This Program Announcement expires on April 1, 2004, unless reissued. SINGLE MOLECULE DETECTION AND MANIPULATION SBIR/STTR Release Date: February 12, 2001 PA NUMBER: PA-01-050 National Institute of General Medical Sciences ( National Institute of Diabetes and Digestive and Kidney Diseases ( National Human Genome Research Institute ( SBIR/STTR Application Receipt Dates: April 1, August 1, and December 1 PURPOSE The purpose of this SBIR/STTR program announcement (PA) is to encourage research on the technology and methodology related to the detection and manipulation of single molecules. Recent advances in optical imaging and biomechanical techniques have demonstrated that it is possible to make observations on the dynamic behavior of single molecules, to determine mechanisms of action at the level of an individual molecule, and to explore heterogeneity among different molecules within a population. These studies have the potential to provide fundamentally new information about biological processes and are critical for a better understanding of cellular function. Current high-resolution methods, such as x-ray crystallography and NMR, have provided a vast array of structural detail for biological molecules, yet the output of these methods is limited by its static molecular view and ensemble averaging. Single molecule methods provide an alternative set of approaches that will lead to a more direct view of the action of molecules without the need to infer process or function from static structures. Real-time measurements on the spatial and temporal fluctuations of single molecules in living cells, which are not possible using other methods, are a major goal of this initiative. Despite the promise of single molecule methods, there are a number of technical challenges that must be met to optimize these studies. Development of the collateral chemistry and instrumentation required to carry out single molecule studies is essential for progress. New tools and strategies, as well as refinement of current methods, are also needed. Single molecule methods are likely to lead to significant advances in understanding molecular movement, dynamics, and function. HEALTHY PEOPLE 2010 The Public Health Service (PHS) is committed to achieving the health promotion and disease prevention objectives of "Healthy People 2010," a PHS led national activity for setting priority areas. This Program Announcement (PA), Single Molecule Detection and Manipulation SBIR/STTR, is related to one or more of the priority areas. Potential applicants may obtain a copy of "Healthy People 2010" at ELIGIBILITY REQUIREMENTS Eligibility requirements are described in the SBIR/STTR Omnibus Solicitation. Any small business concern, independently owned and operated by United States citizens or lawfully admitted permanent resident aliens, which is located in the United States and is organized for profit, may apply. MECHANISM OF SUPPORT Support for the PA is through the National Institutes of Health (NIH) SBIR and STTR mechanisms. Applications can be submitted for support as Phase I STTR (R41) or Phase I SBIR (R43) grants; Phase II STTR (R42) or Phase II SBIR (R44) grants; or under the SBIR/STTR FAST-TRACK option as described in the SBIR/STTR Omnibus Solicitation. Phase II applications in response to this PA will only be accepted as competing continuations of previously funded NIH Phase I SBIR/STTR awards. The Phase II proposal must be a logical extension of the Phase I research. Information on the FAST-TRACK process and the SBIR/STTR Omnibus Solicitation is available at A similar program announcement for single molecule methodology and technology development to be supported by individual research project (R01) and program project (P01) applications is available at: ( A related program designed for the development of bioengineering SBIR/STTR projects on nanotechnology, as part of the trans-NIH BECON (Bioengineering Consortium) can be found at: RESEARCH OBJECTIVES Background Recent advances in the detection and manipulation of single molecules offer great promise for enhancing our understanding of the behavior of individual biological macromolecules in the living cell. Scanning probe techniques allow imaging of single molecules on surfaces, and specialized optical techniques enable their characterization in complex environments. Single molecule biomechanical studies have been used to manipulate individual molecules and to measure the force generated by molecular motors or covalent bonds. The development of new probe technologies, such as quantum dots and high-resolution laser fluorescence microscopy, allow real-time observations of molecular interactions and trafficking within living cells. These tools enable individual members of a population to be examined, identified, and quantitatively compared within cellular sub-populations and substructures. Single molecule studies have the potential to provide spatial and temporal information that is impossible to obtain using other, more static techniques. X-ray crystallography, nuclear magnetic resonance, and electron microscopy have provided a wealth of information on molecular structure, yet none of these methods can be used to make measurements on the in vivo dynamic movements of single molecules in intracellular space or to observe the behavior of single molecules over extended periods of time. Using single molecule methods, it should be possible to study time trajectories and reaction pathways of individual members in a cellular assembly without averaging across populations. Cellular processes, such as exocytosis, flux through channels, or the assembly of transcription complexes, could be visualized. Individual differences in structure or function generated by allelic polymorphisms should be detectable at the level of the single molecule. Monitoring the coordinated expression of a gene or group of genes in specific tissues, or at certain developmental stages, is within reach using these technologies. Thus, single molecule methods are recognized as an important new set of tools that can be applied to high resolution studies in many areas of biology. On April 17-18, 2000, the National Institute of General Medical Sciences (NIGMS) sponsored a workshop to explore the progress and potential for targeted research in single molecule detection and manipulation. Topics that were discussed included single molecule fluorescence studies, imaging and manipulation of single molecules with Atomic Force Microscopy (AFM), studies of single channels, biomechanical studies on single molecules using optical tweezers, and computational studies based on biological machines. In addition to making presentations on their most recent work, the participants were asked to discuss how to develop further the technologies to facilitate progress in this field. A summary of the workshop can be found at This program announcement is issued in response to the findings and recommendations of the workshop, as endorsed by the National Advisory General Medical Sciences Council at its May, 2000, meeting. It recognizes the powerful impact that single molecule research may have on research in biology, and it addresses the needs of potential research programs in this area. Scientific Objectives: The goals of single molecule research are to observe the dynamic behavior of individual molecules, to explore heterogeneity among molecules, and to determine mechanisms of action. Single molecule studies are uniquely designed to yield information about molecular motion, behavior and fluctuations over time and space. An important aspect of the research will be to measure features of individual molecules that are masked by ensemble measurements. Real-time observation of single molecules in live cells, relative to in vitro studies, is an important goal. Targets for study Potentially any biological molecule is a target for study. Typical molecules are members of multi-component systems that change in response to environmental cues or specific cellular signals. Examples of experimental systems currently under study at the single molecule level include but are not limited to: o Protein folding: pathways, existence of intermediates, kinetics, heterogeneity o Enzyme catalysis: mechanism of catalysis, conformational changes o Ion channels: local structural changes, kinetics o Signaling: formation of multimers, kinetics of cascades, phosphorylation dynamics o DNA, DNA binding proteins, RNA: binding constants, regulation of gene expression o Membrane structure: restricted diffusion, phase changes o Molecular motors: motility, processivity, directionality o Complex cellular structures (e.g., transcription complexes): assembly, dynamics The specific objectives of this program announcement are: (1) To encourage investigators to develop and extend existing single molecule technologies to examine molecular motion, behavior, heterogeneity, and fluctuations over time and space; (2) To devise new tools and strategies for studying single molecules; (3) To validate the methodology used to study single molecules to establish the reliability of the observations. Differences between ensemble and single molecule measurements need to be clarified so that the contributions of the single molecule to the ensemble behavior are understood. (4) To encourage studies on the 3-D visualization of cellular processes in real-time, in the living cell, at high resolution. Many complex cellular processes, such as signaling or translation, are amenable to analysis using single molecule methods. (5) To develop the collateral chemistry to facilitate the detection and handling of target molecules. The categories of greatest need in chemistry are to: o Improve the photophysical properties of fluorophores and other labels used for single molecule spectroscopy, including the synthesis of probes with improved luminescent characteristics that are compatible with intracellular conditions; optimization of quantum dots, plasmon and Raman probes, and G/C/Y/R- fluorescent proteins; o Develop new classes of probes or new strategies for labeling single molecules, particularly those that can be used for in vivo studies; o Develop better methods for insertion of site-specific labels for detection of single molecules, and better mechanical handles for their manipulation; o Design better surface attachment protocols to immobilize single molecules or cells for in vitro measurements. In order to achieve these goals it will be necessary to create strong collaborations with chemists with the goal of testing new chemistry on single molecule problems. (6) To develop improved instruments to optimize high-resolution single molecule measurements. The goals are to: o Refine currently used techniques such as high resolution laser microscopy, near-field scanning optical microscopy, confocal microscopy, wide-field microscopies such as TIR (total internal reflection microscopy) or epifluorescence, optical tweezers and AFM; o Develop instruments with the capability to carry out higher resolution measurements, such as time-resolved/time-gated CCDs for faster, more sensitive detection; higher resolution AFM; optical traps to measure forces in the femtoNewton range; multiphoton spectroscopy optimized in the 50 nm range; flow chambers designed for 0.01 msec measurements; o Design instrumentation using principles to enable commercialization so that more investigators will have access to it and will not be required to build their own; Applicants are encouraged to include physicists, engineers, and computational scientists in the strategies to solve instrumentation problems related to single molecule studies. Scope This PA emphasizes the need to encourage the participation, in addition to biologists and biophysicists, of chemists, engineers, physicists, and computational scientists in single molecule research. Because of the level of experience and skill required, support may include career track, senior postdoctoral scientists with expertise in chemistry, physics, and instrument development. The funding Institutes may provide a substantial contribution for the purchase or development of instruments, when the instrument is justified as part of the supported research effort. In all cases, the cost of the instrument and associated operating support must be consistent with the scope of the research project(s) with which it is associated. Summary The purpose of this SBIR/STTR program announcement is to stimulate research projects on the development of methodology and technology related to single molecule studies. It has become increasingly clear that state-of-the-art single molecule methods offer a powerful new approach to understanding subcellular structure and function. These methods have significant advantages over more static methods since they are designed to make observations on molecules as they move in time and space. Goals of this initiative are to observe the dynamic behavior of individual molecules, to explore heterogeneity between molecules, and determine mechanisms of action. A long-range goal of these studies is to extend the measurements to the intracellular environment where individual molecules will be viewed as they move inside the cell, carry out specific functions, or behave as components of larger systems. In order to fully realize the potential of these tools, there are technical barriers that must be overcome. Development of the chemistry and instrumentation that support single molecule studies is emphasized as well as innovative new methods and tools that will facilitate single molecule approaches. INCLUSION OF WOMEN AND MINORITIES IN RESEARCH INVOLVING HUMAN SUBJECTS It is the policy of the NIH that women and members of minority groups and their sub-populations must be included in all NIH-supported biomedical and behavioral research projects involving human subjects, unless a clear and compelling rationale and justification are provided indicating that inclusion is inappropriate with respect to the health of the subjects or the purpose of the research. This policy results from the NIH Revitalization Act of 1993 (Section 492B of Public Law 103-43). All investigators proposing research involving human subjects should read the UPDATED "NIH Guidelines for Inclusion of Women and Minorities as Subjects in Clinical Research," published in the NIH Guide for Grants and Contracts on August 2, 2000 (; a complete copy of the updated Guidelines are available at The revisions relate to NIH defined Phase III clinical trials and require: a) all applications or proposals and/or protocols to provide a description of plans to conduct analyses, as appropriate, to address differences by sex/gender and/or racial/ethnic groups, including subgroups if applicable; and b) all investigators to report accrual, and to conduct and report analyses, as appropriate, by sex/gender and/or racial/ethnic group differences. INCLUSION OF CHILDREN AS PARTICIPANTS IN RESEARCH INVOLVING HUMAN SUBJECTS It is the policy of NIH that children (i.e., individuals under the age of 21) must be included in all human subjects research, conducted or supported by the NIH, unless there are scientific and ethical reasons not to include them. This policy applies to all initial (Type 1) applications submitted for receipt dates after October 1, 1998. All investigators proposing research involving human subjects should read the "NIH Policy and Guidelines on the Inclusion of Children as Participants in Research Involving Human Subjects" that was published in the NIH Guide for Grants and Contracts, March 6, 1998, and is available at the following URL address: Investigators also may obtain copies of these policies from the program staff listed under INQUIRIES. Program staff may also provide additional relevant information concerning the policy. URLS IN NIH GRANT APPLICATIONS OR APPENDICES All applications and proposals for NIH funding must be self-contained within specified page limitations. Unless otherwise specified in an NIH solicitation, internet addresses (URLs) should not be used to provide information necessary to the review because reviewers are under no obligation to view the Internet sites. Reviewers are cautioned that their anonymity may be compromised when they directly access an Internet site. APPLICATION PROCEDURES This PA must be read in conjunction with the Omnibus Solicitation of the Public Health Service for Small Business Innovation Research (SBIR)(PHS 2001-2) and Small Business Technology Transfer (STTR) (PHS 2001-2). All of the instructions within the Omnibus Solicitation apply. Omnibus Solicitations for both the SBIR and STTR programs are available electronically through the NIH, Office of Extramural Research "Small Business Funding Opportunities" web site: Helpful information in preparation of the application can be obtained at: and Applications in response to this PA are to be submitted on the grant application form PHS 6246-1 (1/98) for SBIR Phase I and PHS 6246-3 (1/98) for STTR Phase I [], PHS 6246-2 (1/98)for SBIR Phase II [], and PHS 6246-4 (1/98) for STTR Phase II []. The title and number of this PA must be typed in line 2 on the face page of the application. Potential applicants are encouraged to contact program staff for guidance and to read the advice and information on the web sites. However, responsibility for planning, direction, and execution of the proposed research will be solely that of the applicant. As stated in the MECHANISMS OF SUPPORT section, applications may be submitted for Phase I alone (R41/43), or Phase II (R42/44) alone if there has been previous and successful Phase I support, or through the FAST-TRACK mechanism. Application instructions specified in the SBIR/STTR Omnibus Solicitation for each mechanism must be followed. The normal level of support and period of time for a Phase I SBIR award is $100,000 and six months; for a Phase II SBIR award, $750,000 and two years. The normal level of support and period of time for a Phase I STTR award is $100,000 and one year; for a Phase II STTR award is $500,000 and two years. However, applicants may propose longer periods of time and greater amounts of funds if necessary for completion of the project. (See NIH Guide for Grants and Contracts, February 12, 1998; FAST-TRACK applications must specify clear, measurable goals that should be achieved prior to Phase II funding. Failure to provide measurable goals in sufficient detail in the Phase II application may be a reason for the peer review committee to exclude the Phase II application from consideration. Phase II applications submitted in response to this PA will only be accepted as continuations of previously funded Phase I grants. The Phase II proposal must be a logical extension of the Phase I research but not necessarily a Phase I supported in response to this PA. All Phase II applications must contain a succinct product development plan. The National Institutes of Health (NIH) is employing features of the Modular Grant Application and Award procedures under its SBIR program. SBIR Phase I grant applications requesting up to $100,000 total costs (direct costs, indirect costs, and fixed fee) will request direct costs in a budget narrative format rather than being compiled from detailed and separate categories. A summary of the unique features of the SBIR and STTR modular grant applications and award processes can be found within the SBIR/STTR Omnibus Solicitation. For applications requesting in excess of $100,000 total costs (direct costs, indirect costs, and fixed fee) the Modular Grant features do not apply. The completed original application and two legible copies must be sent or delivered to: CENTER FOR SCIENTIFIC REVIEW NATIONAL INSTITUTES OF HEALTH 6701 ROCKLEDGE DRIVE, ROOM 1040, MSC 7710 BETHESDA, MD 20892-7710 BETHESDA, MD 20817 (for express/courier service) Applications must be received by the receipt dates listed at the top of the first page of this PA. REVIEW CONSIDERATIONS Upon receipt, applications will be reviewed by the Center for Scientific Review (CSR) for completeness and by the NIH institute for responsiveness. Applications not adhering to application instructions described above and those applications that are incomplete or non-responsive will be returned to the applicant without review. Applications will be assigned on the basis of established NIH referral guidelines. Applications will be evaluated for scientific and technical merit by an appropriate scientific review group convened in accordance with the standard NIH peer review procedures. As part of the initial merit review, all applications will receive a written critique and undergo a process in which only those applications deemed to have the highest scientific merit, generally the top half of applications under review, will be discussed, assigned a priority score, and receive a second level review by the appropriate national advisory council or board. Review Criteria The goals of NIH-supported research are to advance our understanding of biological systems, improve the control of disease, and enhance health. In the written comments reviewers will be asked to discuss the following aspects of the application in order to judge the likelihood that the proposed research will have a substantial impact on the pursuit of these goals. Each of these criteria will be addressed and considered in assigning the overall score, weighting them as appropriate for each application. Note that the application does not need to be strong in all categories to be judged likely to have major scientific impact and thus deserve a high priority score. For example, an investigator may propose to carry out important work that by its nature is not innovative but is essential to move a field forward. Review criteria are described in the NIH Omnibus Solicitation and are available on the Web at the following URL address: The goals of NIH- supported research are to advance our understanding of biological systems, improve the control of disease, and enhance health. The reviewers will comment on the following aspects of the application in their written critiques in order to judge the likelihood that the proposed research will have a substantial impact on the pursuit of these goals. Each of these criteria will be addressed and considered by the reviewers in assigning the overall score, weighting them as appropriate for each application. Note that the application does not need to be strong in all categories to be judged likely to have a major scientific impact and thus deserve a high priority score. (1) Significance: Does this study address an important problem? Does the proposed project have commercial potential to lead to a marketable product or process? What may be the anticipated commercial and societal benefits of the proposed activity? If the aims of the application are achieved, how will scientific knowledge be advanced? Does the proposal lead to enabling technologies (e.g., instrumentation, software) for further discoveries? Will the technology have a competitive advantage over existing/alternate technologies that can meet the market needs? (2) Approach: Are the conceptual framework, design, methods, and analyses adequately developed, well-integrated, and appropriate to the aims of the project? Is the proposed plan a sound approach for establishing technical and commercial feasibility? Does the applicant acknowledge potential problem areas and consider alternative strategies? Are the milestones and evaluation procedures appropriate? (3) Innovation: Does the project challenge existing paradigms or employ novel technologies, approaches, or methodologies? Are the aims original and innovative? (4) Investigators: Is the Principal Investigator capable of coordinating and managing the proposed SBIR/STTR? Is the work proposed appropriate to the experience level of the Principal Investigator and other researchers, including consultants and sub-awardees (if any)? (5) Environment: Is there sufficient access to resources (e.g., equipment, facilities)? Does the scientific and technological environment in which the work will be done contribute to the probability of success? Do the proposed experiments take advantage of unique features of the scientific environment or employ useful collaborative arrangements? In accordance with NIH policy, all applications will also be reviewed with respect to the following: o The adequacy of the proposed protection for animals or the environment to the extent they may be adversely affected by the project proposed in the application. o The appropriateness of the proposed budget and duration in relation to the proposed research. The following evaluation criterion will be presented in an administrative note in the Summary Statement and will not factor into the numerical score: o The adequacy of plans to make the methods and materials generated in the project widely available in a timely fashion to the scientific community, given the proposed plan to exercise (or not to exercise) intellectual property rights. Phase II Applications In addition to the above criteria, to what degree was progress toward the Phase I objectives met and feasibility demonstrated in providing a solid foundation for the proposed Phase II activity? Phase I/Phase II Fast-Track Applications For Phase I/Phase II Fast-Track applications, the following additional criteria will be applied: Does the Phase I application specify clear, measurable goals (milestones) that should be achieved prior to initiating Phase II? Did the applicant submit a concise Product Development Plan that adequately addresses the four areas described in Section VI, item G, of the Omnibus Solicitation? To what extent was the applicant able to obtain letters of interest, additional funding commitments, and/or resources from the private sector or non-SBIR/STTR funding sources that would enhance the likelihood for commercialization? The phase I application should specify clear, measurable goals (milestones) that should be achieved prior to initiating Phase II. Failure to provide clear, measurable goals may be sufficient reason for the study section to judge the application non-competitive. AWARD CRITERIA Applications will compete for available funds with all other recommended SBIR and STTR applications. Funding decisions for Phase I or Phase II applications will be based on quality of the proposed project as determined by peer review, availability of funds, and program priority. Particular attention will also be given to whether or not the development of methods and technologies described in the application are likely to increase high throughput structure determination and provide the underpinning for structural genomics projects. In addition, the application must include plans for rapid dissemination of the results, and, if applicable, rapid deposition and release of all protein coordinates into the Protein Data Bank, i.e., holds on release are not permitted. FAST-TRACK Phase II applications may be funded following submission of the Phase I progress report and other documents necessary for continuation. Phase II applications will be selected for funding based on the initial priority score, NIGMS' assessment of the Phase I progress and determination that Phase I goals were achieved, the project's potential for commercial success, and the availability of funds. INQUIRIES Inquiries are encouraged. The opportunity to clarify any issues or questions from potential applicants is welcome. Direct inquiries regarding programmatic issues to: Catherine Lewis, Ph.D. Division of Cell Biology and Biophysics National Institute of General Medical Sciences 45 Center Drive, Room 2AS.13C Bethesda, MD 20892-6200 Telephone: (301) 594-0828 FAX: (301) 480-2004 Email: Joan Harmon, Ph.D. Senior Advisor for Diabetes Division of Diabetes, Endocrinology and Metabolic Diseases National Institute of Diabetes and Digestive and Kidney Diseases 2 Democracy Plaza, Room 697 6707 Democracy Blvd, MSC 5460 Bethesda, MD 20892-5460 301-594-8813 Jeffery A. Schloss, Ph.D. Division of Extramural Research National Human Genome Research Institute Bldg. 31, Room B2-B07 Bethesda, MD 20892-2033 TEL: (301) 496-7531 FAX: (301) 480-2770 Email: Direct inquiries regarding fiscal matters to: Ms. Linda Roberts Grants Management Office National Institute of General Medical Sciences 45 Center Drive, Room 2AS.55E Bethesda, MD 20892-6200 Telephone: (301) 594-5141 FAX: (301) 480-2554 Email: Cheryl Chick Supervisory Grants Management Specialist National Institute of Diabetes and Digestive and Kidney Diseases 2 Democracy Plaza, Room 606 6707 Democracy Blvd, MSC 5460 Bethesda, MD 20892-5460 Telephone: 301-594-8825 Email: Jean Cahill Grants Administration Branch National Human Genome Research Institute Bldg. 31, Room B2-B34 Bethesda, MD 20892-2031 TEL: (301) 402-0733 FAX: (301) 402-1951 E-mail: AUTHORITY AND REGULATIONS This program is described in the Catalog of Federal Domestic Assistance No. 93.821 for NIGMS, 93.847 for NIDDK, and 93.172 for NHGRI. Awards are made under authorization of the Public Health Service Act, Title IV, Part A (Public Law 78- 410, as amended by Public Law 99-158, 42 USC 241 and 285) and administered under the NIH Grant Policy Statement (10/1/98) and Federal Regulations 42 CFR 52 and 45 CFR Part 74 and Part 92. This program is not subject to the intergovernmental review requirements of Executive Order 12372 or Health Systems Agency review. The PHS strongly encourages all grant and contract recipients to provide a smoke-free workplace and promote the non-use of all tobacco products. In addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain facilities (or in some cases, and portion of a facility) in which regular or routine education, library, day care, health care or early childhood development services are provided to children. This is consistent with the PHS mission to protect and advance the physical and mental health of the American people.

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