Precise quantification of transcription factors in a surface-based single-molecule assay.

Biosensors have recognized a rapid development the last years in both industry and science. Recently, a single-molecule assay based on alternating laser excitation has been established for the quantitative detection of transcription factors. These proteins specifically recognize and bind DNA and play an important role in controlling gene expression. We implemented this assay format on a total internal reflection fluorescence microscope to detect transcription factors with immobilized single-molecule DNA biosensors. We quantify transcription factors via colocalization of the two halves of their binding site with immobilized single molecules of a two-color DNA biosensor. We could detect a model transcription factor, the bacterial lactose repressor, at different concentrations down to 150pM. We found that robust modeling of stoichiometry derived TIRF data is achieved with Student's t-distributions and nonlinear least-squares estimation with weights equal to the inverse of the expected number of bin entries. This significantly improved transcription factor concentration estimates with respect to distribution modeling with Gaussians without adding notable computational effort. The proposed model may enhance the precision of other single-molecule assays quantifying molecular distributions. Our measurements reliably confirm that the immobilized biosensor format is more sensitive than a previously published solution based approach.

Quenched substrates for live-cell labeling of SNAP-tagged fusion proteins with improved fluorescent background.

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.