Overview

DNA Probe Design for Pre-Flight and In-Flight Microbial Monitoring

Principal Investigator:
George E. Fox, Ph.D.

Organization:
University of Houston

Technical Summary

Crew health is a dominant issue in manned space flight. Microbiological concerns, in particular, have repeatedly emerged as determinants of flight readiness. Microbial infection is prominently featured as a possible risk in the Critical Path Roadmap document, and means of monitoring the microbial environment are an important class of countermeasures to be developed. It is essential to the success of long-term missions that systems that deliver acceptable quality of air and water during the anticipated lifetime of the spacecraft be available. As mission duration and re-supply intervals increase, it will be necessary to rely on advanced life support systems which incorporate both biological and physical-chemical recycling methods for air and water as well as provide food for the crew. It therefore is necessary to develop real-time, robust, in-flight monitoring procedures. It would also be desirable if the monitoring system could be readily "reprogrammed" to identify specific pathogens if an in-flight incident were to occur. Thus, the monitoring technology must simultaneously detect many organisms of interest, be subject to miniaturization and be highly automated. The long-range goal of the project was to develop such monitoring systems. In the shorter term, it would be possible to use the technology being developed to obtain a better understanding of the effects of the space environment on microorganisms

Our underlying hypothesis was that the most appropriate target is either ribosomal RNA or the DNA that encodes it. The small subunit rRNA sequence (16S rRNA) in particular has been determined in several thousand bacterial species. Each of these sequences contains short sub-sequences that are widely conserved throughout the data set as well as other sub-sequences totally unique to, and characteristic of, a particular species. This pattern of sequence conservation made it possible to design oligonucleotide hybridization probes that can distinguish individual organisms, or groupings of organisms. Once an appropriate set of target sub-sequences have been identified for a desired assay, any of a variety of formats can be used to implement the assays. Thus, the final assay system may utilize PCR-amplified nucleic acids or, because rRNAs are high copy number molecules, direct detection systems such as chemiluminescence or fluorescence. Both types of assay are compatible with miniaturization and data can be processed automatically or returned to Earth by telemetry. The project objectives included an examination of alternative implementations and development of space craft compatible methods for sample processing.

Project Accomplishments

We initially demonstrated the feasibility of a DNA chip based assay for monitoring of water quality using probes that target 16S rRNA. This result also validated our probe designs for several species of spaceflight interest. It was at the same time clear that three major issues needed to be addressed to make the approach truly useful. These are (1) development of an appropriate set of hybridization probes with minimal cross-reactivity, (2) the development of spacecraft compatible procedures for extracting and purifying the target nucleic acids and (3) increasing the sensitivity and ease of execution of the assay. The work undertaken in subsequent years focused on these issues and considerable progress was made towards resolving them. This progress was achieved in part through the development of advanced software for probe design, and also through other accomplishments described below.
We established specific probes for essentially all the organisms needed to devise an assay system for monitoring spacecraft water quality. This included probes for total bacteria, Gram negative bacteria, enteric bacteria, Escherichia coli, Vibrio proteolyticus, Burkholderia cepacia, and Acinetobacter. Several of these probes will also be required for an air analysis system. Several of these probes have now been successfully utilized in multiple assay formats, including molecular beacons as discussed below.
We developed compaction precipitation for purifying DNA and RNA. The new technique, which has significant Earthbound spin-off potential, will be particularly useful in developing and possibly in performing spacecraft-based nucleic acid probe assays. A patent application has been filed, and a Nature Biotechnology article on the technique generated a high level of inquiries from outside laboratories. The method is being utilized in research on plasmid-based DNA vaccines for HIV, and the email protocols we have sent out appear to be spreading from user to user. The method has the potential for broad use in molecular biology for cloning, sub-cloning, genomics, DNA sequencing, etc. UH has identified a likely licensee for the technology, and as licensing terms are being finalized plasmid miniprep kit design has advanced to the point that the licensee now has packaging mockups for the commercial spin off product.
We found that a well-known method of protein purification is also very effective for many nucleic acid separations. Immobilized-metal affinity chromatography (IMAC) is the basis of the ubiquitous six-histidine purification "tag" for recombinant proteins. We hypothesized that chelated metals might also form ligand interactions with the exposed aromatic base nitrogens of single-stranded nucleic acid molecules. Surprisingly, this prospect has not been previously investigated. IMAC proves to be extremely effective at capturing RNA from mixtures with other molecules, and also for stripping primers e.g., from PCR and sequencing reactions. At least some (possibly all) single-base mismatches can be detected, raising the possibility of developing IMAC-based hybridization assays for microbial identification, SNP scoring, etc. A publication and a patent application are in preparation, and the UH licensing office is in negotiation with at least five prospective licensees, including the dominant companies in the field.
We applied molecular beacons to rapid, low-labor detection of organisms of spaceflight interest. These DNA hairpin probes bear a fluorophore at one end and a quencher at the other. The beacon becomes highly fluorescent when bound to target sequences in an extended configuration. The resulting homogeneous assay also has the advantage of minimal waste generation and reduced danger of cross-contamination, especially when used with amplification methods such as PCR or NASBA. We have converted several probes for organisms of space flight interest into beacon formats, and demonstrated simultaneous multiplex detection of several organisms using fluors with non-overlapping spectral properties ("colors"). In preliminary results toward highly parallel detection we have also demonstrated the feasibility of arrays of immobilized beacons, in which positive signals are identified by the position, rather than the color, of fluorescence emission.

These results have significant implications for future work. At this stage enough progress has been made in probe design that it would be possible to begin actual instrument development. This should, however, be accompanied by further refinement and testing of the already validated probes. Additional probes can also be designed and validated such that a prototype instrument could examine either air or water samples. Probe design can also now focus on the identification of possible pathogens or otherwise problematic bacteria without preconceived notions of what these organisms will be.

The two main methods under consideration for microbial monitoring are array hybridization and molecular beacon technology. Both are attractive approaches because they might also be usable with samples from blood, urine, and other crew-derived specimens, as well as water and bio-regenerative life-support system samples. This possibility has been further facilitated by the development of space-craft compatible methods for handling samples. Finally, if a hybridization array instrument were developed for microbial monitoring it would also be useable for in-flight studies of global gene expression. This approach might be an excellent way to determine whether key properties such as growth rates, mutation rates or pathogenicity are likely to be affected by the space environment.

This project's funding ended in 2000