.sub.3 --H.sub.2 O 
(20:14:66), or a commercial buffered oxide etch (BOE 10:1, Olin Hunt). The 
etch rate is determined with an Alpha-step profilometer (Tencor, Ind., 
Mountain View, Calif.), and the channel depth is then controlled by timing 
the etch period. The photoresist and metal masks are then removed with the 
etches described above. 
The etched glass plate is then cut into individual dies of selected size 
using a Model 1100 wafer saw (Microautomation, Fremont, Calif.) with a 
spindle speed of 20,000 rpm, cutting speed of 0.64 mm/s, and a depth 
increment of 0.51 mm. 
The etched plate and cover plate are cleaned as described above, aligned 
under a microscope, and thermally bonded in a Model 6-525 programmable 
furnace (J. M. Ney Co., Yucaipa, Calif.). The temperature program is as 
follows: 40.degree. C./min to 550.degree. C. for 30 min total, 20.degree. 
C./min to 610.degree. C. for 30 min, 20.degree. C./min to 635.degree. C. 
for 30 min, and 10.degree. C./min to 650.degree. C. for 6 h, followed by 
natural cooling of the furnace to room temperature. Unbonded regions are 
evidenced by interference fringes and differences in optical clarity. The 
bonding cycle is repeated once or twice with weights (.about.90 g) placed 
over the bonded regions. References relating to bonding of the cover plate 
to the substrate plate are as follows: S. C. Jacobson et al., 66 Anal. 
Chem. 1107 (1994); D. J. Harrison et al., 64 Anal. Chem. 1926-32 (1992), 
which are hereby incorporated by reference. Prior to bonding, the cover 
plate is drilled ultrasonically with about 0.5-mm holes to provide channel 
access points. Pipet tips are glued into these holes, as shown in FIG. 4. 
Fluid is added and withdrawn from the reservoir through the access holes 
with microsyringes. 
EXAMPLE 6 
In this example, a method of immobilizing and aligning microtubules in the 
microchannel of a micromachined device according to Example 5 is 
illustrated. It is desirable to immobilize microtubules only at the 
entrance to and within the microchannel of the device while minimizing the 
number of microtubules immobilized in the loading reservoir. Immobilizing 
microtubules in the loading reservoir reduces the efficiency of active 
separation. To immobilize microtubules only in the microchannel, the 
surfaces of the microchannel are derivatized, and then the microtubules 
are bonded to the derivatized surfaces, as illustratively shown in FIGS. 
6A-E. In an illustrative embodiment of this procedure, the channel 
surfaces are covalently derivatized with amino-silane (1% 
trimethoxysilylpropyldiethylenetriamine, United Chemical Tech.), FIG. 6A. 
A fraction of the surface amine groups is then activated for 
photo-crosslinking with low concentrations of 
sulfosuccinimidyl-(4-azidosalicylamido)-hexanoate (Pierce Chemical Co.), a 
heterobifunctional crosslinker that has an amine-reactive group at one end 
thereof and a photoactivatable azido group at the other end thereof, FIG. 
6B. Microtubules reassembled in vitro from purified cow brain tubulin have 
a net negative surface charge at neutral pH and can be immobilized on 
positively charged amino-silanized surfaces (FIG. 6C), as described in K. 
Svoboda et al., Direct Observation of Kinesin Stepping by Optical Trapping 
Interferometry, 365 Nature 721 (1993), which is hereby incorporated by 
reference. Microtubules are strongly bound by electrostatic interactions 
at physiological pH and ionic strength, but can be exchanged off the 
positively charged surface with moderately high salt concentrations 
(300-500 mM KCl). Thus, the electrostatically bound microtubules are 
selectively and covalently immobilized in the microchannel by irradiation 
of the microchannel with a UV microbeam to covalently crosslink the 
microtubules to the amino-silanized surfaces, FIG. 6D. Uncrosslinked 
microtubules in the reservoirs are then washed out with a solution of 
moderately high salt concentration, such as 0.5 M KCl, as shown in FIG. 
6E. 
In this example, the microtubules are aligned in the channel by flow 
alignment prior to immobilization (FIG. 7A). Flow alignment is the 
simplest method of aligning microtubules in the channel. Because 
microtubules are rod-shaped, the process of flowing a microtubule solution 
through the device causes the microtubules to align in the microchannel. 
Thus, a solution containing microtubules is loaded into the loading 
reservoir through the loading port. The solution flows throught the 
channel, aligning the microtubules parallel to the longitudinal axis of 
the channel. A consequence of simple flow alignment is that the 
microtubules are aligned with random polarities. 
EXAMPLE 7 
In this example, the procedure of Example 6 is followed, with the exception 
that alignment of the microtubules is by nucleated assembly inside the 
device (FIG. 7B). During in vitro reassembly of microtubules, microtubules 
are assembled with their plus-ends distal to isolated centrosomes or 
axonemal fragments. Thus, use of such centrosomes or axonemal fragments as 
nucleating sites for microtubule assembly, wherein such centrosomes or 
axonemal fragments are immobilized in the loading reservoir, results in 
microtubules assembling in parallel orientation through the channel with 
the plus-ends distal to the nucleating sites. 
EXAMPLE 8 
In this example, the procedure of Example 6 is followed, except that 
alignment of the microtubules is accomplished with the use of fletchings 
(FIG. 7C). Biotin-labeled tubulin seeds are used to nucleate microtubule 
assembly with unmodified tubulin. Fletchings of streptavidin or 
streptavidin-coated particles orient the microtubules in parallel as they 
flow through the channel of the device. 
EXAMPLE 9 
In this example, separation of a selected nucleic acid molecule in a 
heterogeneous mixture of nucleic acid molecules is illustrated. A 
miniature separation device with immobilized microtubules is prepared 
according to the procedures of Examples 5 and 6. A solution containing a 
mixture of phage .lambda. DNA and pBR322 plasmid DNA is placed in the 
loading reservoir of the device in a buffer comprising 50 mM MgSO.sub.4, 
50 mM ATP, 0.1 M PIPES, pH 6.9, 2.5 mM MgSO.sub.4, 0.5 mM EDTA, 5 mM EGTA, 
and protease inhibitors as described above. The 441 amino acid residue 
kinesin fragment (SEQ ID NO:3) coupled to the cos oligonucleotide (SEQ ID 
NO:1) prepared according to Example 1 is then added to the loading 
reservoir. The cos oligonucleotide (SEQ ID NO:1) hybridizes with the cos 
site on .lambda. DNA to form a kinesin/cos-oligonucleotide/.lambda.-DNA 
complex. The kinesin fragment of this complex attaches to the immobilized 
microtubules in the separation device and moves along the immobilized 
microtubules, resulting in a net movement of these complexes into the 
receiving reservoir. The pBR322 plasmid DNAs fail to hybridize to the 
kinesin/cos-oligonucleotide compositions, and thus are not actively 
transported to the receiving reservoir. There is a separation of .lambda. 
DNA from pBR322 DNA because active transport of .lambda. DNA to the 
receiving reservoir is more rapid than diffusion of the pBR322 DNA. 
EXAMPLE 10 
In this example, the procedure of Example 9 is followed except that 
glycerol is added to the reservoir solution to inhibit the diffusion of 
pBR322 DNA from the loading reservoir to the receiving reservoir. 
EXAMPLE 11 
In this example, the procedure of Example 9 is followed except that an 
electric field is applied across the reservoir solution such that the 
anode is at the loading reservoir and the cathode is at the receiving 
reservoir. Negatively charged pBR322 DNA is thus inhibited from diffusing 
from the loading reservoir to the receiving reservoir, but the 
actively-transported .lambda. DNA is transported to the receiving 
reservoir. 
EXAMPLE 12 
In this example, the procedure of Example 9 is followed except that the 
loading reservoir is loaded with biotinylated microbeads, which are 
commercially available, instead of a mixture of DNAs, and the kinesin 
motor protein complex is the kinesin/streptavidin fusion protein of 
Example 3. A plurality of kinesin/streptavidin fusion proteins bind to 
each biotinylated microbead by the affinity binding of streptaviding to 
biotin. The kinesin motor portion of the fusion protein attaches to and 
moves along the immobilized microtubules from the loading reservoir to the 
receiving reservoir. 
EXAMPLE 13 
In this example, the procedure of Example 12 is followed except that the 
solution in the loading reservoir also contains complex of bovine serum 
albumin (BSA) and streptavidin. The BSA/streptavidin complex also binds to 
the biotinylated microbeads, thus a plurality of BSA/streptaviding 
complexes are actively transported from the loading reservoir to the 
receiving reservoir along with the biotinylated microbeads. This example 
shows that multiple selected molecules can be separated from other 
molecules per kinesin motor protein according to the present invention. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AGGTCGCCGCCCAT14 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3572 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CGCACTCCAGCGATATCGCCATTTGACCAATTCGACTTTTGTGTCTGGTATAACCACAAA60 
ATTTTCACTGCATCTTCTTATTTGAGCCGCCCCCAACCCAATTTTCTCGAGTGTGTCGGA120 
ATCAGCAGAGAATCGGAACAAAAGGGGCAAAACGGAGTACCAGAATCGGCACAACAACAC180 
AGTGAACAGCAGATAGTGCGGACGATAGCGAGTCCGTTGATTATCCACGTAAACACTGAG240 
CTGCATGCGCAGAGGTCGCGCCCAGAGCTGCAGAAATCCAACAATCTCCGCCAGAAAGAT300 
CTATCTCCGCCCTGTAAGCAATGTCCGCGGAACGAGAGATTCCCGCCGAGGAC353 
MetSerAlaGluArgGluIleProAlaGluAsp 
1510 
AGCATCAAAGTGGTCTGCCGATTCCGACCGCTGAACGACAGCGAAGAG401 
SerIleLysValValCysArgPheArgProLeuAsnAspSerGluGlu 
152025 
AAGGCCGGCTCCAAGTTCGTGGTCAAGTTCCCCAACAATGTGGAGGAG449 
LysAlaGlySerLysPheValValLysPheProAsnAsnValGluGlu 
303540 
AACTGCATATCCATAGCGGGCAAGGTGTATTTGTTCGACAAGGTCTTC497 
AsnCysIleSerIleAlaGlyLysValTyrLeuPheAspLysValPhe 
455055 
AAACCGAATGCATCCCAGGAAAAGGTCTACAATGAGGCGGCCAAGTCC545 
LysProAsnAlaSerGlnGluLysValTyrAsnGluAlaAlaLysSer 
60657075 
ATTGTTACGGATGTCCTGGCCGGGTACAATGGAACGATATTCGCATAT593 
IleValThrAspValLeuAlaGlyTyrAsnGlyThrIlePheAlaTyr 
808590 
GGTCAGACGTCCTCCGGAAAAACGCATACGATGGAGGGCGTGATCGGG641 
GlyGlnThrSerSerGlyLysThrHisThrMetGluGlyValIleGly 
95100105 
GACTCCGTAAAACAGGGTATCATACCACGTATCGTCAACGACATTTTC689 
AspSerValLysGlnGlyIleIleProArgIleValAsnAspIlePhe 
110115120 
AATCACATCTACGCGATGGAGGTGAACCTGGAGTTTCACATCAAGGTC737 
AsnHisIleTyrAlaMetGluValAsnLeuGluPheHisIleLysVal 
125130135 
TCCTACTACGAGATCTACATGGACAAGATTCGAGATCTGTTGGACGTC785 
SerTyrTyrGluIleTyrMetAspLysIleArgAspLeuLeuAspVal 
140145150155 
TCCAAGGTGAACCTTAGTGTGCACGAGGATAAGAACCGGGTGCCGTAC833 
SerLysValAsnLeuSerValHisGluAspLysAsnArgValProTyr 
160165170 
GTCAAGGGCGCTACGGAACGGTTCGTCTCGTCGCCGGAGGATGTTTTC881 
ValLysGlyAlaThrGluArgPheValSerSerProGluAspValPhe 
175180185 
GAGGTGATCGAGGAGGGCAAATCCAATCGTCACATCGCTGTGACAAAC929 
GluValIleGluGluGlyLysSerAsnArgHisIleAlaValThrAsn 
190195200 
ATGAACGAGCATTCTTCGCGATCCCACTCAGTATTCCTTATCAATGTG977 
MetAsnGluHisSerSerArgSerHisSerValPheLeuIleAsnVal 
205210215 
AAGCAGGAGAACCTGGAGAACCAGAAGAAACTATCCGGCAAACTCTAC1025 
LysGlnGluAsnLeuGluAsnGlnLysLysLeuSerGlyLysLeuTyr 
220225230235 
CTGGTGGATTTGGCCGGTTCCGAGAAGGTTTCCAAGACTGGAGCGGAG1073 
LeuValAspLeuAlaGlySerGluLysValSerLysThrGlyAlaGlu 
240245250 
GGAACCGTTCTTGATGAAGCCAAGAACATCAACAAGTCGCTGTCGGCC1121 
GlyThrValLeuAspGluAlaLysAsnIleAsnLysSerLeuSerAla 
255260265 
TTGGGCAACGTAATTTCTGCCCTGGCGGACGGAAACAAAACGCACATC1169 
LeuGlyAsnValIleSerAlaLeuAlaAspGlyAsnLysThrHisIle 
270275280 
CCCTACCGTGATTCCAAGCTAACGCGCATCCTGCAGGAGTCGCTGGGA1217 
ProTyrArgAspSerLysLeuThrArgIleLeuGlnGluSerLeuGly 
285290295 
GGCAACGCACGCACAACCATCGTCATCTGCTGCTCTCCAGCCAGTTTC1265 
GlyAsnAlaArgThrThrIleValIleCysCysSerProAlaSerPhe 
300305310315 
AACGAATCTGAAACGAAGTCAACGCTGGACTTCGGTCGTAGAGCCAAG1313 
AsnGluSerGluThrLysSerThrLeuAspPheGlyArgArgAlaLys 
320325330 
ACAGTGAAGAACGTGGTCTGCGTTAACGAGGAGCTTACTGCCGAGGAA1361 
ThrValLysAsnValValCysValAsnGluGluLeuThrAlaGluGlu 
335340345 
TGGAAGCGACGCTATGAAAAGGAGAAGGAAAAGAACGCCCGACTAAAG1409 
TrpLysArgArgTyrGluLysGluLysGluLysAsnAlaArgLeuLys 
350355360 
GGTAAGGTGGAGAAGCTGGAGATCGAGCTTGCGCGCTGGAGAGCGGGT1457 
GlyLysValGluLysLeuGluIleGluLeuAlaArgTrpArgAlaGly 
365370375 
GAAACTGTTAAGGCGGAGGAGCAAATCAACATGGAGGATCTCATGGAG1505 
GluThrValLysAlaGluGluGlnIleAsnMetGluAspLeuMetGlu 
380385390395 
GCAAGCACGCCCAACCTGGAAGTGGAGGCAGCACAGACGGCGGCGGCC1553 
AlaSerThrProAsnLeuGluValGluAlaAlaGlnThrAlaAlaAla 
400405410 
GAGGCCGCTTTGGCCGCCCAGCGAACGGCTCTCGCCAATATGTCCGCA1601 
GluAlaAlaLeuAlaAlaGlnArgThrAlaLeuAlaAsnMetSerAla 
415420425 
TCGGTTGCCGTGAACGAGCAGGCCAGGCTGGCTACAGAGTGCGAGCGT1649 
SerValAlaValAsnGluGlnAlaArgLeuAlaThrGluCysGluArg 
430435440 
CTCTACCAGCAGCTGGACGACAAGGATGAGGAGATCAATCAGCAGAGC1697 
LeuTyrGlnGlnLeuAspAspLysAspGluGluIleAsnGlnGlnSer 
445450455 
CAGTACGCCGAGCAGCTCAAGGAGCAGGTGATGGAGCAGGAGGAACTC1745 
GlnTyrAlaGluGlnLeuLysGluGlnValMetGluGlnGluGluLeu 
460465470475 
ATCGCTAACGCTCGGCGTGAGTATGAGACTTTGCAGTCGGAGATGGCG1793 
IleAlaAsnAlaArgArgGluTyrGluThrLeuGlnSerGluMetAla 
480485490 
CGAATCCAACAGGAGAACGAGTCCGCCAAGGAAGAGGTTAAGGAGGTG1841 
ArgIleGlnGlnGluAsnGluSerAlaLysGluGluValLysGluVal 
495500505 
CTCCAAGCTCTCGAAGAGCTGACTGTAAACTACGACCAGAAATCCCAG1889 
LeuGlnAlaLeuGluGluLeuThrValAsnTyrAspGlnLysSerGln 
510515520 
GAGATCGATAACAAGAACAAGGATATCGATGCCCTCAACGAGGAGCTG1937 
GluIleAspAsnLysAsnLysAspIleAspAlaLeuAsnGluGluLeu 
525530535 
CAGCAGAAGCAGTCTGTGTTCAACGCCGCCTCCACAGAGCTACAGCAG1985 
GlnGlnLysGlnSerValPheAsnAlaAlaSerThrGluLeuGlnGln 
540545550555 
CTCAATGACATGTCCTCACACCAGAAGAAGCGCATCACGGAAATGCTA2033 
LeuLysAspMetSerSerHisGlnLysLysArgIleThrGluMetLeu 
560565570 
ACCAACCTACTGCGCGACCTCGGCGAAGTGGGCCAGGCCATTGCCCCC2081 
ThrAsnLeuLeuArgAspLeuGlyGluValGlyGlnAlaIleAlaPro 
575580585 
GGCGAGTCCAGCATCGACCTTAAGATGAGTGCTCTGGCTGGCACGGAT2129 
GlyGluSerSerIleAspLeuLysMetSerAlaLeuAlaGlyThrAsp 
590595600 
GCCAGCAAGGTGGAGGAAGATTTCACCATGGCGCGTTTGTTTATCAGC2177 
AlaSerLysValGluGluAspPheThrMetAlaArgLeuPheIleSer 
605610615 
AAGATGAAGACGGAGGCCAAGAACATTGCCCAGCGATGCTCCAACATG2225 
LysMetLysThrGluAlaLysAsnIleAlaGlnArgCysSerAsnMet 
620625630635 
GAAACACAGCAGGCTGACTCCAACAAGAAGATCTCCGAATATGAGAAA2273 
GluThrGlnGlnAlaAspSerAsnLysLysIleSerGluTyrGluLys 
640645650 
GATCTGGGCGAGTACCGGCTACTCATTTCGCAGCACGAGGCACGCATG2321 
AspLeuGlyGluTyrArgLeuLeuIleSerGlnHisGluAlaArgMet 
655660665 
AAGTCGCTGCAGGAGTCGATGCGGGAGGCAGAGAACAAGAAGCGCACG2369 
LysSerLeuGlnGluSerMetArgGluAlaGluAsnLysLysArgThr 
670675680 
CTCGAGGAACAAATCGATTCGCTGCGCGAGGAATGCGCCAAGCTCAAG241 
LeuGluGluGlnIleAspSerLeuArgGluGluCysAlaLysLeuLys 
685690695 
GCCGCCGAGCACGTTTCCGCCGTTAACGCCGAGGAGAAACAGCGGGCT2465 
AlaAlaGluHisValSerAlaValAsnAlaGluGluLysGlnArgAla 
700705710715 
GAGGAGCTGCGCTCCATGTTCGATTCTCAGATGGACGAGCTACGCGAA2513 
GluGluLeuArgSerMetPheAspSerGlnMetAspGluLeuArgGlu 
720725730 
GCCCACACCCGGCAGGTGTCCGAGCTCCGGGACGAAATTGCCGCCAAG2561 
AlaHisThrArgGlnValSerGluLeuArgAspGluIleAlaAlaLys 
735740745 
CAGCACGAAATGGACGAGATGAAGGATGTCCATCAAAAGCTGCTCTTG2609 
GlnHisGluMetAspGluMetLysAspValHisGlnLysLeuLeuLeu 
750755760 
GCGCACCAACAGATGACGGCCGACTACGAGAAGGTGCGCCAGGAGGAT2657 
AlaHisGlnGlnMetThrAlaAspTyrGluLysValArgGlnGluAsp 
765770775 
GCCGAGAAGTCCAGCGAGCTTCAGAACATCATCCTCACCAACGAGCGT2705 
AlaGluLysSerSerGluLeuGlnAsnIleIleLeuThrAsnGluArg 
780785790795 
CGGGAGCAAGCGCGCAAAGACCTCAAGGGCCTGGAGGACACGGTGGCC2753 
ArgGluGlnAlaArgLysAspLeuLysGlyLeuGluAspThrValAla 
800805810 
AAGGAGTTGCAGACGCTACACAACCTGCGAAAACTTTTCGTTCAGGAT2801 
LysGluLeuGlnThrLeuHisAsnLeuArgLysLeuPheValGlnAsp 
815820825 
CTACAGCAACGAATCCGAAAGAATGTCGTAAACGAGGAGAGCGAGGAG2849 
LeuGlnGlnArgIleArgLysAsnValValAsnGluGluSerGluGlu 
830835840 
GACGGTGGATCACTCGCGCAGAAACAGAAGATTTCCTTCTTGGAGAAC2897 
AspGlyGlySerLeuAlaGlnLysGlnLysIleSerPheLeuGluAsn 
845850855 
AACCTCGACCAGCTGACCAAGGTGCACAAGCAATTGGTGCGGGACAAC2945 
AsnLeuAspGlnLeuThrLysValHisLysGlnLeuValArgAspAsn 
860865870875 
GCCGATCTGCGGTGCGAGCTGCCCAAGCTGGAGAAGCGTCTACGCTGT2993 
AlaAspLeuArgCysGluLeuProLysLeuGluLysArgLeuArgCys 
880885890 
ACCATGGAGCGGGTGAAAGCTCTGGAGACAGCGCTCAAGGAGGCGAAG3041 
ThrMetGluArgValLysAlaLeuGluThrAlaLeuLysGluAlaLys 
895900905 
GAGGGCGCAATGCGGGATCGCAAGCGCTACCAATACGAGGTGGACCGC3089 
GluGlyAlaMetArgAspArgLysArgTyrGlnTyrGluValAspArg 
910915920 
ATCAAGGAGGCGGTGCGACAGAAGCATCTGGGCAGACGTGGCCCACAG3137 
IleLysGluAlaValArgGlnLysHisLeuGlyArgArgGlyProGln 
925930935 
GCACAGATCGCAAAGCCGATCCGGTCCGGCCAAGGTGCAATCGCCATT3185 
AlaGlnIleAlaLysProIleArgSerGlyGlnGlyAlaIleAlaIle 
940945950955 
CGTGGTGGTGGCGCCGTTGGAGGACCATCCCCGCTGGCCCAGGTTAAT3233 
ArgGlyGlyGlyAlaValGlyGlyProSerProLeuAlaGlnValAsn 
960965970 
CCTGTCAACTCGTAGATCCAATCACCACCTGTCGCCGCCCAGTTCAGCTCCG3285 
CTTTAAACTAAACTAGTTATACATACTTAACATAACTGATAATTGCCTTCGCTTAGATGA3345 
GATGTGTCGCGATCATGTGCAGCGCTTTAAATATACATACATATAATTTAATTAAATAAA3405 
TGAAAGGAAACCGGAAATTAACTAAATTTTACAAACCGAAAATAATAAAACCCACAGATA3465 
TGTAAGGACATCTATATACGTTAAGAGTATTTATAAACTTTTCAAACATAAACCTAAATA3525 
AAAGTCGCAGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA3572 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 441 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetSerAlaGluArgGluIleProAlaGluAspSerIleLysValVal 
151015 
CysArgPheArgProLeuAsnAspSerGluGluLysAlaGlySerLys 
202530 
PheValValLysPheProAsnAsnValGluGluAsnCysIleSerIle 
354045 
AlaGlyLysValTyrLeuPheAspLysValPheLysProAsnAlaSer 
505560 
GlnGluLysValTyrAsnGluAlaAlaLysSerIleValThrAspVal 
65707580 
LeuAlaGlyTyrAsnGlyThrIlePheAlaTyrGlyGlnThrSerSer 
859095 
GlyLysThrHisThrMetGluGlyValIleGlyAspSerValLysGln 
100105110 
GlyIleIleProArgIleValAsnAspIlePheAsnHisIleTyrAla 
115120125 
MetGluValAsnLeuGluPheHisIleLysValSerTyrTyrGluIle 
130135140 
TyrMetAspLysIleArgAspLeuLeuAspValSerLysValAsnLeu 
145150155160 
SerValHisGluAspLysAsnArgValProTyrValLysGlyAlaThr 
165170175 
GluArgPheValSerSerProGluAspValPheGluValIleGluGlu 
180185190 
GlyLysSerAsnArgHisIleAlaValThrAsnMetAsnGluHisSer 
195200205 
SerArgSerHisSerValPheLeuIleAsnValLysGlnGluAsnLeu 
210215220 
GluAsnGlnLysLysLeuSerGlyLysLeuTyrLeuValAspLeuAla 
225230235240 
GlySerGluLysValSerLysThrGlyAlaGluGlyThrValLeuAsp 
245250255 
GluAlaLysAsnIleAsnLysSerLeuSerAlaLeuGlyAsnValIle 
260265270 
SerAlaLeuAlaAspGlyAsnLysThrHisIleProTyrArgAspSer 
275280285 
LysLeuThrArgIleLeuGlnGluSerLeuGlyGlyAsnAlaArgThr 
290295300 
ThrIleValIleCysCysSerProAlaSerPheAsnGluSerGluThr 
305310315320 
LysSerThrLeuAspPheGlyArgArgAlaLysThrValLysAsnVal 
325330335 
ValCysValAsnGluGluLeuThrAlaGluGluTrpLysArgArgTyr 
340345350 
GluLysGluLysGluLysAsnAlaArgLeuLysGlyLysValGluLys 
355360365 
LeuGluIleGluLeuAlaArgTrpArgAlaGlyGluThrValLysAla 
370375380 
GluGluGlnIleAsnMetGluAspLeuMetGluAlaSerThrProAsn 
385390395400 
LeuGluValGluAlaAlaGlnThrAlaAlaAlaGluAlaAlaLeuAla 
405410415 
AlaGlnArgThrAlaLeuAlaAsnMetSerAlaSerValAlaValAsn 
420425430 
GluGlnAlaArgLeuAlaThrGluCys 
435440 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 411 amino acid residues 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetSerAlaGluArgGluIleProAlaGluAspSerIleLysValVal 
151015 
CysArgPheArgProLeuAsnAspSerGluGluLysAlaGlySerLys 
202530 
PheValValLysPheProAsnAsnValGluGluAsnCysIleSerIle 
354045 
AlaGlyLysValTyrLeuPheAspLysValPheLysProAsnAlaSer 
505560 
GlnGluLysValTyrAsnGluAlaAlaLysSerIleValThrAspVal 
65707580 
LeuAlaGlyTyrAsnGlyThrIlePheAlaTyrGlyGlnThrSerSer 
859095 
GlyLysThrHisThrMetGluGlyValIleGlyAspSerValLysGln 
100105110 
GlyIleIleProArgIleValAsnAspIlePheAsnHisIleTyrAla 
115120125 
MetGluValAsnLeuGluPheHisIleLysValSerTyrTyrGluIle 
130135140 
TyrMetAspLysIleArgAspLeuLeuAspValSerLysValAsnLeu 
145150155160 
SerValHisGluAspLysAsnArgValProTyrValLysGlyAlaThr 
165170175 
GluArgPheValSerSerProGluAspValPheGluValIleGluGlu 
180185190 
GlyLysSerAsnArgHisIleAlaValThrAsnMetAsnGluHisSer 
195200205 
SerArgSerHisSerValPheLeuIleAsnValLysGlnGluAsnLeu 
210215220 
GluAsnGlnLysLysLeuSerGlyLysLeuTyrLeuValAspLeuAla 
225230235240 
GlySerGluLysValSerLysThrGlyAlaGluGlyThrValLeuAsp 
245250255 
GluAlaLysAsnIleAsnLysSerLeuSerAlaLeuGlyAsnValIle 
260265270 
SerAlaLeuAlaAspGlyAsnLysThrHisIleProTyrArgAspSer 
275280285 
LysLeuThrArgIleLeuGlnGluSerLeuGlyGlyAsnAlaArgThr 
290295300 
ThrIleValIleCysCysSerProAlaSerPheAsnGluSerGluThr 
305310315320 
LysSerThrLeuAspPheGlyArgArgAlaLysThrValLysAsnVal 
325330335 
ValCysValAsnGluGluLeuThrAlaGluGluTrpLysArgArgTyr 
340345350 
GluLysGluLysGluLysAsnAlaArgLeuLysGlyLysValGluLys 
355360365 
LeuGluIleGluLeuAlaArgTrpArgAlaGlyGluThrValLysAla 
370375380 
GluGluGlnIleAsnMetGluAspLeuMetGluAlaSerThrProAsn 
385390395400 
LeuGluValGluAlaAlaGlnThrAlaAlaCys 
405410 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GAAGGCCTTGACCCCTCCAAGGACTC26 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GGAATTCAATGATGATGATGATGATGCTGAACGGCGTCGA40 
__________________________________________________________________________