Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels.

Multiplexed, real-time fluorescence detection at the single-molecule level can reveal the stoichiometry, dynamics and interactions of multiple molecular species in mixtures and other complex samples. However, fluorescence-based sensing is typically limited to the detection of just 3-4 colours at a time due to low signal-to-noise ratio, high spectral overlap and the need to maintain the chemical compatibility of dyes. Here we engineered a palette of several dozen composite fluorescent labels, called FRETfluors, for multiplexed spectroscopic measurements at the single-molecule level. FRETfluors are compact nanostructures constructed from three chemical components (DNA, Cy3 and Cy5) with tunable spectroscopic properties due to variations in geometry, fluorophore attachment chemistry and DNA sequence. We demonstrate FRETfluor labelling and detection for low-concentration (<100 fM) mixtures of mRNA, dsDNA and proteins using an anti-Brownian electrokinetic trap. In addition to identifying the unique spectroscopic signature of each FRETfluor, this trap differentiates FRETfluors attached to a target from unbound FRETfluors, enabling wash-free sensing. Although usually considered an undesirable complication of fluorescence, here the inherent sensitivity of fluorophores to the local physicochemical environment provides a new design axis complementary to changing the FRET efficiency. As a result, the number of distinguishable FRETfluor labels can be combinatorically increased while chemical compatibility is maintained, expanding prospects for spectroscopic multiplexing at the single-molecule level using a minimal set of chemical building blocks.

Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels.

Multiplexed, real-time fluorescence detection at the single-molecule level is highly desirable to reveal the stoichiometry, dynamics, and interactions of individual molecular species within complex systems. However, traditionally fluorescence sensing is limited to 3-4 concurrently detected labels, due to low signal-to-noise, high spectral overlap between labels, and the need to avoid dissimilar dye chemistries. We have engineered a palette of several dozen fluorescent labels, called FRETfluors, for spectroscopic multiplexing at the single-molecule level. Each FRETfluor is a compact nanostructure formed from the same three chemical building blocks (DNA, Cy3, and Cy5). The composition and dye-dye geometries create a characteristic F\"orster Resonance Energy Transfer (FRET) efficiency for each construct. In addition, we varied the local DNA sequence and attachment chemistry to alter the Cy3 and Cy5 emission properties and thereby shift the emission signatures of an entire series of FRET constructs to new sectors of the multi-parameter detection space. Unique spectroscopic emission of each FRETfluor is therefore conferred by a combination of FRET and this site-specific tuning of individual fluorophore photophysics. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs statistically selected to minimize classification errors, measured using an Anti-Brownian ELectrokinetic (ABEL) trap which provides precise multi-parameter spectroscopic measurements. The ABEL trap also enables discrimination between FRETfluors attached to a target (here: mRNA) and unbound FRETfluors, eliminating the need for washes or removal of excess label by purification. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs selected to minimize classification errors.

Aurora Kinase A-YBX1 Synergy Fuels Aggressive Oncogenic Phenotypes and Chemoresistance in Castration-Resistant Prostate Cancer.

Multifunctional protein YBX1 upregulation promotes castration-resistant prostate cancer (CRPC). However, YBX1 protein abundance, but not its DNA status or mRNA levels, predicts CRPC recurrence, although the mechanism remains unknown. Similarly, the mechanism by which YBX1 regulates androgen receptor (AR) signaling remains unclear. We uncovered the first molecular mechanism of YBX1 upregulation at a post-translational level. YBX1 was identified as an Aurora Kinase-A (AURKA) substrate using a chemical screen. AURKA phosphorylates YBX1 at two key residues, which stabilizes it and promotes its nuclear translocation. YBX1 reciprocates and stabilizes AURKA, thereby initiating a synergistic loop. Notably, phospho-resistant YBX1 is dominant-negative and fully inhibits epithelial to mesenchymal transition, chemoresistance, drug-resistance and tumorigenesis in vivo. Unexpectedly, we further observed that YBX1 upregulates AR post-translationally by preventing its ubiquitylation, but not by increasing its transcription as reported before. Uncovering YBX1-mediated AR stabilization is highly significant due to AR's critical role in both androgen-sensitive prostate cancer and CRPC. As YBX1 inhibitors are unknown, AURKA inhibitors provide a potent tool to degrade both YBX1 and AR simultaneously. Finally, this is the first study to show a reciprocal loop between YBX1 and its kinase, indicating that their concomitant inhibition will be act synergistically for CRPC therapy.