Patent Application
20250163515
This application contains a Sequence Listing submitted as an electronic text file named “21-1564-WO.xml” having a size in bytes of 21 kb and created on Feb. 24, 2023. The information contained in this electronic file is hereby incorporated by reference in its entirety.
The present invention relates to compositions and methods for amplifying and sequencing circular DNA and, specifically eukaryotic extrachromosomal circular DNA (eccDNA). Specifically, the invention relates to amplifying human extrachromosomal circular DNA for isolating, sequencing, subsequent screening for disease and identifying and providing treatment thereof.
Extrachromosomal circular DNA (eccDNAs) are circular DNA species found in eukaryote genomes ranging from yeast to humans. Although little is known regarding the biological purposes and function of eccDNAs in vivo, it is generally understood that these nucleic acid species carry genetic sequences of protein-coding sequences or repetitive elements with many functions including cell regulation, proliferation, environmental adaptation, genome stability and stimulation of immune responses. It has been postulated that the presence of eccDNAs and their genetic diversity confer fitness advantages. Additionally, cancer studies demonstrate that eccDNAs play a role in driving human tumor malignancy, including metastasis, and drug resistance. Despite these findings in cancerous cells, the specific function of eccDNAs in normal cells remains unknown. The mystery surrounding the role of eccDNA involves multiple factors, including for example lower levels of eccDNA in a cell relative to other forms of DNA, the highly heterogeneous nature of eccDNA itself, and a dearth of known sequences that can used as a basis for further experimentation. Therefore, there exists a need to efficiently amplify and characterize human eccDNA that currently does not exist in the art.
Described herein are compositions, methods, and kits for amplifying and characterizing human extrachromosomal circular DNAs (eccDNA). In some aspects of this invention are provided methods called OREO-seq (OriC-mediated Extrachromosomal Circles (O)).
In one preferred aspect, OREO-seq based sequencing can specifically amplify eccDNAs from biological samples including extracted genomic DNA, permeabilized nuclei, body fluid (including but not limited to plasma, urine, serum, and cerebrospinal fluid) and convert them to templates for long-read sequencing analysis. OREO-seq uses an in vitro assembled transposome carrying a replication origin sequence from the E. coli chromosome (oriC) to specifically tag and exponentially amplify eccDNAs from a eukaryotic cell extract. These methods comprise engineering into eukaryotic DNA a transposon that contains oriC and transposases to catalyze a transposition reaction. Once the transposome (a complete transposon-transposase complex) is introduced to cellular genetic material, it randomly binds and catalyzes a transposition reaction-inserting oriC into both chromosomal DNA as well as eccDNA. However, because eccDNAs are circular by nature and thereby similar to a bacterial genome, exposing the genetic material to the conditions necessary for an isothermal replication cycle reaction causes the eccDNAs that possess the transposed OriC region to replicate while the chromosomal DNA does not.
Additionally, in certain embodiments, treatment with an Exo V nuclease can be used to assist the breakdown of remaining linear products for higher resolution of amplified eccDNA or ecDNA.
Furthermore, in yet another embodiment, the amplification products can be treated with PacI to digest circular background mitochondrial DNA. Mitochondrial DNA digestion permits higher resolution of amplified eccDNA or ecDNA products. These aspects of the inventive methods permit large amounts of eccDNA or ecDNA to be replicated selectively over eukaryotic chromosomal DNA in vitro.
The OriC sequences in the amplified eccDNA can then be used as signatures for targeted capture and subjected for long-read sequencing. Once purified, the eccDNA can be cleaved at engineered predetermined locations by CRISPR/Cas9 or restriction enzymes along the engineered OriC sequences resulting in purified linear strands of eccDNA or ecDNA. Various sequencing methods, such as long-read sequencing, can provide full length eccDNA or ecDNA structure characterization which can assist in elucidating the functions of said DNA products and associated disease product therein.
These and other features, objects, and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention. The description of preferred embodiments is not intended to limit the invention to cover all modifications, equivalents, and alteratives. Reference should therefore be made to the claims recited herein for interpreting the scope of the invention.
For the purposes of explicating and understanding the principles of this disclosure, reference is made to embodiments and specific language used to describe the same. The skilled artisan will nevertheless understand that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would be understood by one skilled in the art to which the disclosure relates.
As used herein, articles “a” and “an” are intended to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element. “About” is used to provide flexibility to a numerical range endpoint by providing that a given value can be “slightly above” or “slightly below” the endpoint without affecting the therapeutically beneficial result. The term “about” in association with a numerical value means that the numerical value can vary by plus or minus 5% or less of the numerical value.
Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements, or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).
Recitation of ranges of values herein are merely intended to serve as a succinct method of referring individually to each separate value falling within the range, unless otherwise indicated herein. Furthermore, each separate value is incorporated into the specification as if it were individually recited herein. For example, if a range is stated as 1 to 50, it is intended that values such as 2 to 4, 10 to 30, or 1 to 3, etc., are expressly enumerated in this disclosure. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this disclosure belongs.
Provided herewith is a more detailed description of the compositions, methods, and kits comprising the invention, which is provided to explain and enhance but not replace or be a substitute for the claims set forth below.
There are multiple names for different types of small circular DNA found in eukaryotes. As discussed above, eccDNA is extrachromosomal circular DNA, typically referring to such extrachromosomal circular DNA found in mammals. By contrast, ecDNA is extrachromosomal DNA typically associated with oncogenesis and tumor proliferation. eccDNA can be either oncogenic, or healthy, but ecDNA is usually associated with cellular disease-states, particularly cancer.
Complete enzymatic replication of plasmids containing the origin of Escherichia coli chromosomal replication has been described extensively in the art. See Funnel et al., 1986, J Biol Chem. 261: 5616-5624. For example, Funnel and colleagues described reconstitution of a complete cycle of oriC plasmid replication in vitro using purified proteins, beginning and ending with supercoiled molecules. However, in the past, such replication resulted in limited products.
A common method for amplifying DNA in vitro is the polymerase chain reaction (PCR). However, PCR is limited to amplifying linear DNA as the proteins involved in the process do not naturally possess the capabilities of replicating circular DNA. In vitro circular DNA amplification has been achieved by a separate mechanism used by phage λ, namely the rolling circle amplification process (RCA). The amplification product obtained by the RCA method is a linear DNA in which the DNA sequence of one round of circular DNA is repeated and is used exclusively for preparing template DNA for additional replication.
Researchers have identified many unique plasma eccDNAs in human blood plasma, but characterization of such eccDNAs has continued to be elusive. Zhu et al., 2017, Sci Reports 7:10968.
Wang and colleagues have identified that eccDNAs are apoptotic products with high innate immunostimulatory activity. See Wang et al., 2021, Nature 599: 308-317. They showed that in mouse cell lines, eccDNA could be isolated and then purified using ligase-assisted minicircle accumulation (LAMA). Wang and colleagues linearized eccDNA products using sequential treatments of nickase fnCpf1 in addition to S1 nuclease and used nanopore sequencing for preparing eccDNA libraries.
Fakruddin and colleagues discuss loop-mediated isothermal amplification as an alternative to PCR. See Fakruddin et al., 2013, J Pharm Bioallied Scie. 5: 245-252.
Peng and Marians further demonstrated that Escherichia coli rely on topoisomerase IV encoded by parC and parE for separation of the daughter chromosomes in chromosomal replication. Peng & Marians, 1993, Proc. Natl. Acad. Sci. USA 90: 8571-8575.
Additionally, decatenation of the linked daughter chromosomes after replication is an important step in oriC-mediated DNA replication. In the oriC replication system, DNA gyrase-catalyzed decatenation of daughter DNA molecules is very inefficient while topoisomerase III is capable of complete decatenation. Hiasa et al., 1994, J. Biol Chem 269: 2093-2099. In contrast topoisomerase I is incapable of decatenation, and instead, only inhibits DNA synthesis.
Researchers have demonstrated that plasmid DNA templates containing oriC and two Ter sites can be used to study the role of Tus in terminating bidirectional replication. See Hiasa et al., J. Biol Chem 269: 26959-26968. Hiasa and colleagues showed that in the absence of Tus, over-replication required DNA ligase and arose via a template strand-switching mechanism.
Researchers have also identified critical enzymes to accomplish the replication cycle of a circular chromosome absent the formation of a linearized product. See Masayuki et al, 2017, Nucleic Acids Research 45: 11525-11534. Masayuki and colleagues were able to reconstitute the entire replication cycle with fourteen purified enzymes that catalyze initiation at oriC, bidirectional fork progression, Okazaki-fragment maturation, and decatenation of the replicated circular products. Using the replication-cycle reaction (RCR) circular DNA was capable of being replicated exponentially as intact covalently closed molecules.
Finally, Nara and Masayuki have shown that it is possible to amplify whole large plasmids via transposon-mediated oriC insertions in vitro. See Nara & Masayuki, 2021, BioTechniques 71: 1-6. Specifically, Nara and Masayuki demonstrated that by using a replication cycle reaction catalyzed by a hyperactive Tn5 transposase, and an oriC-containing transposon, that circular DNA could be replicated efficiently.
The invention as disclosed and claimed herein will be understood by the skilled worker in the art in the context of the understanding in the art of eccDNA, ecDNA, and the ability to replicate bacterial and plasmid DNA using species-specific origins of replication and bacterial enzymes that facilitate such replication.
In one embodiment, the invention comprises a composition further comprising a plurality of eukaryotic extrachromosomal circular DNA (eccDNA) or eukaryotic circular DNA isolated from a cancerous cell, (ecDNA), wherein the circular DNA species have been modified by insertion of a prokaryotic origin of replication.
The composition of circular DNA can be further modified to contain a plurality of circular DNA species that are capable of being amplified by replication in vitro mediated by a prokaryotic origin of replication.
The origin of replication can be a bacterial origin of replication, in particular E. coli OriC. Specifically, the origin of replication can be imbedded into a transposable cassette. The transposable cassette can comprise the following nucleic acid sequence:
5′-CTGTCTCTTATACACATCTgaagatccggcagaagaatggagtatgttgtaactaaagataacttcgtataatgtatgct atacgaagttatacagatcgtgcgatctactgtggataactctgtcaggaagcttggatcaaccggtagttatccaaagaacaactgttgt tcagtttttgagttgtgtataacccctcattctgatcccagcttatacggtccaggatcaccgatcattcacagttaatgatcctttccaggtt gttgatcttaaaagccggatccttgttatccacagggcagtgcgatcctaataagagatcacaatagaacagatctctaaataaatagatc ttctttttaatacccaggatccatctatgtcgggtgcggagaaagaggtaatgaaatggctttagttacaacatactcaggtctttctcaagc cgacAGATGTGTATAAGAGACAG-3′ (SEQ ID NO:7). Other origins of replication can be used as well.
The plurality of circular DNA is produced by enzymatic amplification, in particular isothermal replication cycle reaction (RCR) amplification in vitro. RCR can be carried out by various protocols known to one of ordinary skill in the art. For example, a technique as described by Masayuki and colleagues, Masayuki et al., 2017, Nucleic Acids Research 45: 11525-11534, which is hereby incorporated by reference in its entirety, can be used to amplify eccDNA and ecDNA.
Alternatively, RCR can be carried out using a commercial kit such as the OriCiro® Cell-Free Cloning System provided by OriCirco genomics, the experimental protocol which is herein incorporated by reference in its entirety.
In some embodiments of the invention, the cells from which the eccDNA and ecDNA are isolated are eukaryotic cells. In yet another preferred embodiment, the cells from which the eccDNA or ecDNA are isolated are mammalian cells. In an exemplary embodiment of the present invention, the cells from which the eccDNA or ecDNA are isolated are human cells. Circular DNA obtained from prokaryotic cells can too be utilized in the present invention.
In one embodiment, next generation DNA sequencing, for example, ultra-long read sequencing provided by Oxford Nanopore Technologies can be used to sequence the amplified and isolated eccDNA and ecDNA. See Kazuo et al., 2020, Front. Cell. Infect. Infect. Microbiol. 10: 11. Alternatively, eccDNA or ecDNA can be sheared into 100-300 bp by sonication. The sheared DNA can then be subjected to ThruPLEX® DNA-seq kit provided by Rubicon Genomics. Sequencing can be further assisted with software such as that provided by DNASTAR.
In another embodiment, the eukaryotic circular DNA can be linearized in preparation for sequencing with a CRISPR-associated protein such as Cas9 and an associated guide RNA (crRNA). Other CRISPR associated proteins can be utilized as well such as Cpf1, dCas, dCas9, Mutated Cpf1-dCpf1, and associated crRNAs and trcrRNAs. In yet another embodiment, the eukaryotic circular DNA can be linearized with a restriction enzyme that would be known to one of ordinary skill in the art. In some exemplary embodiments, a restriction site can be engineered into the oriC transposition primers that will later be inserted into the circular DNA sets. In other exemplary embodiments, the restriction site can correspond to SrfI. In other exemplary embodiments, the restriction site can correspond to PmeI.
In some embodiments, the eukaryotic DNA can be cancerous DNA, (ecDNA). ecDNA can be attributable to a plurality of different types of cancers. In other embodiments the circular eukaryotic DNA is not an oncogenic eccDNA but can code for other disease processes.
In some embodiments, the circular DNA is isolated from a tissue. Tissues include but are not limited to neural tissue, thyroid papillary or medullary tissues, esophageal tissues, squamous cell epithelium from the mouth, tongue, nasopharynx, vagina, cervix, or rectum, tissue from the dermis, venous tissue, myocytes, cardiomyocytes, tissue isolated from the thymus, blood, lymphatic fluid, bone, marrow, connective tissues such as fibrocartilage, hyaline cartilage, and other proteoglycan matrix like tissues, smooth muscle tissues such as those isolated from the stomach, or other tissues such as those present in the duodenum, the small intestine, the large intestine, the colon, the prostate, the testes, the ovaries, the cervix or the uterus. Additionally, tissues isolated from the liver, pancreas, gallbladder, lungs, bladder, breasts, mammillary glands, corneas, or sclera can be suitable sources for isolating circular DNA.
Additionally, in yet other embodiments, the tissue is isolated from a tumor or biopsy thereof. The tissue can comprise cancerous cells isolated from blood of a patient with leukemia or another associated cancer of the blood. In other embodiments, the tissue can comprise cancerous cells isolated from the blood of a patient with a lymphoma or other associated cancer of the lymphatic system.
In yet another embodiment, the circular DNA can be isolated from a biofluid. A biofluid can be any fluid secreted or produced from the body during normal physiologic processes as well as disease processes. Biofluids include but are not limited to sweat, blood, plasma, urine, lymph, semen, mucous, tears, colostrum, cerebral spinal fluid, amniotic fluid, bile, chime, puss, sebum, glandular excretions, and waste products such as excrement or feces.
Some embodiments of the invention comprise a kit comprising nucleic acid encoding a prokaryotic origin of replication, protein components specific for the prokaryotic origin of replication and capable of replicating DNA comprising said prokaryotic origin of replication, and a transposase capable of inserting the prokaryotic origin of replication into human-derived eccDNA, and associated reaction buffers and reagents. The prokaryotic origin of replication can be OriC. Alternatively, other prokaryotic origins of replication can be included in the transposon. The protein components specific for the prokaryotic origin of replication, capable of replicating the DNA, comprise: SSB, IHF, DnaG, DnaN, PolIII, DnaB, RNaseH, Ligase, Poll, GyrA, GyrB, Topo IV, Topo III, and RecQ. As set forth herein, SSB indicates SSB derived from E. coli, IHF indicates a combination of IhfA and IhfB derived from E. coli, DnaG indicates DnaG derived from E. coli, DnaN indicates DnaN derived from E. coli, PollIl indicates DNA polymerase III complex consisting of a combination of DnaX, HolA, HolB, HolC, HolD, DnaE, DnaQ, and HolE, DnaB indicates DnaB derived from E. coli, DnaC indicates DnaC derived from E. coli, DnaA indicates RNaseH derived from E. coli, Ligase indicates DNA ligase derived from E. coli, Poll indicates DNA polymerase I derived from E. coli, GyrA indicates GyrA derived from E. coli, GyrB indicates GyrB derived from E. coli, Topo IV indicates a combination of ParC and ParE derived from E. coli, Topo III indicates topoisomerase III derived from E. coli, and RecQ indicates RecQ derived from E. coli.
SSB can be prepared by purifying an E. coli strain expressing SSB by steps that include ammonium sulfate precipitation and ion-exchange column chromatography.
IHF can be prepared by purifying an E. coli strain coexpressing IhfA and IhfB by steps that include ammonium sulfate precipitation and affinity column chromatography.
DnaG can be prepared by purifying an E. coli strain expressing DnaG by steps that include ammonium sulfate precipitation and anion-exchange column chromatography and gel filtration column chromatography.
DnaN can be prepared by purifying an E. coli strain expressing DnaN by steps that include ammonium sulfate precipitation and anion-exchange column chromatography.
PolIII can be prepared by purifying an E. coli strain coexpressing DnaX, HolA, HolB, HolC, HolD, DnaE, DnaQ, and HolE by steps that include ammonium sulfate precipitation, affinity column chromatography and gel filtration column chromatography.
DnaB and DnaC can be prepared by purifying an E. coli strain coexpressing DnaB and DnaC by steps that include ammonium sulfate precipitation, affinity column chromatography and gel filtration column chromatography.
DnaA can be prepared by purifying an E. coli strain expressing DnaA by steps that include ammonium sulfate precipitation, dialysis precipitation, and gel filtration column chromatography.
GyrA and GyrB can be prepared by purifying a mixture of an E. coli strain expressing GyrA and an E. coli strain expressing GyrB by steps that include ammonium sulfate precipitation, affinity column chromatography and gel filtration column chromatography.
Topo IV can be prepared by purifying a mixture of an E. coli strain expressing ParC and an E. coli strain expressing ParE by steps that include ammonium sulfate precipitation, affinity column chromatography and gel filtration column chromatography.
Topo III can be prepared by purifying an E. coli strain expressing Topo III by steps that include ammonium sulfate precipitation, and affinity column chromatography.
RecQ can be prepared by purifying an E. coli strain expressing RecQ by steps that include ammonium sulfate precipitation, affinity column chromatography and gel filtration column chromatography.
Commercially available enzymes derived from E. coli can be used for RNaseH, Ligase and Poll, for example those provided by Takara Bio Inc. In the use of these enzymes as set forth in instructions provided by the manufacturer and as further set forth in U.S. Pat. No. 10,301,672 to Su'etsugu et al. at col. 13, ln 32-50, a 9.6 kb circular DNA can serve as a template having a replication origin sequence oriC and a portion coding for kanamycin resistance. Alternatively, an 80 kb long circular DNA having a replication origin sequence oriC and a portion encoding a kanamycin resistance gene can be used as a template. In yet another alternative, the template can comprise a 200 kb long chain circular DNA having a replication origin sequence oriC and encoding a kanamycin resistance gene. DNA cyclized by in vitro ligation can also serve as a template.
The template long chain circular DNAs can be prepared by in vivo recombination in E. coli cells. Specifically, an in vivo recombination can be performed using E. coli expressing a recombination protein group of a k phage to prepare circular DNA of a desired length that would also include a cassette coding for kanamycin resistance and a region in the E. coli chromosome that includes oriC.
In a preferred embodiment, the kit comprises a hyperactive Tn5 transposase, and an oriC-containing transposon, that catalyze a transposition reaction into target eccDNA and ecDNA.
In yet another embodiment, the kit can further comprise a purified preparation of CRISPR associated proteins (Cas) and include a corresponding guide mRNA or trcrRNA. The Cas protein can comprise Cas9, dCas, mutated Cas9-nickase, Cpf1, mutated Cpf1-dCpf1, C2c2-Cas13a-mutants, other Cas proteins, and other mutated Cas varieties.
In yet another embodiment, the invention provides methods for producing a plurality of extrachromosomal circular DNA (eccDNA) or (ecDNA) isolated from a eukaryotic cell comprising the steps of (1) isolating cellular DNA comprising circular DNA from the cell; (2) modifying the circular DNA comprising eccDNA or ecDNA by inserting the prokaryotic origin of replication into the isolated circular DNA; (3) amplifying in vitro the modified circular DNA comprising eccDNA or ecDNA; and (4) isolating the amplified eccDNA or ecDNA. The method can further comprise inserting a prokaryotic origin of replication into the isolated circular DNA by a transposase.
In yet another embodiment of the invention, the method can utilize a prokaryotic origin of replication such as E. coli's OriC.
In another preferred embodiment of a method of the invention, the method can utilize a transposon that is assembled with a set of linear DNA oligonucleotide primers. The linear DNA oligonucleotide primers can comprise SEQ ID: 1 and SEQ ID: 2. Alternatively, the linear DNA oligonucleotide primers can comprise SEQ ID: 3 and SEQ ID: 4. In yet another embodiment, the linear DNA oligonucleotide primers can comprise SEQ ID: 5 and SEQ: ID: 6.
SEQ ID: 1 in Table 1 above corresponds to a 19 base pair mosaic end used as a primer to synthesize DNA templates of a cassette that contains a prokaryotic transposable origin of replication. In a preferred embodiment, that transposable prokaryotic origin of replication is OriC.
SEQ ID: 2 in Table 1 above corresponds to a 19 base pair mosaic end used as a primer to synthesize DNA templates of a cassette that contains a prokaryotic transposable origin of replication. In a preferred embodiment, that transposable prokaryotic origin of replication is OriC.
SEQ ID: 3 in Table 1 above corresponds to a 19 base pair mosaic end used as a primer to synthesize DNA templates of a cassette that contains a prokaryotic transposable origin of replication that also contains a restriction site. In SEQ ID: 3, that restriction site is SrfI.
SEQ ID: 4 in Table 1 above corresponds to a 19 base pair mosaic end used as a primer to synthesize DNA templates of a cassette that contains a prokaryotic transposable origin of replication that also contains a restriction site. In SEQ ID: 4, that restriction site is SrfI.
SEQ ID: 5 in Table 1 above corresponds to a 19 base pair mosaic end used as a primer to synthesize DNA templates of a cassette that contains a prokaryotic transposable origin of replication that also contains a restriction site. In SEQ ID: 5, that restriction site is PmeI.
SEQ ID: 6 in Table 1 above corresponds to a 19 base pair mosaic end used as a primer to synthesize DNA templates of a cassette that contains a prokaryotic transposable origin of replication that also contains a restriction site. In SEQ ID: 6, that restriction site is PmeI.
In another preferred embodiment of a method of the invention, the method can utilize an isothermal replication cycle reaction (RCR) amplification in vitro to amplify the plurality of eccDNA or ecDNA.
In one embodiment of methods of the invention, the method comprises producing a plurality of extrachromosomal circular DNA (eccDNA) or (ecDNA) isolated from a eukaryotic cell comprising the steps of (1) isolating circular DNA comprising eccDNA or ecDNA from the cell; (2) modifying the circular DNA comprising eccDNA or ecDNA by inserting a prokaryotic origin of replication into the isolated circular DNA; (3) applying an Exo V endonuclease to remove linearized non-circular DNA; (4) applying PacI to linearize the circular mitochondrial DNA; (5) amplifying in vitro the modified circular DNA comprising eccDNA or ecDNA; and (6) isolating the amplified eccDNA or ecDNA. In the described method, the circular DNA comprising eccDNA or ecDNA is human. Additionally, the human eccDNA or ecDNA is isolated from a tissue. The tissue can be a tumor or biopsy thereof. The human eccDNA or ecDNA can also be isolated from a biofluid. As described above, the biofluid can be blood or cerebral spinal fluid (CSF).
In yet another embodiment, the invention provides methods for identifying a nucleic acid sequence of eukaryotic extrachromosomal circular DNA (eccDNA) or (ecDNA) and performing nucleic acid sequencing on the amplified DNA products. The eukaryotic extrachromosomal circular DNA (eccDNA) or (ecDNA) can be isolated from a human cell, tissue, or biofluid. Additionally, the human cell, tissue, or biofluid can be in either a normal physiological state, or alternatively, in a diseased state.
In an exemplary embodiment of a method of the present invention, eukaryotic extrachromosomal circular DNA (eccDNA) or (ecDNA) that has been amplified can be linearized with Cas9 or a restriction enzyme before sequencing, and the sequencing of the amplified DNA product can assist a medical professional or patient to determine whether the patient carries extrachromosomal circular DNA (eccDNA) or (ecDNA) from a cell, tissue, or biofluid sample indicative of a disease state therein. The method of sequencing can be nanopore long-read sequencing. In yet another embodiment the sequencing can serve to screen a cancer patient having metastatic disease for treatment options based on the genetic composition of the patient's diseased sample of ecDNA.
In a particular exemplary embodiment of the methods of the invention, the invention provides a method for identifying a cancer patient having metastatic disease for treatment comprising the steps of (1) isolating circular DNA (ecDNA) from the patient; (2) modifying the ecDNA by inserting a prokaryotic origin of replication into the isolated circular DNA via a transposition reaction; (3) applying an Exo V endonuclease to remove linearized non-circular DNA; (4) applying Pac to linearize the circularized mitochondrial DNA; (5) amplifying in vitro the modified circular DNA comprising ecDNA; (6) purifying the amplified ecDNA; (7) linearizing the amplified ecDNA with a Cas9 enzyme or restriction enzyme; (8) characterizing the amplified product of step 6 using a nanopore long-read sequencing technique; and (9) using the characterization of step 7 to of identify a cancer patient having metastatic disease.
Various exemplary embodiments of compositions and methods according to this invention are now described in the following non-limiting Examples. The Examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims.
To generate an OriC containing transposome, OriC DNA oligos were first amplified using primer pairs SEQ ID: 1 and SEQ ID: 2 listed in Table 1 to introduce 19-bp mosaic ends to the OriC cassette. The OriC cassette was obtained from an OriCiro Cell-Free Cloning System Kit (OriCiro Genomics). Additional OriC DNA oligos aside from SEQ ID:1 and SEQ ID:2 can be used. For example, SEQ ID:3, SEQ ID:4, SEQ ID:5, SEQ ID:6 can also be used in the transposition reaction. SEQ ID:3 through SEQ ID:6 were engineered with restriction sites embedded within the DNA oligo to allow linearization of amplified circular products for long read sequencing treatment after transposition and amplification.
The engineered OriC DNA oligos were then amplified using a polymerase chain reaction (PCR). PCR can be performed by a plurality of protocols that would be known to one of ordinary skill in the art. Specifically. PCR was performed using a KAPA HiFi HotStart ReadyMix kit and protocol (#7958927001, Roche).
The amplified OriC DNA oligo products were then purified. Purification can be accomplished by a plurality of protocols that would be known to one of ordinary skill in the art. Specifically, the OriC DNA oligos were purified using a SPRI bead system (Beckman Coulter).
The transposition reaction was initiated using the following protocol, OriC DNA oligos (2.36 pmol) were loaded with a 1 μl aliquot solution of hyperactive Tn5 transposase, Diagenode Tagmentase (C01070010, Diagenode), and incubated at 30° C. for 30 min in a thermocycler. Additional OriC DNA oligos aside from SEQ ID:1 and SEQ ID:2 can be used. For example, SEQ ID:3, SEQ ID:4, SEQ ID:5, SEQ ID:6 can also be used in the transposition reaction.
A replication cycle reaction can be prepared using general protocols known by one of ordinary skill in the art. For example, the following protocol by Masayuki et al, Nucleic Acid Res 45(20), 11525-11534 (2017), is generally sufficient to accomplish RCR. As detailed by Masayuki, a 5× replication-cycle reaction (RCR) buffer (100 mM Tris-HCl pH8.0, 750 mM potassium glutamate, 50 mM ammonium sulfate, 50 mM Mg(OAc)2, 40 mM dithiothreitol (DTT), 20 mM creatine phosphate, 5 mM each NTP, 0.5 mM each dNTP, 0.25 mg/ml yeast tRNA, and 1.25 mM NAD+) and a 4× Enzyme mix (2 mg/ml bovine serum albumin, 0.08 mg/ml creatine kinase, 0.1 mM adenosine triphosphate (ATP), 1.6 M SSB4, 160 nM IHF2, 1.6 M DnaG, 160 nM DnaN2, 20 nM Pol III, 80 nM DnaB6C6, 400 nM DnaA, 40 nM RNaseH, 200 nM ligase, 200 nM Pol I, 200 nM gyrase (GyrA2B2), 20 nM Topo IV (ParC2E2), 200 nM Topo III and 200 nM RecQ). The reaction mixture (final 10 l, unless otherwise noted) included 5×RCR buffer (2 l) and 4× Enzyme mix (2.5 l) and was assembled on ice. After addition of oriC-containing circular DNA, the reaction was incubated at 30° C. for the indicated times. [−32P]dATP was included at 40-100 cpm/pmol of total deoxynucleotides when indicated. The reaction was stopped by addition of an equal volume of 2× Stop buffer (50 mM Tris-HCl pH8.0, 50 mM ethylenediaminetetraacetic acid, 0.2% sodium dodecyl sulphate, 0.1 mg/ml proteinase K, 10% glycerol and 0.2% bromophenol blue), and further incubated at 37-C for 30 min followed by phenol-chloroform extraction. An aliquot (2 l) was analyzed by 0.5% agarose-gel electrophoresis followed by SYBR Green I staining (Molecular Probes) or by phosphor imaging. The images were acquired with a Typhoon FLA 9500 (GE Healthcare). The 4× Enzyme mix can be stored at −80° C. after rapid freezing with liquid nitrogen and its activity is retained even after five freeze-thaw cycles.
Specifically, the transposition reaction was performed using 100 ng of purified DNA by incubating the reaction mix at 55° C. for 7 min. Subsequently, 1l of transposed product mixture was amplified using the Amplification Reaction specified in the OriCiro Cell-Free Cloning System Kit (OriCiro Genomics). The reaction mixture was then incubated at 33° C. for a period of 6-16 hr. The amplified products (eccDNA or ecDNA) were analyzed by 0.5% agarose gel electrophoresis and staining protocols known to one of ordinary skill in the art, (e.g. ethidium bromide staining or staining with SYBR safe DNA gel stain obtained from Thermo Fisher). Images were acquired using Chemidoc (Biorad).
In certain embodiments the extracted cellular DNA is further treated with PacI and ExoV, wherein PacI cleaves circular mitochondrial DNA and Exo V further digest linear DNA products. For PacI treatment, 1 ug of DNA was treated with 15 U PacI (NEB) for 1 hr at 37° C. For EvoV treatment, 1 ug of DNA was treated with 10 U for 30 min at 37° C. In samples wherein both enzymes were applied. PacI treatment was performed followed by ExoV treatment. Results of these experiments are shown in
The circular DNA products were further purified from amplified products via phenol/chloroform extraction. For sequencing, multiple protocols can be used. For example, Illumina Novaseq 6000 (Illumina sequencing) can be used to sequence the amplified and purified products. For Illumina sequencing, 10-100 ng of DNA were sheared to 400 bp using a LE220 focused-ultrasonicator (Covaris) and then subject to size selection using a SPRI bead system (Beckman Coulter). The fragments were further treated with end-repair, A-tailing, and ligation of Illumina unique adapters (Illumina) using the KAPA Hyper Prep Kit (Illumina) (Roche). The now linear isolated products were further subjected to 5-10 cycles of PCR amplification. Libraries were then prepared by sequencing using 2×l50 bp on an IlluminaNov aseq 6000 sequencing system.
For sequencing, long read sequencing can also be used to characterize the amplified products. For example, the circular DNA products were further purified from amplified products via a phenol/chloroform extraction. 1 μg-5μg DNA samples were digested using the enzymes SrfI (NEB) or PmeI (NEB) to linearize the circular DNA prepared from transposition cassettes that were based on SEQ ID:3 through SEQ ID:6 DNA oligo inserts. The digested DNA was first treated with NEBNext FFPE Repair Mix and then ligated with an adaptor using Ligation Sequencing Kit SQK-LSK110 (Oxford Nanopore Technologies). The linearized DNA were then ligated to an adaptor using a Ligation Sequencing Kit SQK-LSK110 (Oxford Nanopore Technologies). The libraries were then sequenced on the flowcell R9.4.1 (FLO-MIN106, ONT) on a GridION (ONT) using the MinKNOW software for 48 hr.
Alternatively, for long read sequencing, a guide RNA was designed to target the OriC sequences to linearize the circular DNA instead of treatment with restriction enzymes. For this approach, the purified DNA was first dephosphorylated using Quick Calf Intestinal Phosphatase (NEB). After dephosphorylation, the Cas9 complex was introduced where the target OriC site was to be cut. The linearized DNA will be ligated the adaptor using Ligation Sequencing Kit SQK-LSK110 (Oxford Nanopore Technologies). The libraries can be sequenced on the flowcell R9.4.1 (FLO-MIN106, ONT) on a GridION (ONT) using the MinKNOW software for 48 hr.
For sequencing performed using Sequel II from Pacific Biosciences, DNA was prepared as follows. A library of these sequences can be constructed using PacBio SMRTbell Express Template Prep Kit 2.0 and the SMRTbell Enzyme Cleanup Kit (Pacific Biosciences, Menlo Park, CA). Briefly, 1 μg of Srft-digested DNA is treated for DNA damage repair and end repair/A-tailing. The repaired/modified DNA is ligated with SMRTell adapters and purified by Ampure PB using the manufacturer's instruction. The resulting adapter ligated library is subsequently treated with Exonuclease V to remove damaged or non-intact SMRTbell templates. The purified library is then quantified and thereafter sequenced using Sequel II (Pacific Biosciences).
The presence of a nucleic acid the sequence of which was determined according to the methods set forth herein in a sample from a cell or cellular nucleic acid extract was determined as follows. An eccDNA molecule from a cell or cellular nucleic acid extract was converted according to the methods set forth herein to contain an oriC sequence, then amplified and linearized to produce a fragment having portions of the oriC sequence at each end. Following sequence determination from that linearized fragment a pair of primers was produced that, if subject to PCR would produce non-overlapping fragments extended away from one another. Accordingly, any linear fragment subjected to PCR using these primers were not amplified. When these primers were then used for PCR amplification of the eccDNA molecule in the sample from a cell or cellular nucleic acid extract, production of a PCR-amplified product indicated that the sequence was present as an eccDNA in the cell or cellular nucleic acid extract, as illustrated in
Efficient sequencing of eccDNA using the methods of the invention was illustrated as shown.
To further establish that the OriC based transposase methods disclosed herein would amplify circular DNA, plasmid pUC19 was used in the presence (righthand side of
The effect of OrC-mediated amplification on circular and linear DNA was further illustrated in
Amplification of circular DNA isolated from cells transfected with a 5.5 kb plasmid is shown in
To evaluate whether circular DNA could be detected from normal animal tissue, DNA was extracted from heart muscle from 6 C57B1 mice (3 males and 3 females) and subjected to OrnC-mediated amplification as disclosed herein. The results, shown in
Amplification conditions were interrogated under circumstances where a plurality (5) plasmids were amplified together in a single reaction. The plasmids used in this experiment are shown in the table in
Various tissues from two separate mice were analyzed for the presence of eccDNA using the methods set forth herein. As shown in
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application claims priority to U.S. Provisional Patent Application No. 63/313,549 filed on Feb. 24, 2022, which is incorporated herein in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/063268 | 2/24/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63313549 | Feb 2022 | US |
