This Program Announcement expires on February 1, 2004, unless reissued. SINGLE MOLECULE DETECTION AND MANIPULATION Release Date: February 12, 2001 PA NUMBER: PA-01-049 National Institute of General Medical Sciences ( National Institute on Deafness and Other Communication Disorders ( National Human Genome Research Institute ( THIS PA USES THE "MODULAR GRANT" AND "JUST-IN-TIME" CONCEPTS. IT INCLUDES DETAILED MODIFICATIONS TO STANDARD APPLICATION INSTRUCTIONS THAT MUST BE USED WHEN PREPARING APPLICATIONS IN RESPONSE TO THIS PA. PURPOSE The purpose of this program announcement is to encourage basic research on 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 individual 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, is related to one or more of the priority areas. Potential applicants may obtain a copy of "Healthy People 2010" at ELIGIBILITY REQUIREMENTS Applications may be submitted by domestic and foreign, for-profit and non- profit organizations, public and private, such as universities, colleges, hospitals, laboratories, units of State and local governments, and eligible agencies of the Federal government. Foreign institutions are not eligible for program projects grants. Racial/ethnic minority individuals, women, and persons with disabilities are encouraged to apply as principal investigators. MECHANISM OF SUPPORT This PA will use the existing National Institutes of Health (NIH) Research Project Grant (R01) award mechanism. A companion program announcement for methodology and technology development related to this initiative but eligible for the Small Business Innovation Research/Small Business Technology Transfer (SBIR/STTR) program is available at ( Applications for competing supplements to existing grants will also be accepted, if there will be at least one year remaining in the project period at the time of supplemental funding. Responsibility for the planning, direction, and execution of the proposed project will be solely that of the applicant. The total requested project period for an application submitted in response to this PA may not exceed five years. For all competing R01 applications requesting up to $250,000 per year in direct costs, specific application instructions have been modified to reflect MODULAR GRANT and JUST-IN-TIME streamlining efforts being examined by the NIH. Applications that request more than $250,000 in any year must use the standard PHS 398 (rev. 4/98) application instructions. Complete and detailed instructions and information on Modular Grant applications can be found at Some research efforts may be more appropriate for the Program Project (P01) grant mechanism. Investigators considering applying for P01 grants should contact the Institute program staff listed at the end of this announcement. For projects that include a major emphasis on bioengineering, the NIH Bioengineering Research Grants may be the preferred mechanism (PAR-99-009), 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 future 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 and physicists 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. State-of-the-art instruments that are optimized for high-resolution studies on single molecules often require several years to build and are not commercially available. Instrument development is essential for growth in this field and should therefore be recognized as a legitimate research activity on a grant application. As such, this type of research does not have to be hypothesis- driven to be considered worthy of support. The funding Insitutes may provide a substantial contribution for the acquisition 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 This program announcement addresses the need for an expansion in basic research in 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 Applications are to be submitted on the grant application form PHS 398 (rev. 4/98) and will be accepted at the standard application deadlines as indicated in the application kit. Application kits are available at most institutional offices of sponsored research and may be obtained from the Division of Extramural Outreach and Information Resources, National Institutes of Health, 6701 Rockledge Drive, MSC 7910, Bethesda, MD 20892-7910, telephone 301/710-0267, email: Applicants planning to submit an investigator-initiated new (type 1), competing continuation (type 2), competing supplement, or any amended/revised version of the preceding grant application types requesting $500,000 or more in direct costs for any year are advised that he or she must contact the Institute or Center (IC) program staff before submitting the application, i.e., as plans for the study are being developed. Furthermore, the application must obtain agreement from the IC staff that the IC will accept the application for consideration for award. Finally, the applicant must identify, in a cover letter sent with the application, the staff member and Institute or Center who agreed to accept assignment of the application. This policy requires an applicant to obtain agreement for acceptance of both any such application and any such subsequent amendment. Refer to the NIH Guide for Grants and Contracts, March 20, 1998 at Individual Research Project (R01) Grants requesting $250,000 Direct Costs per Year or less. For the R01 mechanism, specific application instructions have been modified to reflect MODULAR GRANT and JUST-IN-TIME streamlining efforts being examined by the NIH. Complete and detailed instructions and information on Modular Grants can be found at R01 applications that request more than $250,000 direct costs per year should follow the instructions in the PHS Form 398. The title and number of the program announcement must be typed on line 2 of the face page of the application form and the YES box must be marked. Submit a signed, typewritten original of the application, including the Checklist, and five signed photocopies in one package 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) SPECIFIC INSTRUCTIONS FOR MODULAR GRANT APPLICATIONS BUDGET INSTRUCTIONS Modular Grant applications will request direct costs in $25,000 modules, up to a total direct cost request of $250,000 per year. (Applications that request more than $250,000 direct costs in any year must follow the traditional PHS 398 application instructions.) The total direct costs must be requested in accordance with the program guidelines and the modifications made to the standard PHS 398 application instructions described below: PHS 398 o FACE PAGE: Items 7a and 7b should be completed, indicating Direct Costs (in $25,000 increments up to a maximum of $250,000) and Total Costs [Modular Total Direct plus Facilities and Administrative (F&A) costs] for the initial budget period Items 8a and 8b should be completed indicating the Direct and Total Costs for the entire proposed period of support. o DETAILED BUDGET FOR THE INITIAL BUDGET PERIOD - Do not complete Form Page 4 of the PHS 398. It is not required and will not be accepted with the application. o BUDGET FOR THE ENTIRE PROPOSED PERIOD OF SUPPORT - Do not complete the categorical budget table on Form Page 5 of the PHS 398. It is not required and will not be accepted with the application. o NARRATIVE BUDGET JUSTIFICATION - Prepare a Modular Grant Budget Narrative page. (See for sample pages.) At the top of the page, enter the total direct costs requested for each year. This is not a Form page. o Under Personnel, list all project personnel, including their names, percent of effort, and roles on the project. No individual salary information should be provided. However, the applicant should use the NIH appropriation language salary cap and the NIH policy for graduate student compensation in developing the budget request. For Consortium/Contractual costs, provide an estimate of total costs (direct plus facilities and administrative) for each year, each rounded to the nearest $1,000. List the individuals/organizations with whom consortium or contractual arrangements have been made, the percent effort of all personnel, and the role on the project. Indicate whether the collaborating institution is foreign or domestic. The total cost for a consortium/contractual arrangement is included in the overall requested modular direct cost amount. Include the Letter of Intent to establish a consortium. Provide an additional narrative budget justification for any variation in the number of modules requested. o BIOGRAPHICAL SKETCH - The Biographical Sketch provides information used by reviewers in the assessment of each individual's qualifications for a specific role in the proposed project, as well as to evaluate the overall qualifications of the research team. A biographical sketch is required for all key personnel, following the instructions below. No more than three pages may be used for each person. A sample biographical sketch may be viewed at: - Complete the educational block at the top of the form page; - List position(s) and any honors; - Provide information, including overall goals and responsibilities, on research projects ongoing or completed during the last three years. - List selected peer-reviewed publications, with full citations; o CHECKLIST - This page should be completed and submitted with the application. If the F&A rate agreement has been established, indicate the type of agreement and the date. All appropriate exclusions must be applied in the calculation of the F&A costs for the initial budget period and all future budget years. o The applicant should provide the name and phone number of the individual to contact concerning fiscal and administrative issues if additional information is necessary following the initial review. REVIEW CONSIDERATIONS Applications will be assigned on the basis of established PHS 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. (1) Significance: Does this study address an important problem? If the aims of the application are achieved, how will scientific knowledge be advanced? What will be the effect of these studies on the concepts or methods that drive this field? (2) Approach: Are the conceptual framework, design, methods, and analyses adequately developed, well-integrated, and appropriate to the aims of the project? Does the applicant acknowledge potential problem areas and consider alternative tactics? (3) Innovation: Does the project employ novel concepts, approaches or method? Are the aims original and innovative? Does the project challenge existing paradigms or develop new methodologies or technologies? (4) Investigator: Is the investigator appropriately trained and well suited to carry out this work? Is the work proposed appropriate to the experience level of the principal investigator and other researchers (if any)? (5) Environment: Does the scientific 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? Is there evidence of institutional support? In addition to the above criteria, in accordance with NIH policy, all applications will also be reviewed with respect to the following: o The adequacy of plans to include both genders, minorities and their subgroups, and children as appropriate for the scientific goals of the research. Plans for the recruitment and retention of subjects will also be evaluated. o The reasonableness of the proposed budget and duration in relation to the proposed research. o The adequacy of the proposed protection for humans, animals or the environment, to the extent they may be adversely affected by the project proposed in the application. AWARD CRITERIA Applications will compete for available funds with all other recommended applications. The following will be considered in making funding decisions: Quality of the proposed project as determined by peer review, availability of funds, and program priority. 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 Building 45, Room 2AS.13C Bethesda, MD 20892 TEL: (301) 594-0828 FAX: (301) 480-2004 Email: Nancy L. Freeman, Ph.D. Scientific Program Director National Institute on Deafness and Other Communication Disorders Executive Plaza South-400C 6120 Executive Blvd. MSC-7180 Rockville, MD 20852 TEL: (301) 402-3458 FAX: (301) 402-6251 Email: 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: Grace Tuanmu Grants Management Office National Institute of General Medical Sciences Building 45, Room 2AS.55J Bethesda, MD 20892 TEL: (301) 594-5520 FAX: (301) 480-2554 Email: Sara Stone Chief, Grants Management Branch National Institute on Deafness and Other Communication Disorders 6120 Executive Blvd, Suite 400B Executive Plaza South, MSC 7180 Bethesda, Maryland 20892-7180 TEL: (301) 402-0909 FAX: (301) 402-1758 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 Email: AUTHORITY AND REGULATIONS This program is described in the Catalog of Federal Domestic Assistance No. 93.821 for NIGMS, 93.173 for NIDCD, and 93.172 for NHGRI. Awards are made under authorization of sections 301 and 405 of the Public Health Service Act as amended (42 USC 241 and 284) and administered under NIH grants policies and Federal Regulations 42 CFR 52 and 45 CFR Parts 74 and 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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