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


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



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.  


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


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.


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),



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
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 

(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

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

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.


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. 

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 


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.


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:

BETHESDA, MD  20892-7710
BETHESDA, MD  20817 (for express/courier service)



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.

of the PHS 398. It is not required and will not be accepted with 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.


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 

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 

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.


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 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

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

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:

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

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 

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, 93.173 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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