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

SPACE FLIGHT COMPATIBLE TECHNOLOGY IS BEING DEVELOPED to monitor microbial flora. During the past year, the feasibility of doing this with medium density DNA hybridization arrays using probes targeted against characteristic regions of 16S ribosomal RNA was demonstrated. We are now designing and testing the probes needed for routine monitoring of the water supply and developing methodology to isolate RNA in space.

Crew health is a dominant issue in manned space flight. Microbiological concerns, in particular, have repeatedly emerged as determinants of flight readiness. In at least one case, suspected contamination of the potable water supply nearly forced a launch delay. In another instance, a member of the crew developed a urinary tract infection that nearly led to early termination of the mission, in part due to the difficulty of accurately diagnosing the nature of the infection in-flight. Microbial problems are an increasing concern with the trend toward longer-duration missions. It is essential to the success of such 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 that are sensitive enough to detect less than 100 CFU (colony forming units) of bacteria per 100 milliliters of water.

Background
Bacterial systematics was revolutionized by the realization in the late 1970s that comparisons of 16S ribosomal RNA (16S rRNA) sequences could be used to discover actual historical relationships between different species and genera in bacteria. This has resulted in major realignments of taxa and renaming of many species and genera. At this point in time, it is essentially mandatory to determine the 16S rRNA sequence of an unknown organism to fully document its identity. As a consequence, 16S rRNA sequence characterizations have been conducted for more than 10,000 bacterial species. Each of these sequences contains short sub-sequences that are widely conserved throughout the data set as well as other sub-sequences, which are essentially unique to, and hence very characteristic of, a particular species. This pattern of sequence conservation makes it possible to design oligonucleotide hybridization probes that can distinguish individual organisms, or groupings of related organisms. Practical diagnostic kits based on this technology (e.g., for Legionella and Chlamydia), have been successfully introduced into the clinical market. These single organism diagnostics rely on the natural amplification associated with ribosomal RNA (as many as 10,000 copies per cell), to provide a probe target and use manual detection procedures. It is believed that a similar strategy can be used in the space environment.

Theory
The long range project goal is to develop an automated hybridization system targeting 16S rRNA that can be used to monitor microbial populations and, when necessary, to identify pathogens in air and water samples during long-duration space missions. In order to do this, it is essential to define what must be detected in order to create a useful monitoring system. For example, in the case of routine water monitoring, it was concluded that a set of probes that would provide information for the levels of total bacteria, fecal coliforms, Escherichia coli, Pseudomonas aeruginosa, Enterococcus and Burkholderia would be sufficient to evaluate a sample. In theory, one would simply design probes specific for each of these groupings or individual species. In practice, bacterial species are more of a continuum of populations than are higher organisms. Thus, it is not usually possible to find a single sequence segment that is totally unique to a particular grouping. Thus, a probe designed for enteric organisms might also react with certain marine bacteria that are actually closely related. This is not necessarily a problem, however, as these organisms are exceedingly unlikely to be present in the samples of interest. Alternatively, a probe for E. coli might react with a few other very closely related organisms and thus, a positive result may actually mean the presence of an "E. coli-like" organism. We therefore anticipate that considerable refinement of probes will be needed. A second theoretical underpinning is the belief that hybridizations of the type described can in fact be performed in space environments in very easy-to-use formats. In addition to automation of the hybridization process itself, this implies a need for sampling and sample preparation procedures that can be used in the space environment.

Experimental or Research Characteristics
The immediate specific aims were: (1) to design a prototype assay system for routine monitoring of the water supply in a space environment and (2) to develop simplified sample preparation procedures that can be used in the space environment. Toward these ends, we (a) designed probes based on known 16S rRNA sequence data; (b) synthesized and began testing the probes needed to implement the prototype system; (c) tested the feasibility of using the prototype assay with a "DNA chip," instrument; (d) evaluated efficiency of existing methods of extracting RNA and (e) studied selective adsorption of RNA as the basis of a one-step isolation procedure.

The essence of the approach used to find species and group specific probes is to identify 16S rRNA sequence segments of significant length (13 or more bases) that are unchanged among all the organisms that one wishes the probe to detect. In addition, this constant segment must be different (preferably in two or more positions) in the sequences of all organisms the probe is not to detect. Two computer programs, find_probe and most_mismatch, were developed to work with aligned sequence files retrieved from the RDP (Ribosomal Data Base Project) 16S rRNA library that is available via the Internet. These programs are used to locate promising probes which subsequently are tested experimentally and refined as needed; e.g., by adjusting length or base composition.

In order to evaluate the specificity of individual probes, a dot blot assay is performed. A sample containing target 16S rRNA is bound by UV cross-linking to a nylon membrane. The probe being tested is synthesized with a biotin at its 5' end. This probe is hybridized with the 16S rRNA and the membrane is washed to remove any RNA that does not hybridize. Strepavidin conjugated with alkaline phosphatase is added to the membrane. If RNA hybridization has occurred, the strepavidin and the conjugated alkaline phosphatase will bind to the biotin carried by the probe that is being tested. Finally, a substrate (NBT/ BCIP) which is cleaved by the alkaline phosphatase is added. The result is a blue precipitate at sites on the membrane where hybridization has occurred.

In order for 16S rRNA targeted hybridizations to be useful for microbial monitoring in space, the technology must be implemented in a format which allows simultaneous detection of many organisms of interest, be subject to miniaturization and be highly automated. Genometrix, Inc. is one of several companies that are developing DNA hybridization array methodologies ("DNA chips") that potentially offer these three capabilities. In collaboration with Genometrix, Inc., we sought to determine if their technology would work in conjunction with 16S rRNA targeted probes. The core approach being investigated by Genometrix Inc. will use fluorescent or chemiluminescence to generate a readily detectable signal. The essence of the idea is to immobilize an array of unique oligonucleotide probes on a solid support. Each probe in the array will be designed to be specific for a particular organism of interest. All probes are simultaneously exposed to the same sample and the chemiluminescence or fluorescence signal that is produced when hybridization events occur is detected. In the Genometrix format this is done with a proximal CCD array. Location of the hybridization signal on the grid serves to identify which probes are responsible for the signal. Since the signal is in discrete locations, the resulting pattern is subject to straightforward computer analysis.

In our work with Genometrix, Inc., a sandwich assay which utilizes both a capture probe and detector probe(s) was tested. First, a surface capture probe selectively acquired the target rRNA from a total RNA preparation. A mixture of detector probes was added. Each detector probe carried a reactive group. If hybridization occurred between the detector and the captured RNA, a fluorescent precipitate was deposited at the site of those probe addresses that contained bound target molecules.

Collection of air and water samples in the space environment by filtration is realistic. However, the methodology to be used for subsequent RNA purification is uncertain. Purification must be accomplished without the use of either hazardous reagents or specialized equipment. It is envisioned that a one- or two-step column chromatography system would be ideal, as this would simplify materials handling procedures. Moreover, these methods offer the special advantages of low toxicity, aerosol generation, and energy consumption.

One of the few chemical differences between RNA and DNA which can serve as the basis of separation is the presence of the vicinal 2',3' cis-diol at the 3' end of the RNA molecule. This feature is absent from the deoxyribose backbone of DNA, and is exploited as the basis of recognition by boronate affinity methods. Boronic acid chromatography has been used for tRNA and (ribo)nucleotide isolation since the 1970s. Several workers have subsequently demonstrated the application of boronic acid chromatography to separation of nucleic acids and their derivatives, and of sugars and glycosylated proteins. In addition, it has been demonstrated that modification of the m-aminophenylboronic acid group to confer a lower pKa on the boronic acid moiety can enhance the range of pH over which these adsorbents are useful. We have used batch equilibrium adsorption isotherm measurement and several supporting techniques to characterize the adsorption of mixed RNAs on m-aminophenylboronic agarose under a variety of conditions.

Special Equipment
The core work, design of probes and their evaluation, is being done with standard methods. Likewise, the testing and development of sample preparation procedures uses essentially standard procedures. However, the eventual implementation of the methodology will require an automated system that allows telemetry of data back to the Earth for full evaluation. A promising approach to doing this is the use of DNA hybridization arrays ("DNA chips"). In this approach, many hybridization probes are attached to a small surface area with the location of each being known. A hybridization signal is then detected electronically and since the location from which the signal arises is known, one knows which probe(s) reacted with the sample. Several companies have prototype instruments of this type under development which differ in details, such as the way in which the probe array is constructed and the way the signal is detected. In our work, we have collaborated with one of these companies, Genometrix, Inc.

Results
Initial designs have been developed for all the capture and detector probes needed for the target organisms and groups of organisms that comprise the water system. Initially, several of these probes were synthesized and tested in conjunction with Genometrix Inc. in the array hybridization ("DNA chip") format. Glass slides carrying 10 arrays, each with 16 probes, were prepared. Each array was loaded with a different sample. A solution carrying all the detector probes was added and hybridization detected by chemiluminescence. Because the individual probes had not yet been optimized, the results for individual probes were in some cases not as specific as we ultimately hope. There was, however, no difficulty whatsoever in terms of cross-reaction between the various detector probes in the hybridization mixture, the ability to prepare arrays, or to detect hybridization. Thus, the feasibility of the DNA chip format was demonstrated. Subsequently, we have begun the refinement of the individual probe designs using the dot blot hybridization procedures.

In terms of the sample preparation issues, we found that the well-known affinity-enhancing influence of divalent cations depends strongly on the precise nature of the cation used, with barium being far more effective than the conventionally-used magnesium. This adsorption-promoting influence of barium is likely to arise primarily from ionic influences on the structure and rigidity of the RNA molecule, as the adsorption of ribose-based small molecules is not similarly affected. The substitution of barium for the standard magnesium counter ion does not greatly promote the adsorption of DNA, implying that the effect is specific to RNA and may be useful in boronate-based RNA separations. RNA adsorption isotherms exhibit a sharp transition as a function of temperature, and this transition occurs at different temperatures with Mg²⁺ and with Ba²⁺. Adsorption affinity and capacity were found to increase markedly at lower temperatures, suggestive of an enthalpically favored interaction process. The stoichiometric displacement parameter, Z, in Ba²⁺ buffer, is three times the value in Mg²⁺ buffer, and is close to unity.

We also examined the selective capture of RNA through single-stranded portions in which bases are exposed to selective adsorptive interactions, with the idea that double-stranded DNA and non-nucleic acid contaminants would not be captured by such interactions. We found that, in agreement with our working hypothesis that base nitrogen heterocycles could mediate an interaction with immobilized (chelated) metals, RNA adsorption is favored over that of DNA.

Discussion
The single most important outcome to date is clearly the demonstration of the feasibility of using the array approach. With that established, the clear next step is to develop useful probe sets. This can be done using more traditional procedures. Toward this end, we have completed the initial design of capture and detector probes for detection of total bacteria, E. coli, Burkholderia, Enterococcus, and Pseudomonas aeruginosa. In general, it is frequently necessary to have multiple probes to obtain specificity. For example, for total bacteria no one capture/detector pair was found in one organism and therefore three capture/ detector pairs are sure of obtaining a positive with essentially all known genera. For individual genera, the problem is distinction from closely related genera. For example, known strains of Burkholderia are very closely related to Neisseria and Ralstonia. No capture/detector probe pair was found that could distinguish Burkholderia uniquely from these other two. Therefore, two pairs were designed; one distinguishes Burkhoderia from Ralstonia whereas the other distinguishes Burkholderia from Neisseria. In practice, however, the probes both bound to Ralstonia. Clearly this distinction will be difficult to make; but is it needed? For practical purposes, "Burkholderia-like" will be sufficient.

In the area of sample preparation, we have established that, in our hands, the widely used Tsai method was found to be the most effective of the standard methods of RNA preparation.

We, therefore, are now using phosphorimager storage plate technology to test novel purification methods based on metal affinity in comparison with the Tsai method. Most recently, we have been exploring the use of nucleic acid conformation-modifying (compaction agents) to facilitate purification by adsorption and by formation of flocs large enough to be separated from RNA by simple coarse filtration, or low-speed centrifugation. We especially are seeking to minimize the number of steps in the procedures. In addition to being spacecraft-compatible, our methods may find eventual spin-off application on Earth because of their potentially greater convenience.

Publications
Eggers, M., W. Balch, L., Mendoza, R. Gangadharan, A. Mallik, M. McMahon, M. Hogan, T. Powdrill, B. Iverson, G. E. Fox, R. C. Willson, K. Maillard, J. L. Siefert, N. and Singh. "Advanced Approach to Simultaneous Monitoring of Multiple Bacteria in Space," Proc., 27th Int'l Conf. Environ. Systems, July 1997.
Fox, G. E. "DNA Probe Design for Preflight and In-Flight Microbial Monitoring," Executive Summarys, Nat'l Space Biomedical Research Inst. Retreat, Del Lago Conf. Center, Montgomery, TX, June 8-11, 1998. D-6
Murphy, J. C. "Nucleic Acid Separations Using Compaction Agents," MS Thesis, Univ. of Houston, Aug. 1998.
Pitulle, C., L. DSouza, and G. E. Fox. "A Low Molecular Weight Artificial RNA of Unique Size with Multiple Probe Target Regions," Syst. Appl. Microbiol. 20 (1997): 133-36.
Singh, N. and R. C. Willson. "Boronate Affinity Adsorption of RNA: Possible Role of Conformational Changes," J. Chromatography. (Submitted for publication.)
Presentations
Fox, G. E. "Microbial Monitoring in Space Environments," Nat'l Space Biomedical Research Inst., Sept. 1997; Rice Univ., Houston, Texas.
--. "Tracking E. coli: Artificial Stable RNAs in Bacterial Monitoring," invited Sigma Chi lecture, Centers for Disease Control, Atlanta, GA, Oct. 30, 1997.
--. Invited symposium speaker, Texas American Soc. of Microbiology Branch Mtg., Houston, TX, Nov. 6-7, 1997.
Maillard, K. I., L. G. Mendoza, M. D. Eggers, D. L. Pierson, R. C. Willson, and G. E. Fox. "Microbial Monitoring in Space Environments," poster, Nat'l Space Biomedical Research Inst. Retreat, Del Lago Conf. Center, Montgomery, TX, June 8-11, 1998.
Murphy, J. C., G. E. Fox, and R. C. Willson. "Nucleic Acid Separations Using Compaction Agents," poster #115, 216th ACS Nat'l Mtg., Boston, MA, Aug. 23-27, 1998.
Singh, N. and R. C. Willson. "Boronate Affinity Purification of RNA," poster, Int'l Symp. on Purification of Proteins, Peptides, and Polynucleotides, Washington, D.C., June, 1998.
Walia, R. P., J. C. Murphy, G. E. Fox, R. C. Willson. "Microbial Monitoring Using Hybridization Assays and Artificial RNA Labels," poster #BIOT-223, 216th ACS Nat'l Mtg., Boston, MA, Aug. 23-27, 1998.
Willson, R. C. "Novel Approaches to Nucleic Acid Isolation," Merck Research Laboratories, Rahway, NJ, Aug. 1998.
Funding
"An Advanced Approach to Simultaneous Monitoring of Multiple Bacteria in Space." Co-Investigators: M. L. Eggers, M. Hogan, and R. C. Willson; NASA 1995-1997, $914,858; subcontract, $240,000.
"Automated Use of DNA Probes for Rapid Detection of Bacteria in Water." Co-Investigator: R. C. Willson; NASA-JSC, July 1, 1998-June 30, 1999, $29,299.00.
"DNA Probe Design for Pre-Flight and In-Flight Microbial Monitoring." Co-Investigator: R. C. Willson; supported by NASA via Co-operative Agreement NCC-9-58 with the National Space Biomedical Research Institute, Oct. 1, 1997-Sept. 1, 1999, $97,152.00.
"Rapid Characterization of Bacterial Biochemistry." Texas ATP, 2 yrs, $173,147; submitted July 1997 but not funded.

Contents
ISSO -- Institute for Space Systems Operations
1997-1998 Annual Report