Patent 10738349 - Polynucleotide based movement, kits and methods related thereto > Description
This application claims priority to U.S. Provisional Application No. 62/245,618 filed Oct. 23, 2015. The entirety of this application is hereby incorporated by reference for all purposes.
This invention was made with government support under R01-GM097399 awarded by NIH. The government has certain rights in the invention.
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 15080US_ST25.txt. The text file is 2 KB, was created on Oct. 24, 2016, and is being submitted electronically via EFS-Web.
Converting chemical energy into controlled motion is useful in applications such as sensors, drug delivery platforms, and computing. Bath et al. report a linear motor built from DNA and a restriction enzyme, which moves a DNA cargo in discrete steps along a DNA track. Angew Chem Int Edit 44, 4358-4361 (2005). DNA-based machines that walk along a track have shown promise in recapitulating the properties of biological motor proteins. See Yin et al. Programming biomolecular self-assembly pathways. Nature, 2008, 451, 318-U314; Cha et al., Nat Nanotechnol, 2014, 9, 39-43; Omabegho et al., Science, 2009, 324, 67-71; Gu et al., Nature, 2010, 465, 202-205; and Lund et al., Nature, 2010, 465, 206-210. However, the maximum distance traveled by the most DNA-based motors is 1 μm. The velocity of these walkers is also limited due to a fundamental trade-off between motor endurance and speed. Thus, there is a need to identify improved architectures.
This disclosure relates DNA based movement of objects. In certain embodiments, particles, pairs of particles, or a rods are conjugated with single stranded DNA that hybridizes to a single stranded RNA that is conjugated to a substrate. When the DNA particle, pair of particles, or rod interacts with the surface RNA in the presence of an endonuclease, such as RNase H and the DNA hybridizes to the RNA, then the particle, pair of particle, or rod moves along the surface. The complementarity of the DNA and RNA affect the velocity. In certain embodiments, this disclosure contemplates amplifying a sample nucleic acid into single stranded DNA and conjugating it to the particle, pair of particles, or a rod. Exposing the particle to complementary surface RNA and measuring the velocity which implicates the nucleic acid sequence in the sample.
In certain embodiments, this disclosure relates to devices comprising, a particle, pair of particles or rod comprising a coating of single stranded DNA; a substrate comprising a coating of single stranded RNA; and an endoribonuclease such as RNase H, wherein the single stranded DNA hybridizes to the RNA on the substrate and the particle, pair of particles or rod is configured on the substrate such that the particle, pair of particles, or rod moves upon mixing the endoribonuclease with the DNA hybridized to the RNA.
In certain embodiment, the substrate comprises channels configure to be slightly greater than the diameter of the particle such that the particle, length of a pair of particles, or length of the rod. In certain embodiments, the channels are separated by a barrier of polyethylene glycol. In certain embodiment, the channels are configured such that the object is capable of moving in the channel but is restricted from isolating itself from RNA on the substrate surface.
In certain embodiments, the DNA is between 5 and 500, or 5 and 50, or 5 and 25, or 10 and 50, or 10 and 25 nucleotides in length. In certain embodiments, the particle comprises silica or a semiconductor material, metal or oxide having a polymer coating.
In certain embodiments, the DNA oligonucleotide is 3′ or 5′ conjugated to the surface of the particle. In certain embodiments, the particle, pair of particles, or rod has a diameter or length of 0.001 micrometers to 1 centimeters, or 0.001 micrometers to 0.1 centimeters, or 0.001 micrometers to 0.01 centimeters, or 0.001 micrometers to 1 micrometer. In certain embodiments, the DNA or the RNA encodes a polynucleotide sequence associates with a polymorphism, SNP, or mutation associated with a genetic disorder. In certain embodiments, the DNA or RNA can encode aptamer or split aptamer sequences associated with binding to aptamer ligand. In certain embodiments, DNA or RNA sequences encodes a catalytic oligonucleotide associated with specific metal cofactors. In certain embodiments, the substrate is a metal surface, glass, polymer, or microscope slide. In certain embodiments, the RNA is conjugated to a fluorescent molecule. In certain embodiments, movement of the particle, pair of particles, or rods are measured for velocity, e.g., random movement or in a single direction.
In certain embodiments, the disclosure relates to methods for moving a particle, pair or particles, or rod comprising DNA, comprising: providing a device comprising, a particle, pair of particles, or rod comprising a coating of single stranded DNA; a substrate comprising a coating of single stranded RNA; and an endoribonuclease such as RNase H, wherein the single stranded DNA hybridizes to the RNA on the substrate and the particle, pair of particles, or rod is configured on the substrate such that the particle, pair of particles, or rod moves upon mixing the endoribonuclease with the DNA hybridized to the RNA; placing the single stranded DNA coated particle, pair or particles, or rod on the surface of the single stranded RNA coated substrate in the presence of the endonuclease under conditions such that the particle, pair of particles, or rod moves on the surface of the substrate.
In certain embodiments, DNA or RNA is a sequence obtained from a sample. In certain embodiments, the speed of the movement of the particle, pair of particles, or rod is correlated to the sequence of the DNA or RNA. In certain embodiments, a maximum speed is associated with complete complementarity. In certain embodiments, a speed of less than the maximum speed is associated with incomplete complementarity.
In certain embodiments, the disclosure relates to kits comprising: a) a pair or primers wherein the primers are configured for amplification of a target DNA sequence, b) a substrate that contains single stranded DNA bound to the surface, and c) an oligonucleotide conjugated to a fluorescent marker wherein the oligonucleotide has a first segment that is complementary to the DNA bonded to the surface of the substrate and a second segment that is RNA and complementary to the target DNA sequence that is to be obtained from a sample by amplification from the pair of primers.
In certain embodiments, the kit further comprises RNase H.
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.
An oligonucleotide is a plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA.
As used herein, biological samples include all clinical samples useful for detection of disease in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as: blood; derivatives and fractions of blood, such as serum; extracted galls; biopsied or surgically removed tissue, including tissues that are, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin; tears; milk; skin scrapes; surface washings; urine; sputum; cerebrospinal fluid; prostate fluid; pus; or bone marrow aspirates. In a particular example, a sample includes blood obtained from a human subject, such as whole blood or serum. In another particular example, a sample includes buccal cells, for example collected using a swab or by an oral rinse.
Amplification of a nucleic acid molecule (such as a DNA or RNA molecule) refers to use of a technique that increases the number of copies of a nucleic acid molecule in a sample. An example of amplification is the polymerase chain reaction (PCR), in which a sample is contacted with a pair of oligonucleotide primers under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. This cycle can be repeated. The product of amplification can be characterized by such techniques as electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing.
A mutation refers to a change of a nucleic acid sequence as a source of genetic variation. For example, mutations can occur within a gene or chromosome, including specific changes in non-coding regions of a chromosome, for instance changes in or near regulatory regions of genes. Types of mutations include, but are not limited to, base substitution point mutations (which are either transitions or transversions), deletions, and insertions. Missense mutations are those that introduce a different amino acid into the sequence of the encoded protein; nonsense mutations are those that introduce a new stop codon; and silent mutations are those that introduce the same amino acid often with a base change in the third position of the codon. In the case of insertions or deletions, mutations can be in-frame (not changing the frame of the overall sequence) or frame shift mutations, which may result in the misreading of a large number of codons (and often leads to abnormal termination of the encoded product due to the presence of a stop codon in the alternative frame).
Polymorphism refers to a variation in a gene sequence. The polymorphisms can be those variations (DNA sequence differences) which are generally found between individuals or different ethnic groups and geographic locations which, while having a different sequence, produce functionally equivalent gene products. Typically, the term can also refer to variants in the sequence which can lead to gene products that are not functionally equivalent. Polymorphisms also encompass variations which can be classified as alleles and/or mutations which can produce gene products which may have an altered function. Polymorphisms also encompass variations which can be classified as alleles and/or mutations which either produce no gene product or an inactive gene product or an active gene product produced at an abnormal rate or in an inappropriate tissue or in response to an inappropriate stimulus. Alleles are the alternate forms that occur at the polymorphism.
A “single nucleotide polymorphism (SNP)” is a single base (nucleotide) polymorphism in a DNA sequence among individuals in a population. Typically in the literature, a single nucleotide polymorphism (SNP) may fall within coding sequences of genes, non-coding regions of genes, or in the intergenic regions between genes. SNPs within a coding sequence will not necessarily change the amino acid sequence of the protein that is produced, due to degeneracy of the genetic code. A SNP in which both forms lead to the same polypeptide sequence is termed “synonymous” (sometimes called a silent mutation)—if a different polypeptide sequence is produced they are “nonsynonymous”. A nonsynonymous change may either be missense or “nonsense”, where a missense change results in a different amino acid, while a nonsense change results in a premature stop codon.
Hybridization refers to the ability of complementary single-stranded DNA or RNA to form a duplex molecule (also referred to as a hybridization complex). Nucleic acid hybridization techniques can be used to form hybridization complexes between a probe or primer and a nucleic acid. Hybridizable and hybridizes are terms which indicate a sufficient degree of complementarity such that stable and specific binding occurs between an oligonucleotide and its DNA or RNA target. An oligonucleotide need not be 100% complementary to its target DNA or RNA sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences.
Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art. The primers disclosed herein can hybridize to nucleic acid molecules under low stringency, high stringency, and very high stringency conditions.
As used herein, Ribonuclease H (RNase H) is a family of non-sequence-specific endonucleases that catalyze the cleavage of RNA via a hydrolytic mechanism. Ribonuclease activity for RNase H cleaves the 3′-O—P bond of RNA in a DNA/RNA duplex substrate to produce 3′-hydroxyl and 5′-phosphate terminated products and the RNase H specifically degrades only the RNA in RNA:DNA complex.
As used herein, conjugated refers to either covalently attaching two or objects together, or creating hydrogen bonding interactions, such as hybridization of two nucleic acids, such that the two objects do not substantially dissociate in a solution of water at room temperature and neutral pH.
In certain embodiments, this disclosure relates to devices comprising, a particle, pair of particles, or rod comprising a coating of single stranded DNA; a substrate comprising a coating of single stranded RNA; and an endoribonuclease such as RNase H, wherein the single stranded DNA hybridizes to the RNA on the substrate and the particle, pair of particles, or rod is configured on the substrate such that the particle, pair of particles or rod, moves upon mixing the endoribonuclease with the DNA hybridized to the RNA.
In certain embodiments, the particle is a conglomerate of matter that is preferably spherical in shape. However, it is contemplated that the particle may not be perfectly spherical, e.g. oval or having imperfections. The diameter of a particle refers to the average size of the diameter. Typically the size of the particle is such that the location of the particle can be readily identified by visual or other spectroscopic means on a substrate. The particle may be made of a core material that has covalent bonds, e.g., polymers or resins, or from semiconductor materials, e.g., CdSe, CdS, CdTe quantum dots, metals or oxides thereof, e.g., iron oxide particles.
In certain embodiments, a “rod” refers two or more particles that are joined together to form a length that is two or more times the smallest diameter of one of the particles. The rod may be straight or have a slight bend, for example when three or more particles are joined but do not exist in an absolute straight line. In certain embodiments, a rod may be nanotube or other structure that can be conjugated with DNA.
In the case of quantum dot, metal particle or rod, the outer surface may have a polymer coating that is chemically crosslinked to prevent the polymers from separating from the particle or rod.
In certain embodiments, the “coating of single stranded DNA” refers to the conjugation of a nucleic acid to the outer surface of a particle, pair of particles, or rod wherein at least a portion of the DNA sequence is single stranded. The DNA may be hybridizing to a complementary strand that is used to conjugate the DNA to the particle providing a portion of the DNA that is double stranded.
In certain embodiments, a “substrate” refers to a surface that is stationary with respect to the RNA conjugated thereto. Conjugation of single stranded RNA to the substrate may be by hybridization or by covalently linking the RNA. The surface may be planar or curved so long as the surface area is sufficiently large in relation to any particles or rods placed thereon such that movement of the particles or rods from different locations on the surface can be detected.
Methods of Use
As illustrated in
In certain embodiments, the disclosure relates to methods for moving a particle, pair of particles, or rod comprising DNA, comprising: providing a device comprising, a particle or rod comprising a coating of single stranded DNA; a substrate comprising a coating of single stranded RNA; and an endoribonuclease such as RNase H, wherein the single stranded DNA hybridizes to the RNA on the substrate and the particle, pair of particles, or rod is configured on the substrate such that the particle, pair of particles, or rod moves upon mixing the endoribonuclease with the DNA hybridized to the RNA; placing the single stranded DNA coated particle on the surface of the single stranded RNA coated substrate in the presence of the endonuclease under conditions such that the particle, pair of particles, or rod moves on the surface of the substrate.
In certain embodiments, DNA or RNA is a sequence obtained from a sample. In certain embodiments, the speed of the movement of the particle is correlated to the sequence of the DNA or RNA. In certain embodiments, a maximum speed is associated with complete complementarity. In certain embodiments, a speed of less than the maximum speed is associated with incomplete complementarity. In certain embodiments, a pair of primers are used to obtain a predetermined DNA sequence from a sample of the subject. The primers may target RNA, e.g., mRNA, or DNA that is in the sample.
In certain embodiments, the disclosure contemplates a kit comprising: a pair or primers, a substrate that contains a DNA bound to the surface, and an oligonucleotide conjugated to a fluorescent marker wherein the oligonucleotide has a first segment that is complementary to the DNA bonded to the surface of the substrate and a second segment that is complementary to a DNA sequence that is to be obtained from a sample by application from the pair of primers. The DNA sequence that is to be obtained from the sample is amplified from a pair of primers. The sequences of the primers may be removed prior to placing the single stranded DNA on the particle through the use of sequence specific restrictions enzymes built into the primers.
Other configurations are contemplated such as the particle, pair of particles, or rod having a capture DNA already attached to the particle. In certain embodiments, the disclosure relates to kits comprising: a particle, pair or particles, or rod comprising a capture DNA; a substrate comprising a coating of single stranded RNA; and a pair of primers configures to amplify a nucleic acid that is complementary to the single stranded RNA and an endoribonuclease. In certain embodiments, the pair of primers is a first primer and a second primer, wherein the first primer is a sequence that has a sequence of five or more nucleotides that are identical or complementary to the single stranded DNA conjugated to the particle, pair of particles, or rod and the second primer has a sequence of five or more nucleotides that are identical or complementary to a sequence of the single stranded RNA.
In certain embodiments, the disclosure relates to kits comprising: a particle, pair or particles, or rod comprising a group reactive the 3′ of 5′ end of single stranded DNA; a substrate comprising a coating of single stranded RNA; and a pair of primers configures to amplify a nucleic acid that is complementary to the single stranded RNA and an endoribonuclease.
Design and Synthesis of Spherical RNase H Powered Motors
In certain embodiments, an embodiment of this disclosure is a motor which consists of a DNA-coated spherical particle (5 μm or 0.5 μm diameter particles) that hybridizes to a surface modified with complementary RNA. The particle moves upon addition of RNase H, which selectively hydrolyses hybridized RNA but not single stranded RNA. Since the driving force for movement is derived from the free energy of binding new single stranded RNA that biases Brownian motion away from consumed substrate (
Highly multivalent motors display greater processivity, thus addressing a major limitation of DNA walkers. The spherical particle template allows for the potential to roll, which is a fundamentally different mode for translocation of DNA based machines.
An RNA-monolayer was generated on a substrate by immobilizing a DNA anchor strand to a thin gold film and then hybridizing a fluorescently labeled RNA-DNA chimera strand to the surface. A Cy3 fluorophore at the 3′ RNA terminus was used to optimize RNA density and to detect RNA hydrolysis using fluorescence microscopy (
Given that particle motion is intimately connected with RNase H efficiency and enzyme rates vary when substrates are immobilized, hydrolysis kinetics were measured for a DNA-RNA duplex monolayer. Initially, when measuring the hydrolysis of surface immobilized RNA, RNase H was completely inhibited. Since RNase H contains multiple cysteine residues, it was suspected that enzyme inhibition was due to irreversible binding of the enzyme to the Au surface. To prevent nonspecific binding, the Au surface was passivated with SH(CH2)11(OCH2CH2)6OCH3 (SH-PEG) in order to reduce nonspecific interactions with surface. To test the assumption that RNase H inhibition was due to Au film binding, the DNA monolayer surface was backfilled with SH-PEG under a range of conditions, where the SH-PEG concentration and the passivation time was varied. It was determined that complete surface passivation occurred after 4 hrs of incubation with a 100 μM SH-PEG solution. This was inferred by observing a saturation in the loss of fluorescence of FAM labeled DNA anchor strand. Next, RNase H hydrolysis of surface immobilized RNA duplexed with DNA was investigated under the various passivation conditions by measuring the loss in fluorescence of Cy3 labeled RNA throughout the channel over time. When the channel was SH-PEG passivated for shorter durations (2 hrs), the fluorescence intensity varied significantly across the length of the well; regions near the port where RNase H was added had the lowest intensities, while regions furthest away from this site showed minimal substrate hydrolysis. In contrast, channels that were blocked for 6 hrs showed homogeneous fluorescence intensities indicating uniform RNase H activity levels.
DNA-functionalized particles with a density of ˜91,000 molecules/μm′ (footprint of 11±3 nm 2 per molecule) were synthesized and hybridized to a substrate presenting the complementary RNA strand. The DNA density matched that of the RNA density on the planar substrate to ensure a high degree of polyvalency (˜104 contacts/μm), therefore minimizing motor detachment from the substrate and maximizing run processivity. Particles remained immobile until RNase H was added, which led to rapid translocation of particles across the substrate. This was quantitatively tracked by finding the centroid of the particles in time-lapse brightfield (BF) microscopy at 5 sec intervals (
Unrestricted Particle Motion
Particle motion could occur through three plausible mechanisms: a) walking/sliding, b) hopping, and c) rolling. The hopping mechanism was immediately ruled out upon examination of the continuous fluorescence depletion tracks (
The particle speed histogram contains two populations (
Particles of 5 μm in diameter were used for the majority of experiments. However, note that the rolling mechanism of translocation can be recapitulated with 0.5 μm diameter particles. Similar maximum velocities up to 5 μm/min and average velocities of 1.8±0.4 and 1.9±0.5 μm/min were observed for both 0.5 and 5 μm particles, respectively, showing that the fundamental cog-and-wheel mechanism of rolling is independent of cargo size within the range tested. The less multivalent 0.5 μm particles roll for shorter average run lengths compared to 5 μm diameter particles (˜3 μm versus ˜200 μm), which continue processively moving throughout the 30 min video and even continue moving for over 5 hrs. Increasing the KCl and Mg concentrations to 75 mM and 3 mM, respectively, enhances 0.5 μm particle endurance such that the majority of particles display processive motion for the entire 30 min video. This provides the 0.5 μm particles with an average run length of greater than 25 μm.
To achieve unidirectional transport resembling motor protein motion along a filament, RNA was spatially micro-patterned into 3 μm wide tracks. Particles were then hybridized to the patterned RNA, and RNase H was added to initiate motion. Using BF time-lapse tracking and RNA fluorescence depletion, a subset of particles moved along the 3 μm substrate corral unidirectionaly deflecting away from the PEG-printed regions was observed (
An alternate strategy to achieve linear motion is to limit lateral particle motion by incorporating multiple monowheels on the body of a single chassis. By happenstance, it was noticed that a 1-10% subset of our particles were fused forming dimers, a common byproduct in silica particle synthesis. These particles travelled linearly for distances that spanned hundreds of μm's at a velocity of ˜0.6±0.5 μm/min, n=68 dimer particles (
SNP Detection by Measuring Particle Displacement
Since monowheel motion is sensitive to kon, koff and kcat, particle motion could provide a readout of molecular recognition. Particles displaying the SNP (5′mAmGTAATTAAmUmC3′) traveled ˜60% slower (0.3 μm/min) than identical particles with a perfect match (5′mAmGTAATCAAmUmC3′). This difference in velocity can be attributed to a slower rate of hydrolysis for RNase H to hydrolyze duplexes possessing a single base mismatch. Due to the μm-sized cargo and large distances travelled, even a smartphone camera equipped with an inexpensive plastic lens could detect motion associated with this SNP by recording particle displacement within a short time interval (t=15 min). SNP detection could also be achieved using unmodified DNA (