This Program Announcement expires on April 1, 2004, unless reissued.


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


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.


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 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.


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 

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:



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 

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,
o Enzyme catalysis: mechanism of catalysis, conformational changes 
o Ion channels: local structural changes, kinetics
o Signaling: formation of multimers, kinetics of cascades, phosphorylation
o DNA, DNA binding proteins, RNA: binding constants, regulation of gene
o Membrane structure: restricted diffusion, phase changes
o Molecular motors: motility, processivity, directionality
o Complex cellular structures (e.g., transcription complexes): assembly,

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 

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.


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 

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.


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.


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.


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 

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.


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.


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 

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:

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.


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 

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 

(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 

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.


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 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

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

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 

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

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

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


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 

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