Gene machines

Abstract:

The “high concentration barrier”, describing the maximum concentration of fluorescent species tolerable, is one of the main limitations of single-molecule fluorescence (SMF) measurements. Addressing this fundamental limit can enable and expand several in vitro and in vivo single-molecule applications, including tracking in crowded environments, fast super-resolution imaging, and single-molecule fluorescence resonance energy transfer (smFRET) experiments.

In this thesis, we develop fluorogenic probes (which become fluorescent upon binding to a target) to address the high-concentration barrier in several SMF applications. The design is based on short ssDNAs fluorescing only upon hybridising to their complementary target sequence. We engineer the quenching efficiency and fluorescence enhancement upon duplex formation through screening several fluorophore-quencher combinations, label lengths, and sequence motifs, which serve as tuning screws to adopt our labels to different experimental designs. With these fluorogenic labels, we can perform SMF experiments at concentrations in excess of 10~µM fluorescent labels – an improvement of two orders of magnitude compared to standard TIRF experiments, without the need for any special optics or nano-fabrication.

We present several experimental applications of our probes, each showcasing a specific feature fluorogenic probes can provide: We demonstrate the ease of implementing these probes into existing protocols by performing super-resolution imaging with DNA-PAINT, employing a fluorogenic 6nt-long imager. Importantly, we did not perform any sequence engineering ourselves, but simply “plugged in” the fluorogenicity feature and reduced the imager length. Through the faster acquisition rate of binding events, the imaging of viral genome segments could be sped up significantly, now only requiring approx. 150~s of imaging to extract physical features in the 20~nm range.

To highlight new experimental paths only possible with fluorogenic labelling species, we performed smFRET measurements where photobleaching is circumvented through a constant exchange of donor- and acceptor- dyes, supplied by fluorogenic ssDNAs (REFRESH-FRET). This process is facilitated by fast the exchange kinetics at probe concentrations far exceeding 100~nM, and allows for observation of smFRET for hours.

Thirdly, we applied our probes in live-cell tracking of individual ribosomes by labelling their 16S rRNA with a complementary probe. The increased signal-specificity that fluorogenicity provides allows for a great signal-to-noise ratio within the cellular environment. Our probes directly hybridise to the rRNA target, so do not require any genetic engineering.

In summary, we characterised the fluorogenic properties of dye-quencher labelled ssDNA probes, and demonstrate several single-molecule applications in vitro and \textit{in vivo}. Through their tuneability and simple implementation, we envision our probes to be widely applicable, beyond what we could demonstrate here.