Determination of association constants from single molecule fluorescence microscopy

MARÍA VICTORIA CAPPELLARI; BELÉN ELGUERO; EDUARDO ARZT; LISSY GROSS; RICARDO BIONDI; ALAN SZALAI; DAVID GONILSKY; PEDRO F. ARAMENDÍA

Viña del Mar

Congreso; XIV Encuentro Latinoamericano de Fotoquímica y Fotobiología; 2019

Singlemolecule fluorescence microscopy can yield molecular resolution witha location precision in the tens of nanometer range in the planeperpendicular to the light propagation. This opens the possibility tocount molecules and correlate their locations, starting from a map ofthe actual positions in a single molecule super resolution image.Inthis work we will present results of three experiments aiming at thecalibration and determination of association constants. The firstexperiment uses complementary oligonucleotides, one strand markedwith Atto-565 and the other with AlexaFluor647. Hybridization iscontrolled by the amount of each strand and measured in steady stateemission. Samples are spread on a coverslip and molecules of eachtype are counted on the microscope by Stochastic OpticalReconstruction Microscopy experiments with split field simultaneoustwo color detection. Actual distributions are compared to random onesto evaluate the association fraction and compare it with the valuesobtained in the correspondent bulk experiment.Inthe second type of experiments we evaluate the dimerization of ....under the presence of different aggregation inhibitors, to test theirefficacy. The protein was marked with SNAP-silicon-rhodamine and thefraction of dimers was estimated by the distribution of emissionintensity of the bright spots obtained in single moleculefluorescence images.Thethird type of experiments was directed to establish the interactionmechanisms between three proteins that are relevant in the hypoxiaadaptation of cancer cells: HIF(Hypoxia inducible factor), VHL (von Hippel-Lindau complex), RSUME(RWD-domain-containing sumoylation enhancer). These proteins weremarked pairwise with SNAP-silicon-rhodamine andCLIP-tetramethylrhodamine. Their association is evaluated as afunction of time in COS-7 cell lines by two color simultaneous STORMexperiments. In all cases the random distribution is used as areference for the pair correlation of locations. We presentpreliminary results from this strategy.p { margin-bottom: 0.25cm; direction: ltr; color: rgb(0, 0, 10); line-height: 120%; text-align: left; }p.western { font-family: "Liberation Serif", serif; font-size: 12pt; }p.cjk { font-family: "Droid Sans Fallback"; font-size: 12pt; }p.ctl { font-family: "FreeSans"; font-size: 12pt; }