Equilibrium and dynamic design principles for binding molecules engineered for reagentless biosensors

Picciotto, Seymour de; Imperiali, Barbara; Griffith, Linda G.; Wittrup, K. Dane

doi:10.1016/j.ab.2014.04.036

Cited by 5 publications

(5 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Equation (11) describes the general form of ( ; , ) which is easily seen to be symmetric with respect to exchange of and and hence consistent with the reaction kinetics-based results mentioned in section 1. Obtaining a closed form expression for ( ; , ) in Eq.…”

Section: Partition Function For a System Containing Receptors And Lig...supporting

confidence: 79%

“…However, we can obtain explicit expressions for p r; L; R ð Þfor special cases, where R ) L or R ( L. For instance, take the case where R ( L as considered commonly in biosensor literature. [16][17][18] As R ( L and maximum of r is the minimum of L and R, it follows that L ) r allowing us to make the approximation:…”

Section: Partition Function For a System Containing R Receptors mentioning

confidence: 99%

“…for ≫ or ≪ . For instance, take the case where ≪ as considered commonly in biosensor literature [11,12,13]. Then, following the derivation in [6], we can write…”

Section: Partition Function For a System Containing Receptors And Lig...mentioning

confidence: 99%

See 2 more Smart Citations

Equilibrium probability distribution for number of bound receptor-ligand complexes

Chakrabortty

Varma

2021

American Journal of Physics

View full text Add to dashboard Cite

The phenomenon of molecular binding, where two molecules, referred to as a receptor and a ligand, bind together to form a ligand-receptor complex, is ubiquitous in biology and essential for the accurate functioning of all life-sustaining processes. The probability of a single receptor forming a complex with any one of L surrounding ligand molecules at thermal equilibrium can be derived from a partition function obtained from the Gibbs-Boltzmann distribution. We extend this approach to a system consisting of R receptors and L ligands to derive the probability density function p r; R; L ð Þ to find r bound receptor-ligand complexes at thermal equilibrium. This extension allows us to illustrate two aspects of this problem which are not apparent in the single receptor problem, namely, (a) a symmetry to be expected in the equilibrium distribution of the number of bound complexes under exchange of R and L and (b) the number of bound complexes obtained from chemical kinetic equations has an exact correspondence to the maximum probable value of r from the expression for p r; R; L ð Þ. We derive the number fluctuations of r and present a practically relevant molecular sensing application which benefits from the knowledge of pðr; R; LÞ.

show abstract

Section: Partition Function For a System Containing Receptors And Lig...supporting

confidence: 79%

Section: Partition Function For a System Containing R Receptors mentioning

confidence: 99%

See 1 more Smart Citation

Equilibrium probability distribution for number of bound receptor-ligand complexes

Chakrabortty

Varma

2021

American Journal of Physics

View full text Add to dashboard Cite

show abstract

“…6 Another approach, which involves labeling with environmentally sensitive fluo-rophores to make reagentless biosensors, has recently extended. 7 Because it is based on the coupling of fluorophore to mutated cysteine (Cys) residue near the antigen-binding site of an antibody, the conjugation site should be carefully designed not to hinder antigen-binding activity; 8 the variation in a suitable and environmentally sensitive dye is limited.…”

mentioning

confidence: 99%

Q-Bodies from Recombinant Single-Chain Fv Fragment with Better Yield and Expanded Palette of Fluorophores

et al. 2015

View full text Add to dashboard Cite

Fluorescence-based immunosensors serve a vital role in biotechnology and diagnostic and therapeutic applications. Our group recently developed a unique fluoroimmunosensor named Quenchbody (Q-body) that operates based on the principle of quenching and the antigen-dependent release of fluorophore, which is incorporated to a recombinant antibody fragment, either the single-chain Fv (scFv) or the Fab fragment of an antibody, using a cell-free transcription-translation system. With the objective of extending the functionality and diversity of the Q-body, here we attempted to make Q-bodies by labeling the recombinant scFv, which was prepared from E. coli using several commercially available dye-maleimides. As a result, we reproducibly obtained larger amounts of antiosteocalcin Q-bodies, with an improved yield and cost-efficiency compared with those obtained from a conventional cell-free system. The fluorescence intensity of each Q-body, including that labeled with newly tested rhodamine red, was significantly increased in the presence of an antigen with a low detection limit, although some differences in response were observed for the dye with different spacer lengths between dye and maleimide. The results indicate the Q-body’s applicability as a powerful multicolored sensor, with a potential to simultaneously monitor multiple targets in a sample.

show abstract

“…First, a binder is engineered against the intended target using established display technologies 9–12 and multiple scaffolds 13–15 , ideally with tailored binding kinetics and affinity 16 . The second step is careful choice of the labeling site.…”

Section: Introductionmentioning

confidence: 99%

Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions

Picciotto

Dickson

Traxlmayr

et al. 2016

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

Quantifying protein location and concentration is critical for understanding function in situ. SuCESsFul biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can in principle detect analytes of interest with high temporal and spatial resolution. However, their adoption has been limited due to the extensive empirical screening required for their development. We sought to establish design principles for this class of biosensor by characterizing over 400 biosensors based on various protein analytes, binding proteins and fluorophores. We found that the brightest readouts are attained when a specific binding pocket for the fluorophore is present on the analyte. Also, interaction of the fluorophore with the binding protein it is conjugated to can raise background fluorescence, considerably limiting sensor dynamic range. Exploiting these two concepts, we designed biosensors that attain a 100-fold increase in fluorescence upon binding to analyte, an order of magnitude improvement over the previously best reported SuCESsFul biosensor. These design principles should facilitate the development of improved SuCESsFul biosensors.

show abstract

Equilibrium and dynamic design principles for binding molecules engineered for reagentless biosensors

Cited by 5 publications

References 35 publications

Equilibrium probability distribution for number of bound receptor-ligand complexes

Equilibrium probability distribution for number of bound receptor-ligand complexes

Q-Bodies from Recombinant Single-Chain Fv Fragment with Better Yield and Expanded Palette of Fluorophores

Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions

Contact Info

Product

Resources

About