Polyphosphate (poly P) is a linear polymer of inorganic phosphates that is found in all living organisms, ranging from tens to hundreds of residues in length. Although its biological functions have not been fully elucidated, recent work has shed light on its role in the virulence of pathogens, the blood clotting cascade, biomineralization, and metal sequestration. Ongoing research suggests that poly P length is correlated with its origin, function, and enzymatic environment. However, existing poly P assays that are facile and require minimal sample purification do not provide information about length. Methods that are length specific are not suitable to a high-throughput format, requiring extensive purification or time-consuming steps. In order to expedite fundamental biochemical studies and the discovery of drugs that target poly P-mediated pathways, facile, sensitive, and length-specific assays are needed. This proposal describes a length-specific, conjugated polymer-based fluorogenic probe for poly P. In the absence of poly P, the conjugated polymer is quenched. Poly P binding to a flexible segment induces a conformational change that restores fluorescence. The length of the poly P- binding region may be tailored, enabling the development of a length-selective fluorescence assay. The mechanism of molecular recognition is inspired by a structurally unique ion channel, found in many bacteria, composed of a polyester, Ca2+, and poly P. The use of a nonionic probe will minimize nonspecific interactions with other biomolecules. Detailed studies of the interaction between poly P and the fluorogenic probe will inform optimization of the assay and provide insights into molecular recognition. The anticipated selectivity of the described probe will enable its use for the analysis of natural samples. In addition to facilitating the study of poly P biochemistry, these studies will provide a framework for the development of chemosensors for other polyelectrolytes.

Public Health Relevance

Inorganic polyphosphate is a long chain of phosphate ions that is found in all living organisms and may have played a role in the chemical origins of life. Little is known about its functions, but researchers have found connections to blood clotting, bone formation, and the virulence of pathogens. The goal of this proposal is to develop a length-sensitive fluorescent probe for polyphosphate that will ultimately aid in both fundamental studies and in the discovery of new therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM106550-01
Application #
8525608
Study Section
Special Emphasis Panel (ZRG1-F04-W (20))
Program Officer
Barski, Oleg
Project Start
2013-08-01
Project End
2016-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
1
Fiscal Year
2013
Total Cost
$47,114
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139