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The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.
A method of discriminating between spore-forming and non-spore-forming bacteria is based on a combination of simultaneous sporulation-specific and non-sporulation-specific quantitative polymerase chain reactions (Q-PCRs). The method was invented partly in response to the observation that for the purposes of preventing or reducing biological contamination affecting many human endeavors, ultimately, only the spore-forming portions of bacterial populations are the ones that are problematic (or, at least, more problematic than are the non-spore-forming portions). In some environments, spore-forming bacteria constitute small fractions of the total bacterial populations. The use of sporulation-specific primers in Q-PCR affords the ability to assess the spore-forming fraction of a bacterial population present in an environment of interest. This assessment can provide a more thorough and accurate understanding of the bacterial contamination in the environment, thereby making it possible to focus contamination- testing, contamination-prevention, sterilization, and decontamination resources more economically and efficiently. The method includes the use of sporulation-specific primers in the form of designed, optimized deoxyribonucleic acid (DNA) oligonucleotides specific for the bacterial spoIVA gene (see table). [In "spoIVA," "IV" signifies Roman numeral four and the entire quoted name refers to gene A for the fourth stage of sporulation.] These primers are mixed into a PCR cocktail with a given sample of bacterial cells. A control PCR cocktail into which are mixed universal 16S rRNA primers is also prepared. ["16S rRNA" denotes a ribosomal ribonucleic acid (rRNA) sequence that is common to all organisms.] Following several cycles of heating and cooling according to the PCR protocol to amplify amounts of DNA molecules, the amplification products can be analyzed to determine the types of bacterial cells present within the samples. If the amplification product is strong, relative to the product of a control PCR sequence, then it is concluded that the bacterial population in the sample consists predominantly of spore-forming cells. If the amplification product is weak or nonexistent, then it is concluded that the bacterial population in the sample consists predominantly or entirely of non-spore-forming cells.
Biochips containing arrays of nanoelectrodes based on multiwalled carbon nanotubes (MWCNTs) are being developed as means of ultrasensitive electrochemical detection of specific deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) biomarkers for purposes of medical diagnosis and bioenvironmental monitoring. In mass production, these biochips could be relatively inexpensive (hence, disposable). These biochips would be integrated with computer-controlled microfluidic and microelectronic devices in automated hand-held and bench-top instruments that could be used to perform rapid in vitro genetic analyses with simplified preparation of samples. Carbon nanotubes are attractive for use as nanoelectrodes for detection of biomolecules because of their nanoscale dimensions and their chemical properties.
The hydrocyclone-filtration extractor (HFE), now undergoing development, is a simple, robust apparatus for processing large amounts of soil to extract trace amounts of microorganisms, soluble organic compounds, and other biomarkers from soil and to concentrate the extracts in amounts sufficient to enable such traditional assays as cell culturing, deoxyribonucleic acid (DNA) analysis, and isotope analysis. Originally intended for incorporation into a suite of instruments for detecting signs of life on Mars, the HFE could also be used on Earth for similar purposes, including detecting trace amounts of biomarkers or chemical wastes in soils.
A system comprising very-large-scale integrated (VLSI) circuits is being developed as a means of bioinformatics-oriented analysis and recognition of patterns of fluorescence generated in a microarray in an advanced, highly miniaturized, portable genetic-expression-assay instrument. Such an instrument implements an on-chip combination of polymerase chain reactions and electrochemical transduction for amplification and detection of deoxyribonucleic acid (DNA).
A small mass spectrometer utilizing a miniature field ionization source is now undergoing development. It is designed for use in a variety of applications in which there are requirements for a lightweight, low-power-consumption instrument that can analyze the masses of a wide variety of molecules and ions. The device can operate without need for a high-vacuum, carrier-gas feed radioactive ionizing source, or thermal ionizer. This mass spectrometer can operate either in the natural vacuum of outer space or on Earth at any ambient pressure below 50 torr (below about 6.7 kPa) - a partial vacuum that can easily be reached by use of a small sampling pump. This mass spectrometer also has a large dynamic range - from singly charged small gas ions to deoxyribonucleic acid (DNA) fragments larger than 104 atomic mass units - with sensitivity adequate for detecting some molecules and ions at relative abundances of less than one part per billion. This instrument (see figure) includes a field ionizer integrated with a rotating-field mass spectrometer (RFMS). The field ionizer effects ionization of a type characterized as "soft" in the art because it does not fragment molecules or initiate avalanche arcing. What makes the "soft" ionization mode possible is that the distance between the ionizing electrodes is less than mean free path for ions at the maximum anticipated operating pressure, so that the ionizer always operates on the non-breakdown side of the applicable Paschen curve (a standard plot of breakdown potential on the ordinate and pressure electrode separation on the abscissa). The field ionizer in this instrument is fabricated by micromachining a submicron-thick membrane out of an electrically nonconductive substrate, coating the membrane on both sides to form electrodes, then micromachining small holes through the electrodes and membrane. Because of the submicron electrode separation, even a potential of only 1 V applied between the electrodes gives rise to an electric field with a strength of in excess of a megavolt per meter strong enough to ionize any gas molecules passing through the holes. An accelerator grid and an electrostatic deflector focus the ions from the field ionizer into the rotating-field cell of the RFMS. The potentials applied to the electrodes of the cell to generate the rotating electric field typically range from 1 to 13 V. The ions travel in well-defined helices within this cell, after which they are collected in a Faraday cup. The mass of most of the molecules reaching the Faraday cup decreases with increasing frequency of rotation of the electric field in the cell. Therefore, the frequency of rotation of the electric field is made to vary in order to scan through a desired range of ion masses: For example, lightweight gas molecules are scanned at frequencies in the megahertz range, while DNA and other large organic molecules are scanned at kilohertz frequencies.
A graphical-user-interface (GUI) computer program has been developed to facilitate research on the damage caused by highly energetic particles and photons impinging on living organisms. The program brings together, into one computational workspace, computer codes that have been developed over the years, plus codes that will be developed during the foreseeable future, to address diverse aspects of radiation damage. These include codes that implement radiation-track models, codes for biophysical models of breakage of deoxyribonucleic acid (DNA) by radiation, pattern-recognition programs for extracting quantitative information from biological assays, and image-processing programs that aid visualization of DNA breaks. The radiation-track models are based on transport models of interactions of radiation with matter and solution of the Boltzmann transport equation by use of both theoretical and numerical models. The biophysical models of breakage of DNA by radiation include biopolymer coarse-grained and atomistic models of DNA, stochastic- process models of deposition of energy, and Markov-based probabilistic models of placement of double-strand breaks in DNA. The program is designed for use in the NT, 95, 98, 2000, ME, and XP variants of the Windows operating system.
A method of ensuring the complete transfer of liquid droplets from nozzles in microfluidic devices to nearby surfaces involves relatively simple geometric modification of the nozzle surfaces. The method is especially applicable to nozzles in print heads and similar devices required to dispense liquid droplets having precise volumes. Examples of such devices include heads for soft printing of ink on paper and heads for depositing droplets of deoxyribonucleic acid (DNA) or protein solutions on glass plates to form microarrays of spots for analysis. The main purpose served by the present method is to ensure that droplets transferred from a nozzle have consistent volume, as needed to ensure accuracy in microarray analysis or consistent appearance of printed text and images. In soft printing, droplets having consistent volume are generated inside a print head, but in the absence of the present method, the consistency is lost in printing because after each printing action (in which a drop is ejected from a nozzle), a small residual volume of liquid remains attached to the nozzle. By providing for complete transfer of droplets (and thus eliminating residual liquid attached to the nozzle) the method ensures consistency of volume of transferred droplets. An additional benefit of elimination of residue is prevention of cross-contamination among different liquids printed through the same nozzle a major consideration in DNA microarray analysis. The method also accelerates the printing process by minimizing the need to clean a printing head to prevent cross-contamination. Soft printing involves a hydrophobic nozzle surface and a hydrophilic print surface. When the two surfaces are brought into proximity such that a droplet in the nozzle makes contact with the print surface, a substantial portion of the droplet becomes transferred to the print surface. Then as the nozzle and the print surface are pulled apart, the droplet is pulled apart and most of the droplet remains on the print surface. The basic principle of the present method is to reduce the liquid-solid surface energy of the nozzle to a level sufficiently below the intrinsic solid-liquid surface energy of the nozzle material so that the droplet is not pulled apart and, instead, the entire droplet volume becomes transferred to the print surface. In this method, the liquid-solid surface energy is reduced by introducing artificial surface roughness in the form of micromachined serrations on the inner nozzle surface (see figure). The method was tested in experiments on soft printing of DNA solutions and of deionized water through 0.5-mm-diameter nozzles, of which some were not serrated, some were partially serrated, and some were fully serrated. In the nozzles without serrations, transfer was incomplete; that is, residual liquids remained in the nozzles after printing. However, in every nozzle in which at least half the inner surface was serrated, complete transfer of droplets to the print surface was achieved.
A method of studying the functions of all the genes of a given species of microorganism simultaneously has been developed in experiments on Saccharomyces cerevisiae (commonly known as baker's or brewer's yeast). It is already known that many yeast genes perform functions similar to those of corresponding human genes; therefore, by facilitating understanding of yeast genes, the method may ultimately also contribute to the knowledge needed to treat some diseases in humans. Because of the complexity of the method and the highly specialized nature of the underlying knowledge, it is possible to give only a brief and sketchy summary here. The method involves the use of unique synthetic deoxyribonucleic acid (DNA) sequences that are denoted as DNA bar codes because of their utility as molecular labels. The method also involves the disruption of gene functions through deletion of genes. Saccharomyces cerevisiae is a particularly powerful experimental system in that multiple deletion strains easily can be pooled for parallel growth assays. Individual deletion strains recently have been created for 5,918 open reading frames, representing nearly all of the estimated 6,000 genetic loci of Saccharomyces cerevisiae. Tagging of each deletion strain with one or two unique 20-nucleotide sequences enables identification of genes affected by specific growth conditions, without prior knowledge of gene functions. Hybridization of bar-code DNA to oligonucleotide arrays can be used to measure the growth rate of each strain over several cell-division generations. The growth rate thus measured serves as an index of the fitness of the strain.
The figure depicts a replaceable microfluidic cartridge that is a component of a miniature biosensor that detects target deoxyribonucleic acid (DNA) sequences. The biosensor utilizes (1) polymerase chain reactions (PCRs) to multiply the amount of DNA to be detected, (2) fluorogenic polynucleotide probe chemicals for labeling the target DNA sequences, and (3) a high-sensitivity epifluorescence-detection optoelectronic subsystem. Microfluidics is a relatively new field of device development in which one applies techniques for fabricating microelectromechanical systems (MEMS) to miniature systems for containing and/or moving fluids. Typically, microfluidic devices are microfabricated, variously, from silicon or polymers. The development of microfluidic devices for applications that involve PCR and fluorescence-based detection of PCR products poses special challenges
A nano-aluminum-oxide fiber of only 2 nanometers in diameter was used to develop a ceramic-fiber filter. The fibers are electropositive and, when formulated into a filter material (NanoCeram(TradeMark)), would attract electro-negative particles such as bacteria and viruses. The ability to detect and then remove viruses as well as bacteria is of concern in space cabins since they may be carried onboard by space crews. Moreover, an improved filter was desired that would polish the effluent from condensed moisture and wastewater, producing potable drinking water. A laboratory- size filter was developed that was capable of removing greater than 99.9999 percent of bacteria and virus. Such a removal was achieved at flow rates hundreds of times greater than those through ultraporous membranes that remove particles by sieving. Because the pore size of the new filter was rather large as compared to ultraporous membranes, it was found to be more resistant to clogging. Additionally, a full-size cartridge is being developed that is capable of serving a full space crew. During this ongoing effort, research demonstrated that the filter media was a very efficient adsorbent for DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and endotoxins. Since the adsorption is based on the charge of the macromolecules, there is also a potential for separating proteins and other particulates on the basis of their charge differences. The separation of specific proteins is a major new thrust of biotechnology. The principal application of NanoCeram filters is based on their ability to remove viruses from water. The removal of more than 99.9999 percent of viruses was achieved by a NanoCeram polishing filter added to the effluent of an existing filtration device. NanoCeram is commercially available in laboratory-size filter discs and in the form of a syringe filter. The unique characteristic of the filter can be demonstrated by its ability to remove particulate dyes such as Metanyl yellow. Its particle size is only 2 nanometers, about the size of a DNA molecule, yet the NanoCeram syringe filter is capable of retaining the dyes as the fluid is passed through the syringe, without much back-pressure. Endotoxins, which are contaminants that are part of the residue of destroyed bacteria, can cause toxic shock and are therefore of major concern in pharmaceutical products. The NanoCeram syringe filter is capable of removing greater than 99.96 percent of the endotoxins.
A method, now undergoing development, of forming nanochannels in planar substrates is intended to enable the fabrication of advanced fluidic devices that could be integrated with complementary metal oxide semiconductor (CMOS) electronic circuits. Such integral combinations of fluidic and electronic components ("laboratory-on-a-chip" devices) could be used, for example, to detect individual molecules of deoxyribonucleic acid (DNA) and proteins. The width of a channel in such a device would be chosen so that molecules of the species of interest would move along the channel in single file. In this method, the fabrication of channels includes the use of such CMOS-compatible processes as chemical-mechanical polishing and oxide deposition. The layout of the channels in the substrate plane is determined by a single photolithographic process, but it is not a nanoscale lithographic process, and this process is not relied upon to define the thickness and width of the channels. Stating it from a slightly different perspective, unlike in the prior fabrication of electronic and fluidic devices involving the use of lithography to define microscale features, this process does not include the use of lithography to define nanoscale features. It is this aspect of the method that enables simplification of the process and, hence, a decrease in cost.
A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of target bacteria in samples are usually amplified by overnight pre-enrichment growth. These tests are usually performed in laboratories by skilled technicians. The present test was designed to overcome the shortcomings of the commercial hybridization tests. The figure summarizes the major steps of the test. It is important to emphasize that the hybridization process used in this test differs from that of other hybridization tests in that it does not extract target nucleic acids. This process is based on intact-cell hybridization (so-called in situ hybridization ), wherein the intact cells act as immobilizing agents. The cells are identified and enumerated by measuring the chemiluminescence emitted from alkaline phosphatase-probe (AP-probe) hybridization; the chemiluminescence is detected or measured by use of photographic film or a luminometer, respectively.
A proposed normally-closed microvalve would contain a piezoelectric bending actuator instead of a piezoelectric linear actuator like that of the microvalve described in the preceding article. Whereas the stroke of the linear actuator of the preceding article would be limited to approximately equal to 6 micrometers, the stroke of the proposed bending actuator would lie in the approximate range of 10 to 15 micrometers-large enough to enable the microvalve to handle a variety of liquids containing suspended particles having sizes up to 10 m. Such particulate-laden liquids occur in a variety of microfluidic systems, one example being a system that sorts cells or large biomolecules for analysis. In comparison with the linear actuator of the preceding article, the bending actuator would be smaller and less massive. The combination of increased stroke, smaller mass, and smaller volume would be obtained at the cost of decreased actuation force: The proposed actuator would generate a force in the approximate range of 1 to 4 N, the exact amount depending on operating conditions and details of design. This level of actuation force would be too low to enable the valve to handle a fluid at the high pressure level mentioned in the preceding article. The proposal encompasses two alternative designs one featuring a miniature piezoelectric bimorph actuator and one featuring a thick-film unimorph piezoelectric actuator (see figure). In either version, the valve would consume a power of only 0.01 W when actuated at a frequency of 100 Hz. Also, in either version, it would be necessary to attach a soft elastomeric sealing ring to the valve seat so that any particles that settle on the seat would be pushed deep into the elastomeric material to prevent or reduce leakage. The overall dimensions of the bimorph version would be 7 by 7 by 1 mm. The actuator in this version would generate a force of 1 N and a stroke of 10 m at an applied potential of 150 V. The actuation force would be sufficient to enable the valve to handle a fluid pressurized up to about 50 psi (approximately equal to 0.35 MPa). The overall dimensions of the unimorph version would be 2 by 2 by 0.5 mm. In this version, an electric field across the piezoelectric film on a diaphragm would cause the film to pull on, and thereby bend, the diaphragm. At an applied potential of 20 V, the actuator in this version would generate a stroke of 10 micrometers and a force of 0.01 N. This force level would be too low to enable handling of fluids at pressures comparable to those of the bimorph version. This version would be useful primarily in microfluidic and nanofluidic applications that involve extremely low differential pressures and in which there are requirements for extreme miniaturization of valves. Examples of such applications include liquid chromatography and sequencing of deoxyribonucleic acid.
When we send humans to search for life on other planets, we'll need to know what we brought with us versus what may already be there. To ensure our crewed systems meet planetary protection requirements-and to protect our science from human contamination-we'll need to assess whether microorganisms may be leaking or venting from our spacecraft. Microbial sample collection outside of a pressurized spacecraft is complicated by temperature extremes, low pressures that preclude the use of laboratory standard (wetted) swabs, and operation either in bulky spacesuits or with robotic assistance. Engineers at the National Aeronautics and Space Administration (NASA) recently developed a swab kit for use in collecting microbial samples from the external surfaces of crewed spacecraft, including spacesuits. The Extravehicular Activity (EVA) Swab Kit consists of a single swab tool handle and an eight-canister sample caddy. The design team minimized development cost by re-purposing a heritage Space Shuttle tile repair handle that was designed to quickly snap into different tool attachments by engaging a mating device in each attachment. This allowed the tool handle to snap onto a fresh swab attachment much like popular shaving razor handles can snap onto a disposable blade cartridge. To disengage the handle from a swab, the user performs two independent functions, which can be done with a single hand. This dual operation mitigates the risk that a swab will be inadvertently released and lost in microgravity. Each swab attachment is fitted with commercially available foam swab tips, vendor-certified to be sterile for Deoxyribonucleic Acid (DNA). A microbial filter installed in the bottom of each sample container allows the container to outgas and repressurize without introducing microbial contaminants to internal void spaces. Extensive ground testing, post-test handling, and sample analysis confirmed the design is able to maintain sterile conditions as the canister moves between various pressure environments. To further minimize cost, the design team acquired extensive ground test experience in a relevant flight environment by piggy-backing onto suited crew training runs. These training runs allowed the project to validate tool interfaces with pressurized EVA gloves and collect user feedback on the tool design and function, as well as characterize baseline microbial data for different types of spacesuits. In general, test subjects found the EVA Swab Kit relatively straightforward to operate, but identified a number of design improvements that will be incorporated into the final design. Although originally intended to help characterize human forward contaminants, this tool has other potential applications, such as for collecting and preserving space-exposed materials to support astrobiology experiments.
Biomarkers for life on Earth are an important starting point to guide the search for life elsewhere. However, the search for life beyond Earth should incorporate technologies capable of recognizing an array of potential biomarkers beyond what we see on Earth, in order to minimize the risk of false negatives from life detection missions. With this in mind, charged linear polymers may be a universal signature for life, due to their ability to store information while also inherently reducing the tendency of complex tertiary structure formation that significantly inhibit replication. Thus, these molecules are attractive targets for biosignature detection as potential "self-sustaining chemical signatures." Examples of charged linear polymers, or polyelectrolytes, include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as well as synthetic polyelectrolytes that could potentially support life, including threose nucleic acid (TNA) and other xenonucleic acids (XNAs). Nanopore analysis is a novel technology that has been developed for singlemolecule sequencing with exquisite single nucleotide resolution which is also well-suited for analysis of polyelectrolyte molecules. Nanopore analysis has the ability to detect repeating sequences of electrical charges in organic linear polymers, and it is not molecule- specific (i.e. it is not restricted to only DNA or RNA). In this sense, it is a better life detection technique than approaches that are based on specific molecules, such as the polymerase chain reaction (PCR), which requires that the molecule being detected be composed of DNA.
When we send humans to search for life on other planets, we'll need to know what we brought with us versus what may already be there. To ensure our crewed systems meet planetary protection requirements-and to protect our science from human contamination-we'll need to assess whether microorganisms may be leaking or venting from our spacecraft. Microbial sample collection outside of a pressurized spacecraft is complicated by temperature extremes, low pressures that preclude the use of laboratory standard (wetted) swabs, and operation either in bulky spacesuits or with robotic assistance. A team at the National Aeronautics and Space Administration (NASA) recently developed a swab kit for use in collecting microbial samples from the external surfaces of crewed spacecraft, including spacesuits. The Extravehicular Activity (EVA) Swab Kit consists of a single swab tool handle and an eight-canister sample caddy. The design team minimized development cost by re-purposing a heritage Space Shuttle tile repair handle that was designed to quickly snap into different tool attachments by engaging a mating device in each end effector. This allowed the tool handle to snap onto a fresh swab end effector much like popular shaving razor handles can snap onto a disposable blade cartridge. To disengage the handle from a swab, the user performs two independent functions, which can be done with a single hand. This dual operation mitigates the risk that a swab will be inadvertently released and lost in microgravity. Each swab end effector is fitted with commercially available foam swab tips, vendor-certified to be sterile for Deoxyribonucleic Acid (DNA). A microbial filter installed in the bottom of each sample container allows the container to outgas and re-pressurize without introducing microbial contaminants to internal void spaces. Extensive ground testing, post-test handling, and sample analysis confirmed the design is able to maintain sterile conditions as the canister moves between various pressure environments. To further minimize cost, the design team acquired extensive ground test experience in a relevant flight environment by piggy-backing onto suited crew training runs. These training runs allowed the project to validate tool interfaces with pressurized EVA gloves and collect user feedback on the tool design and function, as well as characterize baseline microbial data for different types of spacesuits. In general, test subjects found the EVA Swab Kit relatively straightforward to operate, but identified a number of design improvements that will be incorporated into the final design. Although originally intended to help characterize human forward contaminants, this tool has other potential applications, such as for collecting and preserving space-exposed materials to support astrobiology experiments.
Designed for human exploration missions into deep space, NASA's Space Launch System (SLS) represents a new spaceflight infrastructure asset, enabling a wide variety of unique utilization opportunities. While primarily focused on launching the large systems needed for crewed spaceflight beyond Earth orbit, SLS also offers a game-changing capability for the deployment of small satellites to deep-space destinations, beginning with its first flight. Currently, SLS is making rapid progress toward readiness for its first launch in two years, using the initial configuration of the vehicle, which is capable of delivering 70 metric tons (t) to Low Earth Orbit (LEO). On its first flight test of the Orion spacecraft around the moon, accompanying Orion on SLS will be small-satellite secondary payloads, which will deploy in cislunar space. The deployment berths are sized for "6U" CubeSats, and on EM-1 the spacecraft will be deployed into cislunar space following Orion separate from the SLS Interim Cryogenic Propulsion Stage. Payloads in 6U class will be limited to 14 kg maximum mass. Secondary payloads on EM-1 will be launched in the Orion Stage Adapter (OSA). Payload dispensers will be mounted on specially designed brackets, each attached to the interior wall of the OSA. For the EM-1 mission, a total of fourteen brackets will be installed, allowing for thirteen payload locations. The final location will be used for mounting an avionics unit, which will include a battery and sequencer for executing the mission deployment sequence. Following the launch of EM-1, deployments of the secondary payloads will commence after sufficient separation of the Orion spacecraft to the upper stage vehicle to minimize any possible contact of the deployed CubeSats to Orion. Currently this is estimated to require approximately 4 hours. The allowed deployment window for the CubeSats will be from the time the upper stage disposal maneuvers are complete to up to 10 days after launch. The upper stage will fly past the moon at a perigee of approximately 100km, and this closest approach will occur about 5 days after launch. The limiting factor for the latest deployment time is the available power in the sequencer system. Several NASA Mission Directorates were involved in the development of programs for the competition, selection, and development of EM-1 payloads that support directorate priorities. CubeSat payloads on EM-1 will include both NASA research experiments and spacecraft developed by industry, international and potentially academia partners. The Human Exploration and Operations Mission Directorate (HEOMD) Advanced Exploration Systems (AES) Division was allocated five payload opportunities on the EM-1 mission. Near Earth Asteroid (NEA) Scout is designed to rendezvous with and characterize a candidate NEA. A solar sail, an innovation the spacecraft will demonstrated for the CubeSat class, will provide propulsion. Lunar Flashlight will use a green propellant system and will search for potential ice deposits in the moon's permanently shadowed craters. BioSentinel is a yeast radiation biosensor, planned to measure the effects of space radiation on deoxyribonucleic acid (DNA). Lunar Icecube, a collaboration with Morehead State University, will prospect for water in ice, liquid, and vapor forms as well as other lunar volatiles from a low-perigee, highly inclined lunar orbit using a compact Infrared spectrometer. Skyfire, a partnership with Lockheed Martin, is a technology demonstration mission that will perform a lunar flyby, collecting spectroscopy, and thermography data to address questions related to surface characterization, remote sensing, and site selection. NASA's Space Technology Mission Directorate (STMD) was allocated three payload opportunities on the EM-1 mission. These slots will be filled via the Centennial Challenges Program, NASA's flagship program for technology prize competitions, which directly engages the public, academia, and industry in open prize competitions to stimulate innovation. The NASA Science Mission Directorate (SMD) was allocated two payload opportunities on the EM-1 mission. The CubeSat Mission to Study Solar Particles (CuSP) payload will study the sources and acceleration mechanisms of solar and interplanetary particles in near-Earth orbit, support space weather research by determining proton radiation levels during Solar Energetic Particle (SEP) events and identifying suprathermal properties that could help predict geomagnetic storms. The LunaH-Map payload will help scientists understand the quantity of H-bearing materials in lunar cold traps (~10 km), determine the concentration of H-bearing materials with 1m depth, and constrain the vertical distribution of H-bearing materials. The final three payload opportunities for the EM-1 mission were allocated for NASA's international space agency counterparts. The flight opportunities are intended to benefit the international space agency and NASA as well as further the collective space exploration goals. ArgoMoon is sponsored by ESA/ASI and will fly along with the ICPS on its disposal trajectory to perform proximity operations with the ICPS post-disposal, take external imagery of engineering and historical significance, and perform an optical communications demonstration. EQUULEUS, sponsored by JAXA, will fly to a libration orbit around the Earth-Moon L2 point and demonstrate trajectory control techniques within the Sun-Earth- Moon region for the first time by a nano spacecraft. The mission will also contribute to the future human exploration scenario by understanding the radiation environment in geospace and deep space, characterizing the flux of impacting meteors on the far side of the moon, and demonstrating the future deep space exploration scenario using the "deep space port" at Lagrange points. OMOTENASHI, also sponsored by JAXA, will land the smallest lunar lander to date on the lunar surface to demonstrate the feasibility of the hardware for distributed cooperative exploration system. Small landers will enable multi-point exploration, which is complimentary with large-scale human exploration. Once on the lunar surface, the OMOTENASHI spacecraft will observe the radiation and soil environments of the lunar surface by active radiation measurements and soil shear measurements. Following EM-1, Space Launch System will evolve to the more-powerful Block 1B configuration, which uses a new Exploration Upper Stage to increase the vehicle's LEO payload capability from 70 t to 105 t. With that transition, the Orion Stage Adapter, which will carry the secondary payloads on EM-1, will be phased out, and a new Universal Stage Adapter will be introduced, creating opportunities for flying larger secondary payloads. This paper will provide a brief status of SLS progress toward first launch; an overview of smallsat accommodations, integration, and operations on EM-1; information about the specific payloads flying on that launch; and a discussion of future accommodations and opportunities for secondary payloads on SLS for Exploration Mission-2 and beyond.