The specific aims of the work proposed here are limited to proving that peptides can be separated based on their N-terminal residue. We will isolate RNA aptamers that bind to the last three N-terminal residues and then group the aptamers together that share a common N-termini. This group will be called a recognition module (RM) and there will be 20 RMs, one for each residue. If this work is successful, we will be perfectly positioned to use these aptamer based recognition modules to demonstrate our radically new protein analysis method (pRT). The process can be simplified into three basic steps, 1) selectively separate peptides based on their N-termini, 2) covalently couple a short strand of DNA (a codon) that can be used to decode what residue was bound (figure 1), and 3) remove the N-terminal residue and repeat the process until a sufficient number of residues are converted into DNA.
Affectively we will have reverse translated a protein and this can now be treated as complementary DNA, but complimentary to a protein sequence. We believe that this would then allow proteins to be studied quantitatively by PCR. Protein reverse translation PCR (pRT-PCR). Such an advance would have broad implications to the health and research industry as a whole.
We will obtain the aptamers by screening an RNA aptamer library and selectively enriching for highly specific aptamers (SELEX). Our preliminary results suggest that aptamers to tri-peptides with a fixed N-terminus (N,K, and Y) with nM affinities can be achieved. To further expand and rigorously study aptamers isolated toward these three tri-peptide targets will eventually allow us to use the comercial FLAG tag in a series of binding and Edman degradation rounds to provide proof of concept for the reverse translation process. Finally the FLAG tag will be amplified using PCR from a complex protein sample, demonstrating the utility of pRT-PCR.
The Aims of this Phase I project are to:
Use RNA aptamer selection (SELEX) to enrich for aptamers specific for defined N-terminal residues by targeting tri-peptides sharing a common N-terminal residue (400).
Characterize the affinity, specificity, and sequence of the identified aptamers to their respective targets N, K and Y.
MILESTONE:
1) Selection of DNA aptamers specific to the N, K, and Y N-terminal peptides.
2) The specific precipitation or partitioning of a purified peptide degradation product and validation of its sequence by mass spectrometry
3) The sequencing of the aptamer populations that are specific for their positive targets.
4) The characterization of the families by individual binding reaction analysis, leading to a final family concentration that allows the tuning of its individual specificity and reasonable affinity.
Planned Phase II Award (This information is to assist the reviewer in review of the proposal. This is NOT a fast-track application): The work we propose in Phase I will put us in a position to validate the process of "Reverse Translation" in a Phase II project. In Phase II the following parameters will be established:
Select additional RNA aptamers that bind to the full compliment of the 20 known amino acid residues.
Perform reverse translation on tryptic peptide fragments from a well characterized human cell line and PCR amplify pRT targets.
Compare DNA decoded peptide information to mass spectral sequence data to validate the methods usefulness in uses in complex protein samples (e.g. serum, tissues, and cell culture).
Successful completion of Phase I and Phase II will place the organization in a position to initiate commercial development of a kit targeted toward large scale proteomics research and potentially clinical diagnostic.
B. Significance and related R&D
Reverse translation is a proposed process by which a polypeptide is converted into DNA. More accurately, the polypeptide primary sequence information is reverse translated into a sequence carried by a DNA/RNA molecule (an artificial codon).
The proposed reverse translation process shares many similarities with the well known "reverse transcription" reaction. The similarities start with the recognition of a component of a parent molecule followed by the association of this component with a growing daughter molecule. During reverse transcription the parent is a nucleotide and the daughter is a deoxy-nucleotide. In reverse translation the parent is the N-terminal residue of a peptide or protein and the daughter is a polynucleotide carrying a sequence that decodes the residue type (figure 1).
An important characteristic of reverse transcription is that the parent and daughter concentrations are in a one-to-one relationship after synthesis, allowing for an indirect measure of the mRNA concentrations from a complex sample, the transcriptome. This proportional relationship is maintained in reverse translation. Specifically tagging each peptide with a piece of DNA/RNA that decodes the N-terminal residue in a one-to-one relationship is likely to provide an inexpensive and simple tool for the analysis of proteins, the proteome. To complete the process, the tagging event is followed by the cleavage of the N-terminal residue using Edman degradation. The process of separation, coupling, and degradation is repeated until a significant number of amino acid residues are converted to DNA or RNA.
The approach that we will use to identify the "recognition module" will be the systematic evolution of ligands by exponential enrichment (SELEX), a method pioneered in the early 1980's by Larry Gold and Andrew Ellington (1-4). Using SELEX we will identify a family of RNA aptamers that bind to the N-terminal end of peptides in a residue specific manner. For example the tri-peptide sequences NKK, NGL, NLL, and NIL all share a coincidental asparagine at the N-terminus. The selection of aptamers that specifically recognizes one of the 400 possible tri-peptide sequences will be grouped and used for peptide separation. The aptamers will be targeted toward short peptides coupled to a solid support (Y-X-X-support) from a library with a complexity of 5x1016. By identifying aptamers specific for all 400 of the possible tri-peptide combinations for a specific N-terminus, one can then use these as a family for very straight forward separations from complex peptide pools. This process enables the use of "protein based"-PCR and holds the promise of providing a very inexpensive and sensitive approach to protein research and the potential for breakthrough clinical diagnostics.
Proteomics relies on several well characterized instruments and techniques such as; Western blot analysis, the ELISA assay, 2-dimensional electrophoresis, and mass spectrometry. Each of these technologies has its own strengths and weaknesses, and new methods to quickly, accurately, and inexpensively study the complete set of proteins in a research sample, its "proteome," are highly valued. In our Phase I trial we are focused on developing a complimentary proteomics method that we believe is unique and highly complimentary to the existing methods and instruments. Furthermore, we propose that by transforming the primary sequence information contained in a protein to the more stable and amplifiable form of DNA, we can improve the rate of discovery in the area of proteomics.
Figure 2
The concept of reverse translation has been contemplated and discussed by others (5-10) and at least three theoretic models exist by which protein information can be converted into a DNA molecule. Nonetheless, to the best of our knowledge there has been no reported protein reverse translation in any laboratory or natural biological system. We believe that the process has not been attempted in part due to the "Symmetry Argument" proposed and defined by James Watson. It is thought that the very nature and physical characteristics of protein molecules does not allow for the convenient symmetrical reproduction of a polypeptide into another form. In addition to the more complex physical nature of polypeptides, post translational modifications of protein increase the inherent number of possible states of peptides with similar amino acid sequence. This information compounds the reproducibility problem by increasing the number of possible states a peptide might have and the required recognition partners needed to decode the amino acid information.Nonetheless, even though the process of reverse translation is not found in nature, we believe that like the polymerase chain reaction (PCR), a cornerstone of modern molecular biology tools, that it is possible and maybe even practical.
Several researchers have conceived of feasible theoretical models by which the conversion process might be done (see Jack Trevor article for review). In 1983, Biro envisioned a method in which the reverse translation process would occur across a membrane. In 2001, Masyuki Nashimoto proposed that the selective recognition of protein residues by a third molecule carrying the codon complimentary to its c-terminal partner (an RNA a molecule) could be added sequentially to the end of each protein (figure 2). In 2002, Mark Martin of Mirari Biosciences proposed a model in which the N-terminal byproduct of the Edman degradation process is recognized by a recognition substrate (preferably a molecular imprinted polymer known to recognize small molecules). To date, no proof of concept experiments have been attempted (personal communication).
Biochemistry of Protein and DNA
Figure 3
While protein and DNA/RNA are both polymers and are polymerized enzymatically by all cells, the details of protein structure have made it an unfriendly molecule to replicate or manipulate. The selective replication and enzymatic manipulation of DNA specifically has been the single most important contribution to the growing biotechnology field over all. DNA and its "symmetry" allow for the very accurate alignment of complimentary nucleotides within any target strand and therefore serve as a template for identification and manipulation. Protein, unfortunately, has no complimentary interaction and must rely on the elaborate ribosomal architecture for its RNA template dependent synthesis. Because the peptide bond forged in the bowels of the ribosome are energetically favored for a forward reaction, a method by which the translational machinery can be reversed has been elusive, and by some thought to be impossible. Only recently has serious discussion arisen to challenge the central dogma that DNA can be converted back and forth to RNA and RNA only to protein (figure 3). Interestingly, the theoretic approaches for reverse translation do
not include a modification of the ribosomal system. A possible explanation for the absence of theoretic speculations in this area potentially arise due to the non-symmetrical nature of protein and aminoacyl transferase morphology. The very small pockets that make up the heart of the aminoacyl transferase tRNA and free amino acid binding sites are unstable once the peptide bond has been formed and reversal of the process has not been demonstrated in nature nor in any laboratory to our knowledge.However the recognition of short terminal peptides has been demonstrated by two groups. The C-terminal end of peptides can be specifically recognized (Hessleberth J., personal communication) if the adjacent residues are used as a context in which to recognize the end residue. In another example Jeremy Kiburnand and Srinivasan (11) used short peptides that are driven toward the C-terminus through a general affinity to the carboxyl group, and a more specific interaction between flanking peptide aptamer arms secures its binding partner. This "tweezer" like action has allowed Kilburn to specifically target the C-terminus in a sequence dependent manner. In both of these examples the key to specific recognition was dependant on not only the end residue, but also the residues adjacent to it for specific recognition. The success of these two groups to recognize very small features is likely do to the nature of the binding instrument. Because of the small interaction footprint of peptide and RNA aptamers, specific recognition of small features is more likely.
The use of RNA/DNA Aptamers in Protein Research
RNA aptamers have been used for over 20 years and have been successfully implemented in the recognition of various biomolecules as well as other non-biological targets (12-16). Below is a short list of some of the target molecules that have been used as targets and where the specific structure of binding is known (table 1, Hermann, T. and Patel, D.J. 2000. Adaptive Recognition by Nucleic acid Aptamers. Science 287: 820-825).Interestingly, even free amino acids have been specifically bound by RNA aptamers. These RNA aptamers have been used to specifically purify free amino acids as well as many other small molecules. The small footprint of the RNA aptamer make it a good candidate for the specific recognition of the N-termini as we have described it. Notice Arginine in the list of the examples. Aptamers to tyrosine have also been identified (ref). Unfortunately, the affinities of the aptamers to free amino acids are in the uM range.
It is our hope that we can uncover a robust and useful reverse translation system where interactions are fast and specific. We believe affinities in the nM range are more desirable and therefore it is important to point out that our current model of reverse translation 1) does not depend on the recognition of single amino acid residues and 2) requires an affinity in the nM range.
To meet the developmental challenges of our reverse translation model and identify molecules that interact specifically with the N-terminal residue (in the context of its two adjacent residues, a tri-peptide) we have implemented a very stringent alteration to the SELEX method. In the conventional SELEX method the target is bound to a solid substrate while the aptamer pool is allowed to interact with it. After a one (1) hour binding period the non-specific aptamer pool is washed away by dilution. The fractions are then eluted and the stringency is increased by increasing the salt concentration or heating the reaction to release the tightly bound aptamers. In our preliminary experiments it was important to retrieve a broad spectrum of interaction partners and therefore we modified the protocol in that after the one hour incubation, we briefly washed the target bound to magnetic beads and then placed the beads directly into the RT-PCR reaction. This approach has lead to the inclusion of RNA aptamers that collectively bind in the nM range and appear to be specific for tri-peptides with a specific N-terminal residue (see preliminary results). It will be interesting to see how many aptamers in this pool are already useful binding partners for the 400 individual peptides that we will need to make this technique broadly useful, but our preliminary results are very encouraging.
Unmet Commercial Need and Business Opportunity.
The use of antibodies to identify target proteins in biological samples has been a staple for the biotechnology industry for many years and companies that produce, store, and distribute antibodies to the research, diagnostic, and pharmaceutical fields have been very successful. The market opportunity for materials that accurately and inexpensively identify their complimentary targets, and even more so for those technologies that enable users in unique and special ways have been growing dramatically since the recent development and focus on proteomics. Ampliprot will leverage the reverse translation method to supply the research, diagnostic, and pharmaceutical customers with kits that perform the process from any protein sample from any biological system. The kits will contain the RNA aptamer recognition modules, reagents, and instructions required for the pRT process. The kit will enable the customer to amplify low concentration proteins from a complex sample, a task which is impossible today. The value of this proposition is three fold, 1) protein amplification, 2) compatibility with DNA microarray analysis, 3) library construction. We believe that this will be highly valued by the biotechnology industry, and potentially positioned for the production of ultra-sensitive diagnostic applications.
Preliminary Studies
Publications: The P.I. has extensive experience in new product and method development, specifically related to the area of genomics and proteomics.
Figure 4
SELEX
Figure 5
To begin our search for N-terminal specific aptamers we constructed a SELEX library by synthesizing a template molecule that contains a central region of 40nt that are randomized during synthesis, flanked by a 5' and 3' constant region that serve as RT-PCR amplifiable adaptors that allow for the selective enrichment of the affinity purified RNA aptamers. The library template also contains the sequence recognized by the T7 polymerase. The RNA aptamers are synthesized by this polymerase and is then purified using a Qiagen RNA purification kit. The template contains these four (4) elements;
T7 promoter - 5' primer region - 40mer randomized - 3'primer region
This SELEX template was used to create a starting aptamer pool with the final complexity of 5x1016 and was screened against the tri-peptide sequences mentioned above bound to magnetic beads through a biotin-streptavidin interaction. Four rounds of selection and amplification were performed followed by an additional three rounds that included a preliminary negative selection step. The incubation is performed in a buffer that contains MgCl2. Magnesium appears to be crucial for the recognition of aptamers to small peptide targets. It has been suggested that magnesium and other metals play a role in stabilizing the three dimensional structure of the RNA-peptide complex (17).
Figure 6
After a total of seven (7) rounds of the SELEX process the remaining pool of aptamers were expanded using RT-PCR and two pools of RNA was synthesized to study the selectivity and affinity of the pool. A binding study was performed on one half of the material that was radio labeled with P32 UTP. 50 ul of a magnetic bead slurry coated with the specific or non-specific peptides was used during the aptamer screen. A serial dilution of radio labeled "round 7" aptamers were placed in contact with the beads in a 96 well thin walled PCR plate and allowed to incubate for one hour and then washed twice with binding buffer to remove non-specific interactions. The plate was placed on a sheet of photographic film for 20 minutes to image the pattern of binding among the wells. Figure 5 is a scan of this film which indicates that the three aptamer pools, (selected for their binding to N, K, and Y N-termini), bind to there complimentary peptides that contain the correct N-terminus. The same pools were also incubated with peptides containing all but the correct N-terminus and in these wells it is clear that the binding to these peptides is significantly reduced.To better understand the relationship between positive and negative peptides we used the aptamer pool for asparagines and placed 20ul into 1ml of scintillation fluid and scanned the samples for counts per minute (figure 6). The results suggest that the binding of peptides containing an asparagine was being bound more specifically than to peptides that do not contain asparagines at the N-termini. In addition, this preliminary binding study suggests that the affinity of some aptamers is in the nM range. There is still background observed from these pools and therefore we will continue our selection scheme to increase the representation of aptamers that specifically bind our 400 targets.
Figure 7
In addition to the binding radioactive study we wanted to better understand what peptides are actually being bound by the aptamers. Therefore the second sample of RNA synthesized was used to bind to free peptides that contained all possible tri-peptide sequences. 100ug of RNA aptamer from the three pools (N, K, and Y) was incubated with 30ug of free peptide in 1 ml of binding buffer. This was allowed to incubate for 30 minutes and was then filtered through a 30kd MWCO Amicon filter. Since the RNA aptamers are retained by this filter and the free peptides are not, any peptides that are found in the retentate, after filtration, are assumed to be bound to the aptamer. Figure 7 is the MALDI spectrum from the retentate and the flow through. Notice that there are clear peptide spikes in the retentate sample, indicating that free peptides are being bound by the aptamers. They are also not the same peaks for the three pools.
Collectively these observations suggest that the concept of N-terminal specific recognition modules can be created and optimized, and then successfully used to then partition free peptides for coupling to DNA tags. This example also illustrates that more than one aptamer can be successfully multiplexed to affinity purify targets with complex features.
D. Experimental Design and Methods
Rationale in the context of plans for a Phase II award: The methods are outlined based on our quantifiable MILESTONE: the creation of an RNA aptamer family that will specifically recognize peptides sharing a common N-terminal end with an affinity close to 500 nM.
.. Selection of N-termini Specific Aptamers
The binding and partitioning of peptides based on the content of their N-terminal residue is in itself very interesting as a tool to reduce the complextity of complex protein samples, however in the context of an iterative DNA tagging method, followed by the Edman degradation process, the concept of protein amplification becomes far more important and realistic. Nashimoto's theoretical model for how reverse translation may have occurred in nature is very insightful and when the target of the reverse translation mechanism is 1) changed to the N-terminal end, 2) the targets are expanded to include the adjacent residues, and 3) Edman degradation is added, a real and practical path to reverse translation and amplification from a protein substrate become reasonable and clear. Consequently, our objective is to identify and characterize molecules, in this case RNA aptamers, that specifically and avidly recognize the N-terminus.
1) Selection of DNA aptamers specific to the N, K, and Y N-terminal peptides.
2) The specific precipitation or partitioning of a purified product and validation of its sequence by
mass spectrometry
3) the sequencing of the aptamer populations that are specific for there positive targets.
4) The characterization of the families by individual binding reaction analysis, leading to a final family
concentration that allows the tuning of its individual specificity and reasonable affinity.
The empirical measurement of an aptamer binding to a single residue is likely to be similar to that of Arginine (uM). Therefore it is likely that by targeting the N-terminal residue in the context of its adjacent residues we will improve the specificity, affinity, and possibly the rate of the process. The recognition of most objects is based on the interaction at three points of contact. It is possible that the recognition of small targets reach certain three dimensional limitations. It will be interesting to characterize the final pool of aptamers and determine the structural aspects of their recognition, yet that will be considered as a Phase II aim.
Figure 8
To better understand the details of the Phase I aims proposed here it is important to consider the electrostatic morphology of amino acids of the N-terminal residues specifically. Each amino acid residue contributes to the overall pH, hydrophobicity, 3-dimensional boundaries, and how these properties change over time. The partial charge, bond strength and size of atoms in each amino acid residue participate in the recognition of the macromolecule polypeptide. Figure 8 is a simplified model illustrated by Biro (8, 9). Notice that over time, as well as influences by the local micro-environment, the electrostatic morphology of peptide residues can be defined, yet susceptible to minor fluctuations.
It is this physical information that we are attempting to reverse translate into a DNA/RNA molecule of a predefined sequence.
By identifying "recognition modules" (specifically using DNA/RNA aptamers) that recognize the N-terminal structure of interest, we can separate peptides that carry this structure from a complex mixture of unrelated molecules. This separation stage is then followed by the covalent coupling of DNA carrying the appropriate codon (becoming the "leader," or coupling target in successive rounds of pRT) to the carboxyl end of that peptide. This codon label can then be either amplified and sequenced immediately, or allowed to remain attached to the peptide for further polynucleotide extension following Edman degradation of the N-terminal residue.
Figure 9
The process of N-terminal morphology recognition coupled to the selective degradation of the N-terminal end will provide the fidelity over the decoding of the amino acid sequence tag into DNA.
In the phase I experiment the "Flag" tag used will be a purified peptide alone in solution, and the tag sequence will be known. Therefore one would expect that for each degradation product of the tag a different and unique epitope would exist. For each epitope that exists, a separate and unique recognition particle (in this case RNA aptamer obtained using the SELEX method) will be required to pull down the specific structure.
The ultimate goal of our work is to convert the information of a specific polypeptide, with a defined post translational form (FLAG, Asn-Tyr-Lys-Asn-Asn-Asn-Asn-Lys), into a DNA tag that can be 1) sequenced to decode the amino acid sequence, and 2) amplified proportionally to parent molecule's starting concentration (figure 9).
To achieve our future goals it is necessary to collect all of the aptamers that recognize a specific N-terminal specific residue and to couple this group to a solid substrate. This substrate can then be used to partition and tag the peptides of interest. The purpose of this Phase I work is to demonstrate that the physical structure of the leading N-terminal residues can act as a specific recognition epitope in the context of its adjacent residues. Afterwards we will use this aptamer set to convert the FLAG tag into a DNA molecule of a predefined sequence in a reproducible and predicable manner. The N-terminal end of many peptides and proteins have been used as epitopes for the specific recognition of pre and pro-peptides by countless researchers, yet in no case to our knowledge has this information been converted into a polynucleotide that can act as a specific and amplifiable tag.
The structural recognition of N-terminal peptide fragments without sequencing and based purely on the recognition by classical epitope binding agents such as antibodies, show some variability in the amino acid sequence, leading to cross reactivity among protein family members and misrepresentation of the N-terminal peptide sequence.
The pRT model proposed here may possess significant advantages by selectively recognizing an N-terminal epitope by an antibody. Selectively degraded products potentially offer additional information to improve the signal to noise ratios of decoded information, and hopefully to then provide a more defined sequence and peptide/protein identification.
1.1 Selection of RNA aptamers.
The steps listed here in Aim 1 are straightforward in the Smith laboratory at The Burnham Institute. We anticipate few, if any, difficulties and expect the work can be completed within 2-4 months.
The affinity of the aptamers will be considered during and after the selection process. By altering the selection environment we intend to target a high affinity aptamer by including the bound substrate into the RT-PCR reaction. By placing the bead, tightly bound to high affinity aptamer partners, we can include them in the enrichment process. An estimate of this affinity can be deduced from the preliminary results obtained from our first attempt at selecting for this sort of target.
The results of the binding study suggest that at least some portion of the aptamer population is actually binding to peptides that contain the correct N-terminal residue, and most importantly that the aptamers are above background. Although it is reasonable to assume that not all of the aptamers to all of the 400 targets of a specific residue will share this valuable characteristic, we intend to modify the binding conditions (salt concentration, pH, and blocking agents) to refine the process and allow for the most robust reaction achievable.
As mentioned above, it is important for the reverse translation process that all 400 targets for any one of the 20 specific N-termini be bound to a reasonable extent so as to partition or specifically target that peptide of aDNA tagging. To ensure that the balance of individual aptamers to their respective targets is appropriate, we will sequence the aptamer population and characterize the aptamers individually. The aptamer population
Grant Summary
Results obtained with this STTR funding will generate a pool of aptamers specific for peptides that contain specific N-termini, namely N,K, and Y, and these reagents will then allow for the testing of the reverse translation and the first known method of protein amplification (pRT-PCR). With such a set in hand, we can begin testing of its efficiency and practicality of pRT (Phase II).
E. Human Subjects
No human subjects are involved in this study.
Vertebrate Animals
Vertebrate animals are not used in this study.
Citations
1. Ellington, E.P., Wood, J.G., and Hill, E.O. 1982. Disease caused by a marine vibrio -- Vibrio vulnificus. N Engl J Med 307:1642.
2. Gold, L. 1995. The SELEX process: a surprising source of therapeutic and diagnostic compounds. Harvey Lect 91:47-57.
3. Gold, L. 1995. Conformational properties of oligonucleotides. Nucleic Acids Symp Ser:20-22.
4. Gold, L., Brown, D., He, Y., Shtatland, T., Singer, B.S., and Wu, Y. 1997. From oligonucleotide shapes to genomic SELEX: novel biological regulatory loops. Proc Natl Acad Sci U S A 94:59-64.
5. Trevors, J.T. 2001. Molecular evolution: first enzymes, gases as substrates and genetic templates. Riv Biol 94:105-122.
6. Trevors, J.T. 2003. Genetic material in the early evolution of bacteria. Microbiol Res 158:1-6.
7. Trevors, J.T., and Abel, D.L. 2004. Chance and necessity do not explain the origin of life. Cell Biol Int 28:729-739.
8. Biro, J. 1983. Information transfer in biological systems. Part II: Transfer of molecular information through biological membranes. The "reverse-translation". Med Hypotheses 12:31-40.
9. Biro, J. 1983. Information transfer in biological systems. Part I: Calculation of the information content of some amino acids, lipids, and nucleic acid bases. Med Hypotheses 12:21-30.
10. Nashimoto, M. 2001. The RNA/protein symmetry hypothesis: experimental support for reverse translation of primitive proteins. J Theor Biol 209:181-187.
11. Srinivasan, N., and Kilburn, J.D. 2004. Combinatorial approaches to synthetic receptors. Curr Opin Chem Biol 8:305-310.
12. Klug, S.J., and Famulok, M. 1994. All you wanted to know about SELEX. Mol Biol Rep 20:97-107.
13. Lee, J.F., Hesselberth, J.R., Meyers, L.A., and Ellington, A.D. 2004. Aptamer database. Nucleic Acids Res 32:D95-100.
14. Clark, S.L., and Remcho, V.T. 2002. Aptamers as analytical reagents. Electrophoresis 23:1335-1340.
15. Brody, E.N., and Gold, L. 2000. Aptamers as therapeutic and diagnostic agents. J Biotechnol 74:5-13.
16. Famulok, M., and Mayer, G. 1999. Aptamers as tools in molecular biology and immunology. Curr Top Microbiol Immunol 243:123-136.
17. Patel, P.K., Bhavesh, N.S., and Hosur, R.V. 2000. Cation-dependent conformational switches in d-TGGCGGC containing two triplet repeats of Fragile X Syndrome: NMR observations. Biochem Biophys Res Commun 278:833-838.
Contractual Arrangements
Ampliprot has three arrangements with The Burnham Institute; 1) Ampliprot has a sub-contract with the Burnham Institute to conduct 70% of the STTR work in Dr. Smiths laboratory. 2) Ampliprot will also negotiate a separate contractual arrangement with the Burnham Institute to rent laboratory space for Ampliprot personnel, and to give that individual access to shared equipment at the Institute. The leasing of this space and access to shared equipment will be funded by indirect costs requested by Ampliprot. 3) Ampliprot will negotiate a license to the patent on "protein reverse translation" which claims the sequences of the aptamers for reverse translation.
Resource Sharing – N/A
Letters of Support
N/A
Commercialization Plan
While not required for the Phase I STTR application, a section dealing with commercialization strategy of Ampliprot is provided for information.
Value and Expected Outcomes
Ampliprot, LLC, a California limited liability company ("Ampliprot", the "Company" or the like), has developed a novel method that converts protein into DNA to enable researchers and physicians to quantitatively analyze proteins with greater sensitivity, throughput, and ease. The research, pharmaceutical, and diagnostic markets currently enjoy the powerful technique of DNA based technologies like quantitative PCR, microarray, and nucleic acid library analysis. These DNA techniques amplify and record the abundance and sequence information contained in nucleic acid molecules from biological samples, but to the best of our knowledge, there is no comparable method for proteins. Ampliprot's new method "reverse translation" is an enabling technology that has several unique characteristics compared to the current methods of protein research that are projected to radically improve A) the sensitivity by a factor of 10,000X, B) the throughput of research by at least a factor of 100X, and C) ease-of-use by allowing researchers to use conventional DNA based equipment already installed in virtually every laboratory. By improving the sensitivity, throughput, and ease-of-use of these activities at a comparable price, Ampliprot will reduce the inherent costs of its customers' research and development, the central theme of the Company's value proposition.
Company
Ampliprot LLC is an early stage biotech Company owned entirely by David O'Hagan, its chief executive officer. The Company was founded on the development of a method for Reverse Translation and continues to be the Company's only focus.
At this juncture Ampliprot is focusing on three near-term tasks. First, we have begun the process of securing our intellectual property position through our partnership with Jeffery Smith from the Burnham Institute, and we will continue to expand and refine the intellectual property base by filing patents on aptamer sequences found to participate in the reverse translation process. Second, we are working to generating a wider portfolio of pRT examples to underscore the value and utility of the technology. Third, we are seeking diagnostic partners for collaborative projects and an initial seed investment. Given the widespread interest in protein based diagnostics we believe that seed funding can be obtained from a partnership with two or three biotech companies.
This STTR is of great importance to Ampliprot because the outcome will provide proof of concept for the more general and broadly useful pRT method. The study is also likely to yield interested commercial partners. We believe that to stimulate interest in co-development of such a technology, Ampliprot will need to show that pRT has a accurate aptamer based separation, and has efficiency in doing so. We fully anticipate that these objectives can be met by a subsequent Phase II award.
Market
Drug discovery research and other related efforts have substantially increased the visibility of genomics and proteomics, and the possibilities these fields hold for science and humankind. Now uncovered, these possibilities bring to light the need for sensitive, quantitative, and high-throughput methods to realize the new potential. Speed, sensitivity, quality of output, and costs are the most critical issues that face the industry. While current technologies have been continuously improved to support the ever-increasing needs of this market, they are reaching the limitations of their capacity. There are inherent glass ceilings for the current methods, especially for the detection and analysis of human proteins as compared to the very successful DNA analysis efforts. Ampliprot has developed a solution that breaks these barriers for the first time, by converting the sequence and abundance information contained in proteins to a DNA molecule that can be studied using conventional DNA techniques. To the right is how we believe we are positioned in the market as compared to current methods.
According to industry reports, the global market for tools used to develop and perform bioassays is estimated to have been approximately $24 billion in 2004 and is expected to grow at an annual rate of 5.7%. A very important segment of this market is targeted to the study of proteins. According to NIH funding reports there were 52 proteomics based research projects in 2000 that grew to 970 in 2003.
Outside of the basic research market, billions of dollars are spent on individual drug development research that focus on proteins as drug targets, and the product development timeline can be 10 years or more. Companies are seeking any advantages in time or cost in this high risk, high reward business. Products that can provide these advantages are highly valued and demanded.
In addition to the basic research performed by the biotech and pharmaceutical industry, the clinical diagnostic markets also strive to improve the predictive quality of their assays and invite technology innovations that provide a competitive advantage. The clinical diagnostic market revenues topped 24.6 billion in 2003 and is growing at a more modest annual rate of 3.7%
Ampliprot's enabling technology will be developed to provide useful alternatives to the current protein detection methods used by both industries in the form of kits and analysis software.
Unlike conventional protein analysis technologies, Ampliprot's proposed protein analysis technology will take advantage of "reverse translation." reverse translation is the conversion of proteins or peptides into a predetermined DNA molecule that decodes the sequence of the peptide, providing an identification signature and abundance metric that allows for the sensitive analysis of proteins from research samples and most importantly complex bodily fluids that serve as important biomarkers of disease.
It is expected that the technology will improve the detection sensitivity by at least 10,000 fold. Through this technology, Ampliprot will provide a better solution to the marketplace than is currently available, one that speeds the process, providing higher sensitivity and greater accuracy. Ampliprot's proprietary technology is also more flexible than most other solutions, providing a more versatile platform for future market needs.
An important and novel characteristic of the reverse translation method is that the protein sequence information is retained. In nature DNA is converted to mRNA, and that into the proteins that make up the superstructure of our bodies. The mechanism that the cell uses to code for proteins is that each DNA molecule contains a series of three nucleotides (the "codon", the building blocks of the DNA molecule) that code for a particular amino acid (the building blocks of proteins). In our method we reverse the process by converting each amino acid contained in the protein to a predefined DNA codon. Furthermore, the conversion process may also be engineered to recognize modifications of amino acids in protein (post translational modifications) that are important for modulating the activity of certain proteins.Ampliprot is dedicated to establishing a dominant intellectual property position in the area of reverse translation. All forms of reverse translation intellectual property will be filed or licensed over the next 18 months. Ampliprot has developed special expertise by blending the talents of clinical, molecular biology, and proteomics expertise from its participation with the Burnham Institute. This expertise is coupled with significant strategic management qualities. The table to the right summarizes the features/benefits of the planned Ampliprot reverse translation technology.
Competition: Ampliprot's technology meets two important needs in the protein diagnostic market, 1) the ability to amplify protein, and 2) the ability to multiplex protein study inexpensively. The primary competition are the protein array and mass spectrometry markets. Ampliprot's technology is believed to be cheaper than mass spectrometry and more accurate and sensitive than protein microarrays produced today.
Intellectual Property
Financing Plan
At this early stage Ampliprot has several options for financing the company, including venture capital, investment by independent investors (angles and higher levels of funding), and partnerships and investment by larger biotech and diagnostic companies. Having gone the venture route previously (Streamline Proteomics), our intent is to focus on raising funds through investment by our biotech and diagnostic partners.
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