EP1721000A4 - Use of rna polymerase as an information-dependent molecular motor - Google Patents
Use of rna polymerase as an information-dependent molecular motorInfo
- Publication number
- EP1721000A4 EP1721000A4 EP04718069A EP04718069A EP1721000A4 EP 1721000 A4 EP1721000 A4 EP 1721000A4 EP 04718069 A EP04718069 A EP 04718069A EP 04718069 A EP04718069 A EP 04718069A EP 1721000 A4 EP1721000 A4 EP 1721000A4
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- European Patent Office
- Prior art keywords
- dna
- sequence
- rnap
- binding domain
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
Definitions
- RNA polymerases are ubiquitous in nature and used extensively in the biotechnology industry. RNA polymerases are employed in nucleic acid amplification reactions with reverse transcriptase and RNaseH to amplify an RNA target using a methodology known as nucleic acid sequence based amplification. They are also widely used to synthesize messenger RNA
- RNA DNA from a DNA template
- DNA polymerases are used to catalyze the formation of complementary DNA in the presence of DNA templates.
- Single and multi-subunit RNA polymerase enzymes exist in nature.
- the multi-subunit RNA polymerase enzymes are found in bacteria, archaea, and eukaryotes.
- the single subunit RNA polymerase enzymes are found in some bacteriophages, mitochondria, some eukaryotic organelles and may be encoded by some eukaryotic plasmids. Although they share no apparent sequence or structural homology, both types of enzymes carry out the basic steps of transcription in an identical manner.
- the enzyme binds to a specific promoter sequence in the DNA template that lies upstream of the start site for transcription.
- the enzyme then separates (melts) the two strands of the template near the start signal to form a transcription "bubble", and begins RNA synthesis using the coding strand of the downstream DNA as a template and a single ribonucleotide as a primer.
- contacts between the RNA polymerase and the upstream promoter sequences are maintained while the active site translocates (extends) downstream. This results in the formation of a short RNA-DNA hybrid and extension of the transcription bubble.
- T7 RNAP Bacteriophage T7 RNA polymerase
- T7 RNAP EC This overall organization of T7 RNAP EC bears a significant resemblance to that of the multi- s ubunit RNAPs and the structural mechanism by which T7 RNAP achieves the EC configuration is similar to the steps observed in bacterial RNAPs.
- RNAPs DNA-dependent RNA polymerases
- Advancement of the complex at each polymerization step depends upon the availability of a ribonucleotide substrate that is complementary to the next base in the DNA template. If the required substrate is not present, the progress of the polymerase is halted. The halted transcription complexes are usually quite stabile, and transcription is resumed upon addition of the missing substrate. See Gopal , et al., / Mo/. Bio/.: 41 1 -31 (1 999). Immobilization of transcription complexes on a solid surface permits repeated cycles of transcription to be carried out in the presence of limited mixtures of substrate- Single molecule studies of the multi-subunit RNAP of £ coli have shown that RNAP can exert considerable force as it moves along the DNA template.
- Biological motors have been described that can exert linear or rotary forces, for example kinesin, myosin and Fi -ATPase. However, none of these may be precise ly controlled, especially in an information-dependent manner. Whi le the multi-subunit £ c ⁇ // RNAP has been shown to be able to exert force, its use as a biological motor apparently has not been suggested or attempted , perhaps due to the following difficulties. Because the endogenous multi-sub unit RNAP is required for cell growth, any modifications deemed desirable may prove lethal or make purification of a homogeneous RNAP population from the bacterial cell culture difficult or unwieldy. In addition, experiments with the multi-subunit E.
- coli RNAP demonstrate that when halted at certain sites, the enzyme can enter into an irreversibly arrested state ("dead end” complex) or may slide back along the DNA template and cleave the nascent RNA before recovering (“backtracking"). Furthermore, single molecul e studies of E. coli RNAP have indicated that movement of the enzyme along the template is not regular, and that progressive transcript elongation is often interrupted by pauses of an apparently random nature. These stochastic interruptions in enzyme activity may be related to the backtracking and arrest phenomena noted above. The formation of dead ends or backtracked complexes has also been observed with other multi-subunit RNAPs.
- the invention is a molecular motor for actuating macromolecules or molecular devices in the manner of "cargo".
- the motor comprises a nucleotide polymerase (NP) enzyme having a high affinity-binding domain capable of allowing the enzyme to bind to a ligand or ligands (which could be the "cargo" for example, or a structural element) and/or to a solid surface and having the ability to exert movement and force in an information dependent manne r.
- NP nucleotide polymerase
- RNAP and the bound macromolecule or parti cle or molecular device depends upon the availability of the next ribonucleotide substrate(s) to be incorporated by the polymerase motor as directed by the sequence of the template strand. Because advancement of the polymerase depends upon the availability of a ribonucleotide that is complementary to the DNA template, polymerase movement can be controlled in a sequence-specific, information-dependent manner by provid ing or withholding appropriate substrates during each elongation cycle.
- high-affinity binding do ain we mean an amino acid, polypeptide or protein sequence that is fused to the RNAP and has or confers the ability to attach the NP to another substrate , either a solid support or particle or another sequence or molecule. In some cases, the binding may be reversible. Such a sequence also has to be able to bi d tightly enough such that it is released only upon addition of a releasing agent or a modification in reaction conditions. Sequences comprising binding domains exhibiting affinities in the range of nM to pM Kd are exemplary of those that may be employed. Many high-affinity binding domains are known in the art.
- Exemplary are the yeast GAL4 binding protein, the zinc finger domains of DNA-binding proteins such as Zif268, the heavy metal binding domain of metallothionein, the DNA-binding domain of transcription factor Spl 30 , or the streptavidin binding domain of the streptavidin protein.
- DNA or RNA aptamers may be employed as adapters to provid e high-affinity, stereo-specific bindi ng domains.
- an aptamer constructed to include a defined recognition sequence such as the Zif268 binding motif may be attached to the NP enzyme:Zif268 fusion protein.
- the RNAP may also be modified in vivo to a biotinylated form, which has high affi nity to streptavidin or to streptavidin-conjugated molecules. More than one bind ing domain may be attached to the NP.
- the high-affinity bi riding domain can be reversibly or irreversibly bound by attachment or fusion to the NP at any site along its length as long as the functional ability of the NP or the DNA template is not disrupted or deleteriously affected.
- the binding domain is bound at or near the N-terminus of the NP and in an irreversible manner.
- the high-affinity bi nding domain also must be able to bind another entity, structure, substance or device, (the "cargo").
- the cargo may be virtually anything: another solid su pport or bead, a biological small molecule or macromolecule, a peptide ligand, a polypeptide sequence, a protein or a portion of a protein, a DNA or RNA sequence, a typically inanimate substance or object, including inorganic objects or structures which may have useful properties such as semi-conducting materials, heavy metals, magnetic particles, or materials which exhibit desirable optical properties, i.e., anything that has the ability to bind to the high-affinity b inding domain and be actuated by the information- dependent movement of the modified NP enzyme can be employed as the cargo.
- RNAPs encoded by bacteriophage s T7, T3, SP6 and Kl 1 are structurally simple, single subunit RNAPs that are easily manipulated.
- RNAPs RNA molecules having distinct promoter specificities are readily available; this permits use of multiple RNAP motors each of which may be directed to a unique position on the template and separately controlled.
- T7 RNAP is used because it the most well studied and understood nucleotide polymerase enzyme.
- any nucleotide polymerase enzyme may be employed as a molecular motor if they contain or are modified to contain a high-affinity binding domain in such a manner that the functional ability or activity or the enzyme is not disrupted or deleteriously affected.
- single subunit nucleotide polymerases appear to lack the dead ending and backtracking proclivities of the m ulti-subunit nucleotide polymerases, they are preferred. See, He et al., Protein Expression & Purification .2 142-51 (1997). Both single subunit and multi-subunit nucleotide polymerases are readily and publicly available through a variety of sources.
- a high-affinity binding domain which allows the enzyme to bind to a. solid surface or to a ligand (or to both).
- bind to we mean form a high affinity attachment with. Binding to would thus also include a covalent attachment.
- link to and “fuse to” or “fuse with” in the same manner and with the same meaning and intent as "bind to”.
- the binding domain is f sed to the enzyme at a position where it does not affect function activity, i.e., where it does not affect the enzyme's ability to synthesize RNA.
- the binding domain may be fused to T7 RNAP at or near its N-terminus.
- the invention comprises a method of making a molecular motor.
- a NP enzyme is fused to a high-affinity binding domain capable of bindin g to a surface support or a ligand.
- the enzyme may be reversibly or irreversibly attached to this binding domain.
- the enzyme may be a single subunit or a multi -subunit nucleotide polymerase enzyme.
- the high-affinity binding domain can be a DNA sequence, a RNA sequence or an amino acid sequence.
- Exemplary DNA and RNA sequences include DNA or RNA aptamers.
- Exemplary amino acid sequences include the yeast GAL4 DNA binding polypeptide sequence , the Zif268 zinc-finger DNA- binding polypeptide sequence, a streptavidin-binding polypeptide sequence, a metallotheionein binding polypeptide sequence, a sequence-specfic DNA binding polypeptide from the transcription factor Spl 30 and a 6 to 1 5 residue histidine sequence.
- any peptide sequence may be employed as long as it has a length and character so as to be able to form a strong bond with the cargo.
- the bonds formed may each be reversible or only the bond between the polymerase and the cargo may be reversible.
- the high-affinity binding domain can be bound, linked or fused to the NP anywhere along its length, so long as its enzymatic activity is retained.
- the high-affinity binding domain will be bound, linked or fused to the NP at or near its N-terminus.
- Other attributes of the high-affinity binding domain may be readily determined by the skilled artisan and will depend on the desired substance, ligand, cargo to be actuated or moved by the motor.
- the invention comprises a method for moving substances or actuating cargo in an information-dependent manner.
- a solution containing a nucleotide triphosphate (NTP) substrate or a mixture of nucleotide triphosphate substrates is combined with a starting solution containing (a) a DNA template having a promoter sequence containing a polymerase binding site and a start site of transcription and (b) a nucleotide polymerase molecular motor of the invention.
- the nucleotide triphosphate substrates comprise GTP, CTP, UTP and ATP.
- NTPs complementary to the nucleic acids of the DNA template are added in a combination that allows the formation of a stable EC "start-up complex".
- the invention comprises an array of molecular motors composed of a plurality of identical phage nucleotide polymerases.
- Each of these polymerases may be fused to a different high-affinity binding domain and each may be arranged in a linear manner to form the array.
- the array may be composed of a plurality of different phage nucleotide polymerases, resulting in the construction of an linear array of RNAP motors each with an unique promoter specificity.
- a plurality of such linear arrays may be arranged and positioned in a two dimensional grid.
- Fig. 1 illustrates the sequence-dependent, controll d movement of His ⁇ - tagged T7 RNAP as described in detail in Example 1 .
- Fig. 2 illustrates the loading and release of bound ligand during controlled walking of T7 RNAP as described in detail in Example 2.
- Fig. 3 illustrates a simple DNA device, the construction and use of which is disclosed in Example 3.
- Fig. 4 illustrates the rotary movement of polymerase enzyme along the DNA template during transcription as discussed in Example 4.
- Fig. 5 illustrates the results of the experiment measuring th e rotation of a magnetic bead by immobilized T7 RNAP as described in detail in Example 4.
- Fig. 6 illustrates the construction of a parallel array of multi pie RNAP motors on DNA bridges as discussed in detail in Example 5.
- Fig. 7 illustrates the construction of two dimensional grids of DNA containing promoters for RNAP motors as discussed in detail in Example 6.
- nucleotid e polymerase enzyme advances 0.34 nm along the DNA template and exerts linear forces up to 30 pN. Since the progress of the polymerase depends upon the availability of the next ribonucleotide substrate(s) to be incorporated as directed by the sequence of the template strand, its movement can be restricted by providing or withholding appropriate substrates. While other biological motors can generate similar forces, none of them are controllable with the level of precision or in an information-dependent manner. In this manner, the nucleotide polymerase transcription complex may be stepped through multiple cycles of as few as one nucleotide or as great as hundreds or thousands of nucleotides pe r cycle.
- RNAP hexahistidine
- the His ⁇ tagged enzyme was odified to include a 38 amino acid SBP peptide tag that has a high affinity for streptavidin.
- the SPB peptide is compatible with a variety of streptavidin-conjugated fluorescent and enzymatic reporter systems and its binding to a I ⁇ gand is readily reversible by the addition of biotin.
- NP can be linked to ligand-specific apt mers of RNA or of DNA; it can be fused directly to ligand-specific peptide domain s from other proteins such as streptavidin binding protein or metallothionein.
- NP can be use to carry a variety of biological, organic and/or inorganic cargo.
- the N terminus is solvent exposed and projects away from the surface of the enzyme. Sousa, et al., Natirre 364: 593-99
- Such peptides may be incorporated using a flexible 1 1 amino acid linker sequence as described in Kim and Pabo, Proc. l ⁇ at. Acad. Sci. 94: 2812-1 7 (1 998). This results in a six zinc finger fusion protein with extremely tight DNA binding. Additional biological macromolecules comprise fusion proteins composed of other DNA binding domains from transcription factor Spl and the heavy metal binding domain of metallothionein. Methods and materials that can be used in the construction of these fusion proteins are disclosed in Kadanage, et al, Cell 52: 4851 -57 (1990) and Sano et al., Proc. Nat. Acac. Sci. 89. 1 534-38 (2002).
- Aptamers of DNA or RNA are additional high-affinity binding domains that can be used as "adapter” or "linker” molecules.
- Apatamers are small nucleic acids selected from random libraries that are able to bind to other molecules with high affinity and specificity because of their ability to fold into unique structures. See Ellington and Szostak, Nature 346: 81 8-828 (1990); Tuerk and
- evolved peptides that have high affinity for a variety of ligands may also be derived by selection methods such as expressed phage peptide screening.
- selection methods such as expressed phage peptide screening.
- aptamers are typically produced by repeated cycles of selection and nucleic aid amplification
- the most straightforward way to link an aptamer with the desired specificity to a RNAP motor is to incorporate a defined recognition sequence into the amplification primer.
- the Zif268 binding sequence could be used for this purpose.
- the recognition sequence would provide a "handle" for subsequent binding by a Zif268:T7RNAP fusion protein.
- the primer could include a specific single stranded region complementary to a DNA oligomer that contains the Zif268 recognition sequence; hybridization of the complementary regions of the aptamer and the DNA oligomer would permit the fusion protein to capture the aptamer.
- RNAP Because of its ability to be precisely controlled by virtue of its dependence on the presence or absence of ribonucleotides, RNAP provides unique capabilities in its potential applications as a molecular motor. Coupling of RNAP to other materials finds utility in nanorobotics, the positioning of ligands or altering of structures with subnanometer precision, and in the assembly and movement of complex structures.
- RNAP motors could be assembled on a DNA grid immobilized on a solid surface.
- the movement of each RNAP in the array would depend upon the sequence of the DNA template to which it is bound and could be independently controlled.
- the following examples employ bacteriophage T7 RNAP as a prototype
- RNAP molecular motor because T7 RNAP is the prototype of a class of single subunit enzymes that also includes RNAPs encoded by bacteriophages T3, SP6,
- each phage RNAP is specific for its own promoter sequence.
- RNAP motors with unique promoter specificities, each of which may be fused to a different ligand-binding domain. Accordingly, this invention should not be limited to bacteriophage T7; RNAPs from any other bacteriophage could have been used. Engineered mutants such as those disclosed in the articles already cited can be used and are included in the scope of this disclosure. Modified bacteriophage RNAP from other bacteriophages, such as T3 bacteriophage can be used and are included in the scope of this disclosure.
- T3, plasmids for its production, transcription vectors carrying its promoter and promoter cassettes containing T3 and other phage RNAP promoters are described in U.S. Patent Nos. 5,01 7,488 issued May 21 , 1991 ;
- any single subunit RNAP such as mitochondrial or organellar single subunit RNAPs and plasmid RNAPs, from a variety of non-bacteriophage sources can be equally employed.
- the flexibility inherent in the single subunit RNAPs affords great potential in designing arrays of RNAP motors for specific applications.
- the following examples illustrate and present preferred embodiments of the intention. They are not to be construed as a limitation on the scope of the invention, as the skilled artisan will be able without undue experimentation to modify or make variants of the invention.
- One way in which this may be accomplished is to modify the enzyme to include a hexahistidine, His ⁇ , tag.
- the tag allows the enzyme to bind tightly to Ni ++ agarose beads or columns, without affecting enzyme performance. See for example, Van Dyke, et al., Gene 111: 99-104 (1 992).
- the methodology for modifying T7, T3 and SP6 phage RNAP to fuse histidine residues to the amino terminus, the oligonucleotides and other materials used in the modification, and the plasmid vectors containing the His-tagged RNAP are disclosed in detail in He, et al., Protein Expression and Purification 9. 142-1 51 (1 997), specifically incorporated by reference here.
- the number of histidine residues added to the amino terminus is not critical, between 6 and 1 2 are preferred.
- the His leader sequence can also include a thrombin cleavage site.
- These modifications do not affect the properties or performance of the enzyme and other modifications may be made as long as the modifications do not affect the properties or performance of the enzyme.
- the His-tagged RNAP is then advanced stepwise along a DNA template in a controlled, sequence-dependent manner as follows. His-tagged T7 RNAP was first incubated with a DNA template in the presence of a limited mixture of ribonucleotide substrates complementary to those of the DNA template beginning with the start of the promoter sequence and comprising a sufficient number and type to cause the formation of a stable EC. In our hands at least 14 nucleotides composed of those sequentially downstream of the start of the promoter sequence were necessary. This resulted in the formation of a halted,
- EC start-up complex The ECs were then adsorbed to Ni++ beads and washed to remove unincorporated substrates. Advancement of the RNAP along the DNA template requires the presence of ribonucleotides complementary to those specified by the template. The ECs in their halted state, the start-up complex, are quite stable and may be sequentially moved along the template during multiple cycles of washing and elongation. Elongation is strictly dependent upon the presence of the suitable substrate and may be controlled in increments of as little as 1 nucleotide, i.e., 0.34 nm advancement along the template.
- RNA synthesis was initiated by the addition of GTP, ATP and
- step 3 Successive cycles of transcription were carried out in the presence of the substrates indicated in the lower right of Fig. 1 .
- step 3 the addition of U, C and A advanced the polymerase to position +20. Washing again followed by the addition of G and A moved the polymerase to position 22 (step 2).
- RNA products were analyzed by electrophoresis in 20% polyacrylamide gels in the presence of 0.1 % SDS.
- a composite SDS-page gel is illustrated in the upper right of Fig. 1 . Reaction and incubation conditions, volumes, amounts, buffers and enzyme preparations were as disclosed in Temiakov, et al., ProteimDNA Interactions: A Practical Approach (Travers, AA & Buckle, M, eds.), pp 351 -64, Oxford University Press, Oxford, 2000.
- SBP-tag is compatible with a wide variety of streptavidin-conjugated fluorescent and enzymatic reporter systems and its binding to ligand is readily reversible by the addition of biotin.
- auxiliary sequence-specific DNA binding domain the GAL4 binding domain
- T7 RNAP T7 RNAP
- movement of the RNAP along the template changed the disposition of the target DNA relative to the tem plate.
- a 1009 bp template DNA containing a T7 promoter 1 95 bp from one end and a 244 bp target DNA containing a GAL4 binding site near the terminus were prepared by PCR amplification of appropriate plasmids using standard techniques known in the art.
- the two DNA fragments were incubated with GAL4:T7 RNAP in the presence of G, A and U, which allowed transcription to proceed 22 nt downstream from the promoter in the template DNA.
- the samples were fixed with formaldehyde and visualized by AFM (tapping mode).
- the result of the AFM is shown in Panel A, Fig .3.
- a linearlized plasmid containing a T7 promoter and the GAL4 binding site separated by 1 kb were treated in the same manner and visualized by AFM.
- the result is shown in Panel B, Fig. 3.
- the target sequence may be in a second DNA molecule (A) or in the same molecule (B).
- RNAP When the targeted sequence is in the same molecule, transcription results in the formation of a loop whose dimensions may be increased or decreased, depending upon the direction of transcription.
- the target sequence and the transcribed sequence are placed in the same DNA molecule; simultaneous binding of the fusion protein to the target sequence and to the transcribed region results in looping out of the intervening portion. Movement of the RNAP either enlarges or diminished the size of the loop, depending upon the orientation of the promoter in the transcribed region. See Fig. 3, B. EXAMPLE 4: FORCE MEASUREMENT OF THE RNAP MOTOR UNDER LOAD Earlier studies with E.
- coli RNAP showed that as the enzyme moves along the DNA it can exert a linear force up to 30 pN (stall force) and a rotary force of 5 pN-nm.
- stall force a linear force up to 30 pN
- rotary force 5 pN-nm.
- single subunit RNAPs will exert similar or larger forces.
- the methods and approaches developed for the multi-subunit RNAPs were employed. These methods and approaches are disclosed in detail in Davenport 20O1 , supra; Wang 1 998, supra; Yin 1995, supra.
- Example 2 we used gel electrophoresis to demonstrate the ability of T7 RNAP to capture a ligand, move along the template, and release the bound ligand.
- RNAP As the RNAP advances along the DNA it must unwind the two DNA strands at the lead ing edge of the bubble and reanneal the strands at the trailing edge. See Fig. 4. Each step of nucleotide incorporation correspo nds to 0.34 nm of linear translocation and 36° of rotation, and depends upon the availability of the next incoming substrate nucleotide as directed by the seq uence of the DNA template. Movement of the RNAP is controlled in an information-dependent manner by withholding or adding appropriate substrates.
- T7 RNAP As a rotary motor, an elongation complex of T7 RNAP was immobilized on a solid surface using a modified form of T7 RNAP (histidine-tagged T7 RNAP) that has a high affinity for Ni++, and the downstream end of a 4 kb DNA template was tethered to a magnetic bead by means of a biotin-streptavidin linkage.
- the downstream end of the template ws tethered to a streptavidin-conjugated 2.8 ⁇ m magnetic bead attached to a smaller, 1 .0 ⁇ m bead.
- the complexes were injected between two sealed cover glass slides and immobilized on the bottom slide, wh ich was pre-coated with Ni++ -NTA. These procedures are illustrated in Fig. 5A.
- the assembly was placed in a magnetic tweezers device that allowed the bead to be positioned above the surface and to be visualized by microscopy.
- 5B corresponds to an interval of 2.4 seconds.
- the helical template rotates as it passes through the immobilized RNAP, and this force is transmitted to the magnetic bead via the downstream DNA.
- the bead was observed to rotate steadily at a rate of ⁇ 30 rpm.
- the RNAP exerts a rotary force of ⁇ 60 pN • nm, which is within the range reported for FiATPase. Templates can readily be designed that allow for control of the rotation of the bead in incremental steps according to the sequence of the DNA.
- RNAP motor can be determined over a wide range of applied tensions to examine how the motor behaves during active transcription, particularly during walking in one nt intervals, or when halted. This is accomplished by repeating the initial experiments but with the modification that one or more NTPs are withheld as in Examples 1 -2 above. The actual behavior of the RNAP motor at different stages during the transcription process can then be assessed.
- EXAMPLE 5 ASSEMBLY OF ARRAYS OF RNAP MOTORS
- Example 3 above we described the construction of simple DNA nanodevices involving inter- or intramolecular interactions between an RNAP fusion protein and a target DNA sequence.
- RNAP motors on DNA templates, i.e., "bridges" that have been immobilized on a solid surface.
- Each motor may have a distinct ligand specificity, could be addressed to a specific location on the bridge, and could be independently moved along the template in a sequence-dependent manner.
- Two techniques can be used to do this. In the first, DNA bridges are assembled between two locations on a surface by annealing single-stranded regions at the ends of the
- DNA bridges are assembled between two locations on a surface by annealing single stranded regions at the ends of the DNA to previously deposited oligonucleotides ("anchor oligos"). Separate oligomers for each end of the DNA are used to ensure that all templates in the bridge share the same orientation. Phage promoters are included in the bridge DNA so that the RNAP motors can be directed to bind to the bridge at specific locations and with a particular orientation.
- each RNA.P motor may be placed at a particular location on the bridge. By fusion to different binding domains, each RNAP motor may be engineered to bind to a specific ligand.
- the DNA population in the bridge can be homogeneous (homogeneous parallel arrays) or heterogeneous (heterogeneous parallel arrays). Many separate bridges can be deposited on the same surface, in adjacent arrays. Alternatively, DMA can be immobilized via interactions with hydrophobic patches or strips deposited on a glass surface.
- RNAP tagged with the SBP domain as detailed in Examples 1 -3 above is used and the RNAP is labeled by binding to streptavidin-conjugated fluorescent beads or nanodots.
- the assembly of bridge molecules used DNA tern plates of at least ⁇ 10 kb in length , e.g., the 40 kb bacteriophage T7 genome, wh ich contains 1 7 phage promote rs all oriented in the same direction. See Gueroui et al., EPJ ' m press (2003), supra.
- templates may be constructed having only 1 or 2 phage promoters, positioned upstream from cassettes that allow controlled movement in increments of 50-100 bp.
- the promoters are separated by 1 kb and are arranged in either tandem or opposing orientations. By these means the density of loading of the RNAP motors and their relative motions in either orientation may be explored.
- Templates are constructed using standard recombinant methods and employing bacteriophage lambda or other well known plasmids or vectors. To construct cassettes suitable for walking in 50-100 bp increments, DN ⁇ modules that direct the synt hesis of C-less runs of RNA having a composition of (GAU)so are synthesized.
- the modules are ligated in tandem using linkers that contain unique runs of ...CCC... By alternating cycles in which either C or G, ⁇ , and U are provided as substrate(s), the polymerase will be limited to a 50 bp walk, or excursion, during each cycle.
- the internal sequence of the GAvU module is randomized during synthesis.
- EXAMPLE 6 CONSTRUCTION OF TWO DIMENSIONAL DNA GRIDS In some circu mstances it may be desirable to construct more complex DNA platforms upon whi ch RNAP motors can be placed and moved.
- Such patches of oligomers may be used to anchor DNA molecules with complementary ends, allowing the directed assembly of DNA grids.
- immobilized DNA molecules may be used as a scaffold on which to deposit or assemble secondary substances such as colloidal gold or other compounds with desirable electrical or mechanical properties.
- connecting molecules could be engineered to allow the assembly of two-dimensional DNA grids in a sequence specific manner.
- three-finger zinc protei ns of the Cys 2 -His2 type may be engineered to bind to a wide variety of DNA seq uences with high affinities (Kd ⁇ 10 ⁇ 12 M) following the methods and employing the materials disclosed in Choo and Isalan supra, Kim, supra, Liu supra, and Smith supra.
- Three-finger domains can be fused to each other, resulting in six finger peptides that bind to an 18 bp recognition sequence with even h igher affinities (Kd ⁇ l O -1 5 M) following the methods and employing the materials disclosed in Kim, supra.
- peptides having divalent bindi ng capacities can be manufactured.
- a linker peptide should be employed.
- a flexible linker spacer or connector
- This linker is used to construct a bivalent "connector" molecule having two three-finger domains, one with specificity for the 9 bp Zif268 DNA sequence and the other with specificity for the 9 bp sequence recognized by transcription factor Sp 1 . Binding of the fusion protei n to these target sequences, either separately when only one target DNA is present, or together when both target molecules are present, is then determined by means of gel-shift assays known in the art.
- the length of the peptide linker may be increased or an intervening protein domain may be inserted.
- Such a two dimensional grid is illustrated in Fig. 7 wherein connector molecules that contain dual zinc finger bindi g domains, each with a separate sequence-specific binding capacity, are used to link target sequences engineered into immobilized bridge DNA mo lecules (horizontal lines) and cross grid molecules (vertical lines). Promoters for RNAP motors may be engineered into the bridge and cross grid DNA molecules, allowing the placement and controlled movement of the motors within th e grid.
- T7 RNAP transcription complexes displace bound proteins such as the lac repressor with great efficiency
- T7 RNAP will be able to displace divalent three-finger connector molecules such as those described above. See Giordano et al, Gene 84:209-] 9 (1 989).
- standard transcription assays well known in the art are employed on templates that contain such binding sites in the presence and absence of the connector proteins and/or in the presence of the junction (non-template) DNA.
- Six-finger zinc finger proteins with higher DNA affinities may be employed in the same anner.
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Abstract
Description
Claims
Applications Claiming Priority (1)
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PCT/US2004/006896 WO2005098038A2 (en) | 2004-03-05 | 2004-03-05 | Use of rna polymerase as an information-dependent molecular motor |
Publications (2)
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EP1721000A2 EP1721000A2 (en) | 2006-11-15 |
EP1721000A4 true EP1721000A4 (en) | 2008-08-20 |
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EP04718069A Withdrawn EP1721000A4 (en) | 2004-03-05 | 2004-03-05 | Use of rna polymerase as an information-dependent molecular motor |
Country Status (3)
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EP (1) | EP1721000A4 (en) |
JP (1) | JP2008507952A (en) |
WO (1) | WO2005098038A2 (en) |
Families Citing this family (2)
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WO2012116191A1 (en) * | 2011-02-23 | 2012-08-30 | Eve Biomedical, Inc. | Rotation-dependent transcriptional sequencing systems and methods of using |
CA2859913C (en) * | 2011-12-22 | 2021-02-23 | Centre National De La Recherche Scientifique (Cnrs) | Method of dna detection and quantification by single-molecule hybridization and manipulation |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6210896B1 (en) * | 1998-08-13 | 2001-04-03 | Us Genomics | Molecular motors |
WO2003094973A1 (en) * | 2002-05-08 | 2003-11-20 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Agent and method for transporting biologically active molecules in cells |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0956347A4 (en) * | 1996-12-03 | 2002-11-27 | Anadys Pharmaceuticals Inc | M. tuberculosis rna polymerase alpha subunit |
AU2107199A (en) * | 1998-01-08 | 1999-07-26 | Regents Of The University Of California, The | Kinesin motor modulators derived from the marine sponge (adocia) |
DE69917322T2 (en) * | 1998-12-11 | 2005-05-04 | bioMérieux B.V. | RNA POLYMERASE MUTANTS WITH INCREASED STABILITY |
JP2003334070A (en) * | 2002-05-22 | 2003-11-25 | Japan Science & Technology Corp | Modified type f0f1-atp synthase molecule and use thereof |
JP4367825B2 (en) * | 2002-07-31 | 2009-11-18 | 国立大学法人群馬大学 | Recombinant myosin |
-
2004
- 2004-03-05 JP JP2007501757A patent/JP2008507952A/en active Pending
- 2004-03-05 WO PCT/US2004/006896 patent/WO2005098038A2/en not_active Application Discontinuation
- 2004-03-05 EP EP04718069A patent/EP1721000A4/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6210896B1 (en) * | 1998-08-13 | 2001-04-03 | Us Genomics | Molecular motors |
WO2003094973A1 (en) * | 2002-05-08 | 2003-11-20 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Agent and method for transporting biologically active molecules in cells |
Non-Patent Citations (2)
Title |
---|
TEMIAKOV D ET AL: "The specificity loop of T7 RNA polymerase interacts first with the promoter and then with the elongating transcript, suggesting a mechanism for promoter clearance", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 97, no. 26, 19 December 2002 (2002-12-19), pages 14109 - 14114 * |
VALE RONALD D: "The molecular motor toolbox for intracellular transport.", CELL, vol. 112, no. 4, 21 February 2003 (2003-02-21), pages 467 - 480, XP002486482, ISSN: 0092-8674 * |
Also Published As
Publication number | Publication date |
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EP1721000A2 (en) | 2006-11-15 |
WO2005098038A3 (en) | 2008-01-24 |
JP2008507952A (en) | 2008-03-21 |
WO2005098038A2 (en) | 2005-10-20 |
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