Molecular motors - Patent Review 6210896

I claim:

1. A method for analyzing a polymer of linked units, comprising:

exposing a plurality of individual units of a polymer of linked units to an agent selected from the group consisting of an electromagnetic radiation source, a quenching source, and a fluorescence excitation source by causing a molecular motor to move the polymer relative to the agent, and detecting signals resulting from an interaction between the linked units of the polymer and the agent.

2. The method of claim 1, wherein the molecular motor is tethered to a support.

3. The method of claim 2, wherein the agent is attached to the support.

4. The method of claim 1, wherein the agent is attached to the molecular motor.

5. The method of claim 1, wherein the signal is electromagnetic radiation.

6. The method of claim 4, wherein the agent emits electromagnetic radiation.

7. The method of claim 6, wherein a portion of the plurality of individual units of the polymer are labeled with a fluorophore.

8. The method of claim 6, wherein the plurality of individual units of the polymer are sequentially exposed to electromagnetic radiation by bringing the plurality of individual units in proximity to a light emissive compound and exposing the light emissive compound to electromagnetic radiation, and wherein the plurality of individual units of the polymer detectably affect emission of electromagnetic radiation from the light emissive compound.

9. The method of claim 6 wherein the plurality of individual units of the polymer are sequentially exposed to electromagnetic radiation, and wherein the electromagnetic radiation detectably affects emission of electromagnetic radiation from the plurality of individual units of the polymer to produce the detectable signal.

10. The method of claim 8, wherein the individual units detectably affecting emission of electromagnetic radiation from the light emissive compound are labeled with a fluorophore.

11. The method of claim 1, wherein the polymer is a nucleic acid and the molecular motor is a nucleic acid molecular motor.

12. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein the molecular motor is a nucleic acid molecular motor that is a polymerase.

13. An article of manufacture, comprising:

a support,

a molecular motor tethered to the support, and

an agent selected from the group consisting of an electromagnetic radiation source, a quenching source, and a fluorescence excitation source positioned in interactive proximity with a signal station of the molecular motor.

14. The article of claim 13, wherein a plurality of molecular motors is tethered to the support.

15. The article of claim 14, wherein the plurality of molecular motors is tethered to the support in an organized array.

16. The article of claim 13, wherein the support is selected from the group consisting of a slide, a chip, and a wall material having a channel.

17. The article of claim 16, wherein the support is a wall material having a channel and wherein the molecular motor is positioned at an end of the channel.

18. The article of claim 13, wherein the agent is a fluorophore.

19. The article of claim 13, wherein the molecular motor is selected from the group consisting of polymerase, helicase, kinesin, dynein, actin, and myosin.

20. A nucleic acid molecular motor, wherein the nucleic acid molecular motor includes an agent selected from the group consisting of an electromagnetic radiation source, a quenching source, and a fluorescence excitation source positioned in interactive proximity with a signal station of the nucleic acid molecular motor.

21. A solution comprising the nucleic acid molecular motor of claim 20.

22. The solution of claim 21, wherein the nucleic acid molecular motor is a polymerase.

23. A method for analyzing a polymer of linked units comprising:

(1) causing a labeled polymer of linked units to move relative to a molecular motor;

(2) detecting sequentially polymer dependent impulses from linked unit labels of less than all the linked units, and

(3) storing a signature of said polymer dependent impulses detected to analyze the polymer.

24. The method of claim 23, wherein the method is performed on a plurality of polymers simultaneously.

25. The method of claim 23, wherein the signature of polymer dependent impulses is at least 10 polymer dependent impulses.

26. The method of claim 23, wherein the signature of polymer dependent impulses defines the order of unit labels.

27. The method of claim 23, wherein the signature of polymer dependent impulses defines the distance between unit labels.

28. The method of claim 23, wherein the signature of polymer dependent impulses defines the number of unit labels.

29. The method of claim 23, wherein all of the unit labels are detected.

30. The method of claim 23, wherein only a portion of the unit labels are detected.

31. The method of claim 23, wherein the polymer is partially and randomly labeled with unit labels.

32. The method of claim 30, wherein all of the units of the polymer are labeled with a unit label.

33. The method of claim 23, wherein the labeled polymer of linked units is exposed to an agent selected from the group consisting of electromagnetic radiation, a quenching source and a fluorescence excitation source and wherein the polymer dependent impulses are produced by the interaction between a unit label of the polymer and the agent.

34. The method of claim 23, wherein the molecular motor is a nucleic acid molecular motor.

35. The method of claim 23, wherein the unit label of the polymer is an extrinsic label.

36. The method of claim 34, wherein the nucleic acid molecular motor is a polymerase.

37. The method of claim 23, wherein the method is a method for determining the proximity of two unit labels of the polymer wherein the proximity of the two unit labels is the signature of said polymer dependent impulses, the identity of each unit label being indicative of the identity of at least one unit of the polymer, wherein the labeled polymer is moved relative to a station to expose the two unit labels to the station to produce a characteristic polymer dependent impulse arising from a detectable physical change in the unit label or the station, and further comprising the step of measuring the amount of time elapsed between detecting each characteristic polymer dependent impulse, the amount of time elapsed being indicative of the proximity of the two unit labels.

38. The method of claim 23, wherein the method is a method for determining the order of two sequence unit labels of the polymer, the identity of each unit label being indicative of the identity of at least one unit of the polymer wherein the order of the two unit labels is the signature of said polymer dependent impulses, wherein the labeled polymer is moved linearly with respect to a station, to expose one of the unit labels to the station to produce a polymer dependent impulse which is a signature of polymer dependent impulses, and to expose the other of the unit labels to the station to produce a second detectable impulse which is a signature of said polymer dependent impulses, different from the first polymer dependent impulse, and further comprising the step of determining the order of the polymer dependent impulses as an indication of the order of the two unit labels.

39. The method of claim 23, wherein the method is a method for determining the distance between two unit labels of the polymer, the identity of each unit label being indicative of the identity of at least one unit of the polymer wherein the distance between two unit labels is the signature of said polymer dependent impulses, wherein the labeled polymer is moved linearly relative to a station to produce a characteristic polymer dependent impulse generated as each of the two unit labels passes by the station, and further comprising the step of determining the distance between the polymer dependent impulses as an indication of the distance between the two unit labels.

Description Submit all comments and votes

FIELD OF THE INVENTION

The present invention relates to molecular motors and their use in linear analysis of polymers. In particular, molecular motors are used to move polymers with respect to a station such that specific signals arise from the interaction between the polymer and an agent at the station.

BACKGROUND OF THE INVENTION

Polymers are involved in diverse and essential functions in living systems. The ability to decipher the function of polymers in these systems is integral to the understanding of the role that the polymer plays within a cell. Often the function of a polymer in a living system is determined by analyzing the structure and determining the relation between the structure and the function of the polymer. By determining the primary sequence in a polymer such as a nucleic acid it is possible to generate expression maps, to determine what proteins are expressed, and to understand where mutations occur in a disease state. Because of the wealth of knowledge that may be obtained from sequencing of polymers many methods have been developed to achieve more rapid and more accurate sequencing methods.

In general DNA sequencing is currently performed using one of two methods. The first and more popular method is the dideoxy chain termination method described by Sanger et al. (1977). This method involves the enzymatic synthesis of DNA molecules terminating in dideoxynucleotides. By using the four ddNTPs, a population of molecules terminating at each position of the target DNA can be synthesized. Subsequent analysis yields information on the length of the DNA molecules and the nucleotide at which each molecule terminates (either A, C, G, or T). With this information, the DNA sequence can be determined. The second method is Maxam and Gilbert sequencing (Maxam and Gilbert, 1977), which uses chemical degradation to generate a population of molecules degraded at certain positions of the target DNA. With knowledge of the cleavage specificities of the chemical reactions and the lengths of the fragments, the DNA sequence is generated. Both methods rely on polyacrylamide gel electrophoresis and photographic visualization of the radioactive DNA fragments. Each process takes about 1-3 days. The Sanger sequencing reactions can only generate 300-800 nucleotides in one run.

Methods to improve the output of sequence information using the Sanger method also have been proposed. These Sanger-based methods include multiplex sequencing, capillary gel electrophoresis, and automated gel electrophoresis. Recently, there has also been increasing interest in developing Sanger independent methods as well. Sanger independent methods use a completely different methodology to realize the nucleotide information. This category contains the most novel techniques, which include scanning electron microscopy (STM), mass spectrometry, enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA) sequencing, exonuclease sequencing, and sequencing by hybridization.

Further, several new methods have been described for carboxy terminal sequencing of polypeptides. See Inglis, A. S., Anal. Biochem. 195:183-96 (1991). Carboxy terminal sequencing methods mimic Edman degradation but involve sequential degradation from the opposite end of the polymer. See Inglis, A. S., Anal. Biochem. 195:183-96 (1991). Like Edman degradation, the carboxy-terminal sequencing methods involve chemically induced sequential removal and identification of the terminal amino acid residue.

More recently, polypeptide sequencing has been described by preparing a nested set (sequence defining set) of polymer fragments followed by mass analysis. See Chait, B. T. et al., Science 257:1885-94 (1992). Sequence is determined by comparing the relative mass difference between fragments with the known masses of the amino acid residues. Though formation of a nested (sequence defining) set of polymer fragments is a requirement of DNA sequencing, this method differs substantially from the conventional protein sequencing method consisting of sequential removal and identification of each residue. Although this method has potential in practice it has encountered several problems and has not been demonstrated to be an effective method.

SUMMARY OF THE INVENTION

The present invention relates to methods and products for linear analysis of polymers. In particular the invention is based on molecular motors and their use for guiding polymer movement during linear analysis. Recently rapid methods for analyzing polymers using linear analysis techniques have been developed. Such methods are described in co-pending PCT patent application No. PCT/US98/03024 and U.S. Ser. No. 09/134,411, the entire contents of which are hereby incorporated by reference. The method for analyzing polymers described in PCT/US98/03024 is based on the ability to examine each unit of a polymer individually. By examining each unit individually the type of unit and the position of the unit on the backbone of the polymer can be identified. This can be accomplished by positioning a unit at a station and examining a change which occurs when that unit is proximate to the station. The change can arise as a result of an interaction that occurs between the unit and the station or a partner and is specific for the particular unit. For instance if the polymer is a nucleic acid molecule and a T is positioned in proximity to a station a change which is specific for a T could occur. If on the other hand, a G is positioned in proximity to a station then a change which is specific for a G could occur. The specific change which occurs, for example, depends on the station used, the type of polymer being studied, and/or the label used. For instance the change may be an electromagnetic signal which arises as a result of the interaction.

One aspect of linear analysis techniques involves the movement of the polymer past a station in such a manner as to cause a signal that provides information about the polymer to arise. One method by which this movement can be achieved involves the use of molecular motors. A molecular motor is a molecule that interacts with a polymer and moves the polymer, unit by unit, past a station so that the polymer may be analyzed.

In one aspect the invention is a method for analyzing a polymer. The method includes the steps of exposing a plurality of individual units of a polymer to an agent selected from the group consisting of an electromagnetic radiation source, a quenching source, and a fluorescence excitation source by causing a molecular motor to move the polymer relative to the agent, and detecting signals resulting from an interaction between the units of the polymer and the agent. In one embodiment the signal is electromagnetic radiation. In another embodiment the agent is electromagnetic radiation.

In one embodiment the molecular motor is tethered to a support. Preferably the agent is also attached to the support. In another embodiment the agent is attached to the molecular motor.

In a preferred embodiment the agent is an electromagnetic radiation source. A portion of the plurality of individual units of the polymer, in one embodiment, is labeled with a fluorophore. In another embodiment the plurality of individual units of the polymer are sequentially exposed to electromagnetic radiation by bringing the plurality of individual units in proximity to a light emissive compound and exposing the light emissive compound to electromagnetic radiation, and wherein the plurality of individual units of the polymer detectably affect emission of electromagnetic radiation from the light emissive compound. Preferably the individual units detectably affecting emission of electromagnetic radiation from the light emissive compound are labeled with a fluorophore. According to another embodiment the plurality of individual units of the polymer are sequentially exposed to electromagnetic radiation, and wherein the electromagnetic radiation detectably affects emission of electromagnetic radiation from the plurality of individual units of the polymer to produce the detectable signal.

The polymer may be any type of polymer of linked units. The type of molecular motor which can be used, however, will depend on the type of polymer. In one embodiment the polymer is a nucleic acid and the molecular motor is a polymerase. In another embodiment the polymer is a peptide and the molecular motor is a myosin.

The invention in another aspect is an article of manufacture. The article of manufacture includes a support, a molecular motor tethered to the support, and an agent selected from the group consisting of an electromagnetic radiation source, a quenching source, and a fluorescence excitation source positioned in interactive proximity with a signal station of the molecular motor. In a preferred embodiment the agent is a fluorophore.

A plurality of molecular motors is tethered to the support in one embodiment. In another embodiment the plurality of molecular motors is tethered to the support in an organized array.

In one embodiment the support is selected from the group consisting of a slide, a chip, a wall material having a channel. Preferably the support is a wall material having a channel and wherein the molecular motor is positioned at an end of the channel.

The molecular motor tethered to the support may be any type of molecular motor. Preferably the molecular motor is a nucleic acid molecular motor or a peptide molecular motor selected from the group consisting of polymerase, helicase, kinesin, dynein, actin, and myosin.

According to another aspect of the invention a molecular motor is provided. The molecular motor includes an agent positioned in interactive proximity with a signal station of the molecular motor, wherein the agent is selected from the group consisting of an electromagnetic radiation source, a quenching source, and a fluorescence excitation source. In one embodiment the molecular motor is in a solution. In another embodiment, the solution includes only a single molecular motor. Preferably the molecular motor is a nucleic acid molecular motor.

The invention in another aspect is a method for analyzing a polymer of linked units. The method includes the steps of (1) causing a labeled polymer of linked units to move relative to a molecular motor; (2) detecting sequentially polymer dependent impulses from unit labels of less than all of the linked units, and (3) storing a signature of said polymer dependent impulses detected to analyze the polymer. In some embodiments the unit label of the polymer is an extrinsic label.

In one embodiment the method is performed on a plurality of polymers simultaneously. In another embodiment the signature of polymer dependent impulses is at least 10 polymer dependent impulses.

The molecular motor can be a nucleic acid molecular motor or a peptide molecular motor. One type of nucleic acid molecular motor is a polymerase.

In one embodiment the signature of polymer dependent impulses defines the order of unit labels, the identity of each unit label being indicative of the identity of at least one unit of the polymer wherein the order of the two unit labels is the signature of said polymer dependent impulses, wherein the labeled polymer is moved linearly with respect to a station, to expose one of the unit labels to the station to produce a polymer dependent impulse which is a signature of polymer dependent impulses, and to expose the other of the unit labels to the station to produce a second detectable which is a signature of said polymer dependent impulses, different from the first polymer dependent impulse, and further comprising the step of determining the order of the polymer dependent impulses as an indication of the order of the two unit labels.

According to another embodiment the signature of polymer dependent impulses defines the distance between unit labels, the identity of each unit label being indicative of the identity of at least one unit of the polymer wherein the distance between two unit labels is the signature of said polymer dependent impulses, wherein the labeled polymer is moved linearly relative to a station to produce a characteristic polymer dependent impulse generated as each of the two unit labels passes by the station, and further comprising the step of determining the distance between the polymer dependent impulses as an indication of the distance between the two unit labels.

In other embodiments the method is a method for determining the proximity of two unit labels of the polymer wherein the proximity of the two unit labels is the signature of said polymer dependent impulses, the identity of each unit label being indicative of the identity of at least one unit of the polymer, wherein the labeled polymer is moved relative to a station to expose the two unit labels to the station to produce a characteristic polymer dependent impulse arising from a detectable physical change in the unit label or the station, and further comprising the step of measuring the amount of time elapsed between detecting each characteristic polymer dependent impulse, the amount of time elapsed being indicative of the proximity of the two unit labels.

In yet another embodiment the signature of polymer dependent impulses defines the number of unit labels.

In some embodiments all of the unit labels are detected. In other embodiments only a portion of the unit labels are detected. The polymer may be partially and randomly labeled with unit labels. In yet other embodiments all of the units of the polymer are labeled with a unit label. The labeled polymer of linked units is exposed to an agent selected from the group consisting of electromagnetic radiation, a quenching source and a fluorescence excitation source and wherein the polymer dependent impulses are produced by the interaction between a unit label of the polymer and the agent in other embodiments.

According to another aspect the invention is a method for characterizing a test polymer. The method includes the steps of obtaining polymer dependent impulses from unit labels for a plurality of labeled polymers, by making the polymers relative to a molecular motor, comparing the polymer dependent impulses of the plurality of polymers, determining the relatedness of the polymers based upon similarities between the polymer dependent impulses of the polymers, and characterizing the test polymer based upon the polymer dependent impulses of related polymers.

The plurality of polymers may be any type of polymer but preferably is a nucleic acid. In one embodiment the plurality of polymers is a homogenous population. In another embodiment the plurality of polymers is a heterogenous population. The polymers can be labeled, randomly or non randomly. Different labels can be used to label different linked units to produce different polymer dependent impulses.

The polymer dependent impulses provide many different types of structural information about the polymer. For instance the obtained polymer dependent impulses may include an order of polymer dependent impulses or the obtained polymer dependent impulses may include the time of separation between specific signals or the number of specific polymer dependent impulses.

In one important embodiment the polymer dependent impulses are obtained by moving the plurality of polymers linearly past a signal generation station.

A method for sequencing a polymer of linked units is provided according to another aspect of the invention. The method includes the steps of obtaining polymer dependent impulses from a plurality of overlapping polymers, at least a portion of each of the polymers having a sequence of linked units identical to the other of the polymers by moving the polymers linearly past a signal generation