Nanomachines fueled by nucleic acid strand exchange

Details Nanomachines fueled by nucleic acid strand exchange Nanomachines fueled by nucleic acid strand exchange

2000-02-02

2004-02-24

Marschel, Ardin H. (Department: 1631)

Chemistry: molecular biology and microbiology

Apparatus

Mutation or genetic engineering apparatus

C435S006120, C435S286100, C435S289100, C422S050000, C422S129000

Reexamination Certificate

active

06696285

ABSTRACT:

FIELD OF THE INVENTION
The free energy of hybridization of complementary hybridizing oligomers such as nucleic acids and nucleic acid analogs can be directed to effect controlled and reversible changes in the configuration of molecular structures, and so can be used to control a molecular switch, or can serve as fuel to drive the motion of a nanomachine. This invention pertains to methods in which oligomer hybridization/displacement reactions operate a reversible molecular switch, or fuel a nanomachine. The methods of the invention comprise controlling the number and type of hybridizing subunits, e.g., nucleotides, in single-stranded “toehold” regions that extend from double-stranded oligomer complexes attached to the molecular switches or nanomachines. The single-stranded toehold regions enhance the rate with which single-stranded oligomers hybridize to and displace one of the strands of such double-stranded complexes to reversibly alter the configuration of the molecular switches or nanomachines. The invention further includes molecular switches and nanomachines comprising molecular structures which reversibly assume alternate configurations at molecular reaction rates that are controlled by hybridization reactions involving said oligomers comprising single-stranded “toehold” regions.
BACKGROUND OF THE INVENTION
Toehold-Mediated Strand Displacement
It has been shown that a single-stranded region of a nucleic acid extending from the end of a double-stranded (duplex) complex formed by hybridization of two strands of unequal length provides a nucleation site, or “toehold,” for hybridization of a third nucleic acid strand complementary to the longer strand. A toehold-mediated hybridization/displacement reaction is initiated when a portion of the third strand hybridizes to the single-stranded toehold sequence, and proceeds with the remaining portion of the third strand subsequently hybridizing to the longer strand while displacing the shorter strand. The rate of strand displacement by such a toehold-mediated hybridization/displacement reaction is considerably greater than the rate of the strand displacement reaction when there is no toehold region [1-3]. Methods utilizing toehold-mediated hybridization/displacement reactions have been developed for detecting nucleic acids having specific nucleotide sequences [2, 3], and for ligating linker oligonucleotides to nucleic acids having specific nucleotide sequences to facilitate detection, affinity chromatography, and cloning of the nucleic acids [4, 5].
There have been several studies of the effects of varying biochemical and physical parameters of the toehold-mediated hybridization/displacement on the rate of the reaction. The overall rate of a toehold-mediated strand-displacement reaction is limited by the rate of association of the “incoming” displacing strand with the toehold region; the displacement of the shorter strand is not rate-limiting ([2], p. 1635; and [6], p. 4210). Once the displacing strand associates with the toehold region and begins displacing the shorter strand, the shorter strand is displaced via double-strand branch migration with a rate of approximately 12 &mgr;sec per nucleotide (determined in 0.3M NaCl, at 65° C.; see [2], p. 1635, and [7], p. 1911). The overall rate of hybridization/displacement reaction is increased by (1) increasing the is temperature, (2) increasing the concentration of the displacing DNA strand, and (3) adding volume-excluding polymers, presumably by increasing the rate of association of the incoming strand with the toehold region ([2], p. 1635; and [8], p. 20). The hybridization/displacement reaction rate is also increased by (4) modifying the displacing DNA strands so that they contain 5-bromodeoxycytidine (BrdC) or 5-methyl-deoxycytidine (MedC) nucleotides, which increase the affinity with which the displacing strand hybridizes to its complementary DNA strand in the duplex ([5], p. 2251; and [6], p. 4207). The rate of the hybridization/displacement reaction can also be increased by (5) increasing the G+C content of the toehold region ([6], p. 4207), (6) adding Rec A protein to the reaction mixture ([8], p. 25), and (7) using a displacing strand which is double-stranded at its terminal portion adjacent to the single-stranded region that binds the toehold sequence ([6], p. 4210). The effects of the lengths of the displacing strand and the displaced strand on the overall reaction rate are not well understood, and appear to be dependent on the nucleotide sequence of the toehold region, and possibly on the nucleotide sequence of the duplex region adjacent to the toehold region (compare [2], p. 1635, and [6], p. 4211). The effects of mismatches between the bases of the incoming, displacing strand and those of the longer, toehold-linked strand on the rate of the strand displacement are also unclear, and appear to depend strongly on the temperature of the reaction. For example, one study reports that a single mismatch blocks branch migration at 55° C. ([4], p. 8680), whereas another study reports that a cluster of 5 mismatches among 7 bases does not reduce the efficiency of strand displacement at 65° C. ([8], pp. 23-24). Interestingly, the latter study also reports that 27% base mismatch over 85 nucleotides blocks strand displacement at 65° C., but not at 55° C. ([8], pp. 23-24). The latter result indicates that displacing strands can be synthesized having a selected number of mismatches so that displacement does not occur unless the temperature is at or above a selected value.
It has been shown that a toehold-mediated hybridization/displacement proceeds approximately 3 times more rapidly with the 4-nucleotide toehold sequence GGCC- than it does with the 3-nucleotide toehold CCG-([6], p. 4211); however, the precise relationship between the length of the toehold region and the rate of the toehold-mediated hybridization/displacement reaction has not been described prior to the present invention.
Molecular Nanotechnology
Molecular nanotechnology uses molecular engineering and manufacturing capabilities, employing the capabilities of biotechnology in combination with other technologies such as proximate probe technology and supramolecular chemistry, to develop nanometer-scale machines and devices assembled from natural and nonnatural macromolecules and other chemical structures [9]. An example of such a molecular device is a controllable two-state molecular switch, which can be used to store information in the same manner as the counters of an abacus, or the electronically operated switches of a computer ([9], pp. 2012-2014).
DNA nanotechnology takes advantage of the self-organizing properties of DNA polymers, and uses DNA oligomers having selected nucleotide sequences as structural elements in the assembly of complex structures on a molecular scale [10-14]. The advantages of using DNA to construct nanodevices include
(1) double-stranded DNA molecules of 1-3 turns are relatively rigid structural elements, and the intermolecular interactions of DNA are relatively well-understood and predictable, so that DNA polymers can be designed which will self-assemble in a predictable manner;
(2) oligomers of DNA and its analogs having arbitrary subunit sequences can be produced readily using solid support synthesis;
(3) many different methods for chemically modifying DNA have been developed, e.g. to attach linking functions, catalytic or structural polypeptides, or detectable groups such as biotin and fluorescent labels, and to modify properties of the DNA such as resistance to cleavage by nucleases, hydrophobicity, flexibility, and duplex stability;
(4) DNA can be manipulated by an array of enzymes that include DNA restriction endonucleases, DNA ligase, kinases, and exonucleases; and
(5) the external surface of DNA polymers is rich in structural information, and segments of si

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