Polynucleotide motor, a motor system, their preparation and uses
OwnerKeith Firmaninventor
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Abstract
A polynucleotide motor is disclosed, comprising an enzyme capable of binding to a nucleic acid sequence, which enzyme is also capable of translocating the nucleic acid sequence without causing cleavage thereof. The motor may be associated with a substance bound to the nucleic acid sequence so that the bound substance, such as a magnetic bead, biotin, streptavidin, a scintillant or the like, can itself be translocated, relative to the region of binding of the enzyme, during translocation. Such a system has applications in the screening or testing for a pre-determined biological, chemical or physical activity; for example, in screening for new pharmacologically-effective ligands.
Claims28
A type I restriction-modification enzyme capable of binding to a nucleic acid sequence and also capable of translocating the nucleic acid sequence without causing cleavage thereof, said enzyme having HsdR, HsdM and HsdS sub-units, wherein the HsdR sub-unit is of the type IC restriction-modification enzyme EcoprrI or a mutant of said HsdR sub-unit which imparts to the enzyme the property of translocating a nucleic acid sequence without causing cleavage thereof, and without loss of ATPase activity.
A molecular motor system comprising a nucleic acid sequence having bound thereto:
- (1) at a first, proximal, region of the nucleic acid, an enzyme capable of translocating the nucleic acid sequence without causing cleavage of the nucleic acid during the translocation, said enzyme remaining bound to the nucleic acid, as a complex therewith, at said proximal region, during the translocation;
- and
- (2) at a second, distal, region of the nucleic acid, a bound substance capable of remaining bound to the nucleic acid sequence during the translocation, whereby the bound substance becomes translocated, relative to the region of binding of the enzyme to the nucleic acid sequence, as a result of the translocation of the nucleic acid to which it is bound.
A molecular motor system comprising a nucleic acid sequence having bound thereto:
- (1) an enzyme capable of translocating the nucleic acid sequence without causing cleavage of the nucleic acid during the translocation, said enzyme remaining bound to the nucleic acid, as a complex therewith, at said proximal region, during the translocation;
- and
- (2) a solid support.
A method for translocating a substance bound to a nucleic acid sequence from a distal region of the nucleic acid sequence towards a proximal region, which method comprises:
- (a)
- (i) providing at the distal region of the nucleic acid sequence a bound substance, or
- (ii) binding a substance to the distal region of the nucleic acid sequence;
- and
- (b)
- (i) providing at the proximal region a complex of the nucleic acid sequence with an enzyme, or
- (ii) complexing an enzyme to the proximal region of the nucleic acid sequence, which enzyme is capable of translocating the nucleic acid sequence without causing cleavage thereof;
- and
- (c) activating the enzyme, whereby the enzyme translocates the nucleic acid sequence, including the bound substance, from the distal region towards the proximal region.
A method for capturing a test substance in solution, suspension or dispersion and bringing it into association with a solid surface, which method comprises:
- (a) providing a molecular motor system comprising a nucleic acid having:
- (i) a proximal region of the nucleic acid to which is bound an enzyme capable of translocating the nucleic acid sequence without causing cleavage of the nucleic acid during the translocation, said enzyme remaining bound to the nucleic acid, as a complex therewith, at said proximal region, during the translocation;
- (ii) a distal region of the nucleic acid which enables the test substance to be captured, the distal region and/or the test substance being adapted for that purpose;
- and
- (iii) a solid surface attached to the nucleic acid
- (b) bringing the distal region of the polynucleotide-enzyme complex into contact with the test substance, whereby the test substance is captured;
- and
- (c) activating the enzyme, whereby the enzyme translocates the polynucleotide, including the test substance, from the distal region towards the solid surface.
A method for screening a test substance for a predetermined biological, chemical or physical activity, which method comprises:
- (a) providing a solution, suspension or dispersion of the test substance, either (i) itself or (ii) in association with a first interactive substance, capable of providing or inducing a detectable reaction in a second interactive substance;
- (b) providing a polynucleotide motor system comprising a nucleic acid having bound thereto:
- (1) at a first, proximal region of the nucleic acid, an enzyme capable of translocating the nucleic acid sequence without causing cleavage of the nucleic acid during the translocation, said enzyme remaining bound to the nucleic acid, as a complex therewith, at said proximal region, during the translocation;
- and
- (2) at a second, distal region of the nucleic acid, a bound substance capable of remaining bound to the nucleic acid sequence during the translocation, whereby the bound substance becomes translocated, relative to the region of binding of the enzyme to the nucleic acid sequence, as a result of the translocation of the nucleic acid to which it is bound,
- wherein the nucleic acid is attached to a solid support and wherein
- (i) the bound substance is further capable of binding to a test substance exhibiting the predetermined activity;
- and
- (ii) the bound substance is itself or the solid support comprises the second interactive substance;
- (c) activating the polynucleotide motor system to effect translocation;
- and
- (d) monitoring the presence or absence of the detectable reaction during or after translocation.
Dependencies are inferred from the claim text.