Pending Patent-Biosynthetic Plasmid Integrated Capture
Summary of Invention
BioSPLICE is the acronym for Biosynthetic Plasmid Integrated Capture Element. It represents the culmination of basic and applied research to solve the problem of generating a biological system that can detect, identify and neutralize target biological agents in a seamless manner. The final “supersystem” is self-contained, self-replicating and capable of performing the aforementioned functions without extensive reagents or instrumentation. The supersystem has a number of options for reporting and neutralizing the target biological agent that can incorporate sophisticated external instrumentation (Charles River Device, PNNL Green Box, GE DVD Device, GE RFID concept device, microtiter plate reader for visible light absorption or fluorescence, or a luminometer for thermochemiluminescence for “reading” reporters; and cold plasma, pulsed microwave devices or UVA or visible light irradiatiors for neutralization) or employ the simplest physical and chemical means (color or color intensity change, visible to the eye, when bound to a target agent or activation of bound reporter with hydrogen peroxide and bicarbonate for killing with heat or sunlight). This versatility and flexibility combination is the key most desirable advantage of BioSPLICE. BioSPLICE can be used to select new targeting elements in the field and retain this new signature for further replication and utilization and still provide biologically encoded information that can be sent back to the rear echelon.
BioSPLICE involves a recombinant plasmid vector containing a DNA aptamer insert (DNA Capture Element) in the host bacterium species/strain Escherichia coli JM109 or HB101, or a mammalian cell. This plasmid can be extracted and bound specifically to a molecular target (i.e. protein, polysaccharide, lipid, or nucleic acid) immobilized covalently or non-covalently on a paramagnetic metallic nanoparticle. The binding can occur prior to or after plasmid extraction because such nanoparticles can penetrate living microbes or cells. If a random collection of DCEs are present as plasmid inserts, the one that binds best to the molecular target on the nanoparticle will be preferentially collected. After magnetic separation of the nanoparticles, they are added to a parent E. coli that does not contain the plasmid and is, therefore, not resistant to ampicillin or another antibiotic to which the plasmid conveys resistance. After sufficient incubation time for the E. coli to take up the plasmid-coated nanoparticles (uncoated nanoparticles will also be taken up), the bacteria are plated on solid media (agar) or on a media impregnated filter containing ampicillin alone or LB agar plates with 100µg/ml ampicillin. Only the nanoparticles that successfully bound the plasmid specifically through the DCE-molecular target link and transferred an expressible plasmid to the recipient parental host will grow on the ampicillin medium. The surviving clones can be selected and amplified by further culture in liquid growth medium. The DNA can be extracted from this culture and the DCE can be cut out of the plasmid and sequenced to reveal the specific DCE selected. When these surviving clones are grown on 3-AT medium, they will produce diazoluminomelanin (DALM) which will be biosynthetically linked through the plasmid DNA containing the specific DCE to the target microbe. Upon activation with ultraviolet (UVA or UVB) light, sodium bicarbonate and hydrogen peroxide and heat or microwave energy, the target will be killed or neutralized. These DALM/plasmid/DCE complexes can be used also to detect and identify the target microbe by specific binding detected by thermochemiluminescence, slow fluorescence, visible light absorption, or colorimetric means. The magnetic property of the nanoparticles, which bind non-covalently to the DALM/plasmid/DCE, can be used to concentrate the complex bound to the target for detection, identification, or further analysis. Finally, the DALM/plasmid/DCE complex can be used to target the free-radical destruction of target agent facilitated by microwave radiation, UV and visible light, and chemical reductants and oxidants.
Background
Aptamers are currently made by the SELEX method that requires immobilizing a purified target on a chromatographic column or a filter, passing a library of random single-stranded DNA or RNA sequences through the column under binding conditions, separating the bound from the unbound fraction, eluting the bound sequences from the column, and amplifying the sequences by PCR or reverse transcription PCR. The process is repeated as many times as necessary to reduce the number of different sequences by increasing the stringency of binding conditions. Finally, the remaining sequences are cloned into bacterial plasmids and replicated and subsequently cut out by restriction endonucleases and sequenced on an automated sequencing machine. This process differs from the one proposed by requiring sophisticated equipment and laboratory space. Any use of these sequences for reporting the presence of a target ligand requires chemical modification of the bases for linking the fluorescent, luminescent or enzymatic reporting molecules. None of these processes are easily adapted for field use under minimal supporting conditions.
Description, Manner and Process of Making and Using Invention
BioSPLICE involves a recombinant plasmid vector containing a DNA aptamer insert (DNA Capture Element) in the host bacterium species/strain Escherichia coli JM109 (see attached genetic maps). This plasmid can be extracted and bound specifically to a molecular target (i.e. protein, polysaccharide, lipid, or nucleic acid) immobilized covalently or non-covalently on a paramagnetic metallic nanoparticle. If a random collection of DCEs are present as plasmid inserts, the one that binds best to the molecular target on the nanoparticle will be preferentially collected. The binding, in either case, may need to be facilitated by heating the plasmid to 95C to unwind and expose the supercoiled aptamer DNA sequence, contained in the plasmid, to its specific target. After magnetic separation of the nanoparticles, they are added to parent JM109 E. coli that do not contain the plasmid and are, therefore, not resistant to ampicillin. After sufficient incubation time for the JM109 E. coli to take up the plasmid-coated nanoparticles (uncoated nanoparticles will also be taken up), the bacteria are plated on solid media (agar) or on a media impregnated filter containing ampicillin alone or LB agar plates with 100µg/ml ampicillin. Only the nanoparticles that successfully bound the plasmid specifically through the DCE-molecular target link and transferred an expressible plasmid to the recipient parental host will grow on the ampicillin medium. The surviving clones can be selected and amplified by further culture in liquid growth medium. The DNA can be extracted from this culture and the DCE can be cut out of the plasmid and sequenced to reveal the specific DCE selected. When these surviving clones are grown on 3-AT medium they will produce diazoluminomelanin (DALM) which will be biosynthetically linked through the plasmid DNA containing the specific DCE to the target microbe. Upon activation with ultraviolet (UVA or UVB) light, sodium bicarbonate and hydrogen peroxide and heat or microwave energy, the target will be killed or neutralized. These DALM/plasmid/DCE complexes can be used also to detect and identify the target microbe by specific binding detected by thermochemiluminescence, slow fluorescence, visible light absorption, or colorimetric means. The magnetic property of the nanoparticles, which bind non-covalently to the DALM/plasmid/DCE, can be used to concentrate the complex bound to the target for detection, identification, or further analysis.
How would the operator use such a system in the field? A freeze-dried seed stock library of JM109/pIC2OR NR1.1DCE (E. coli host containing a library of random DCE sequences in the plasmid with the genes for ampicillin resistance and the capability to make DALM) would be grown up in liquid growth medium using the standard portable field incubator used currently by BEEs in USAF for water quality assessment (coliforms). This culture would form the stock of plasmid, following lysis with a lysis solution, for selecting the new DNA aptamer sequences for a biological agent discovered in the field. The biological agent source of interest would then be mixed with metallic nanoparticles that chemically (covalently or non-covalently) link to molecular targets on the surface of microbes or directly to molecular-scale toxins. These would be “purified”, extracted from the milieu, with a permanent magnet against the wall of the vial containing the agent in liquid suspension (water or buffer solution). The collected nanoparticle bound target would then be washed several times with water or buffer and then lysed with a lysing buffer or solution (similar to what was used for the library bacteria). This latter process would kill and break up the agent but leave the molecular target bound to the nanoparticle. The nanoparticles would then be magnetically extracted (same as above) and washed with a solution compatible with the binding of the plasmid/DCE. This preparation would then be added to a re-constituted culture of freeze-dried JM109 parent (lacking plasmid). After the appropriate time, the bacteria would be deposited on a culture filter (those used in the standard water quality assay used by the BEEs) and placed over a sponge containing the ampicillin-containing culture medium. This preparation is then incubated in the portable incubator until colonies appear on the filter grid. These can be picked off with a sterile swab or loop and transferred to culture medium for expanding the clones. These clones are the ones that will make DCE against the new target on demand. If they are grown in 3AT liquid medium in the portable incubator and the DALM/plasmid/DCE is extracted into the supernatant (just the spontaneous lysate left when the solids fall out of solution or are frozen out), this crude supernatant can be used to detect, identify and sensitize the target biological to killing as mentioned above (see attachment of slow luminescence results from Dr Parker of the bacteria containing the DCE plasmids, an indication of the detection reporting and killing capability). Also, the DNA from this supernatant contains the sequence fingerprint that can be dried and sent back to the rear echelon for sequencing.
Advantages and New Features
The selected DNA capture element in the selected plasmid is a natural encoding of the agent’s signature. The sequence may match or show homology to one in a look-up data base of aptamer sequences or it can be used to interrogate known agents in rear echelon laboratories to validate identification without ever shipping the agent from the field. Therefore, the DCE is selected and manufactured in the field in the forward area for use against a particular agent. All the reagents (mostly media and lysis solution components) can be kept in un-refrigerated freeze-dried packaged form to be reconstituted with water, as the bacteria themselves are. The cultureware are plastic and disposable and carried in a cooler-sized portable incubator. Pre-placed freeze-dried seed stocks against a given agent can be placed in the field for manufacture of detecting, identifying and neutralizing DCEs, similar to what is described above for selecting DCEs for unknown agents. The non-specific linking of the nanoparticles to the surface of an intact agent before the lysing of the microbe also tends to select molecular targets on the microbe that are accessible and associated with a living or viable agent, other than culture, no existing binding method can determine agent viability.
Alternatives
An alternative selection method would be the uptake of the nanoparticle coated with the molecular target into the living microbe or cell so that its binding to the aptamer occurs intracellularly. After binding, the microbe or cell is lysed and the nanoparticle with the attached aptamer is magnetically extracted. It is then added to a new parent microbe or cell which is selected by the antibiotic resistance marker on the plasmid when successfully incorporated. The same approach as above can be applied to mammalian cells. We already have patented plasmids and vectors for placing and expressing the DALM-producing gene in animal and human cells and this plasmid could potentially harbor the DCEs. Also, it contains a neomycin-resistance gene that is expressed and allows for the selection of gene recombinants in these cells. An E. coli HB101 host, containing this vector plasmid pSV2neoNR101 (American Type Culture Collection #69617; US Patent 5,464,768), can be used to carry and propagate the DCE inserts. This host and plasmid could be used to produce the DCE clones against a specific biological agent as described above. The selected plasmids could be not only transferred to a parent HB101 for further propagation of the plasmid, but also could be transfected by the same nanoparticle carrier technique into a host animal or human cell for the production of DALM and DCEs that would be specifically directed against an intracellular target, through DCE/DALM/DNA, or preferentially the RNA/DALM counterpart’s binding and microwave radiation activation of neutralization. The DNA DCEs would be preferentially converted into RNA already previously selected against a target, or by transcription of the RNA (intracellular) to be selected for binding to intracellular targets. In other words, the DCEs in the plasmids would be cDNA of the actual RNA aptamers that would attach to the target. Current evidence reported in CMI activity reports for July-December, indicate that the process has been successfully repeated and that DALM/DCE plasmids can successfully transfect parental JM109 and HB101 strains of E.coli. “Second generation” transfection of more parental E. coli with the initial transfectants’ DALM/DCE plasmids has not been successfully completed.
References
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14. Johnathan L. Kiel, Eric A. Holwitt, Michael Fan (Fan Maomian) and Shelly D. Roper , METHODS AND COMPOSITIONS FOR PROCESSES OF RAPID SELECTION AND PRODUCTION OF nucleic acid capture elements (or Aptamers), a draft application of the US non-provisional conversion of matters 339459 and 359250 (CMI filing)
15. Mark A. Sloan, Jeevalatha Vivekananda, Eric Holwitt and Johnathan Kiel, METHODS AND COMPOSITIONS FOR NEUTRALIZING ANTHRAX AND OTHER BIOAGENTS, Appln. No.: 10/291,336; Filing Date: November 8, 2002 (CMI filing)
Inventors: Johnathan Kiel, Eric Holwitt, Mark Sloan, Amanda Tijerina, Jill Parker, Maomian Fan and Melanie Woitaske. (Inventors bolded and italicized were CMI employees as of the declaration of IP to the USAF and at the date of filing.)
Provisional Application Serial No. 61/183,453 – Filed June 2, 2009 and June 2, 2010
Our File:75192 – 374201.
Drs Kiel, Holwitt and Parker were US Air Force Civilian employees at the time of both discovery and filing of IP with the USAF and for the provisional application. Ms Woitaske, a CMI team member, and Dr. Fan were added in June 2010 when the completed patent was filed.
Received restriction requirement from USPO 28 Oct 2011. Drafted and responded on 22 November 2011.
This Patent is attributable to work performed by CMI under USAF Contract: FA8650-05-C-6521.