Dr Mirjam Kümmerlin

Abstract:

A main limitation of single-molecule fluorescence (SMF) measurements is the 'high concentration barrier', describing the maximum concentration of fluorescent species tolerable for sufficient signal-to-noise ratio. To address this barrier in several SMF applications, we design fluorogenic probes based on short single-stranded DNAs, fluorescing only upon hybridizing 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 we utilize as tuning screws to adapt our labels to different experimental designs. Using these fluorogenic probes, we can perform SMF experiments at concentrations of 10 μM fluorescent labels; this concentration is 100-fold higher than the operational limit for standard TIRF experiments. We demonstrate the ease of implementing these probes into existing protocols by performing super-resolution imaging with DNA-PAINT, employing a fluorogenic 6-nt-long imager; through the faster acquisition of binding events, the imaging of viral genome segments could be sped up significantly to achieve extraction of 20-nm structural features with only ∼150 s of imaging. The exceptional tunability of our probe design will overcome concentration barriers in SMF experiments and unlock new possibilities in super-resolution imaging, molecular tracking, and single-molecule fluorescence energy transfer (smFRET).

Abstract:

DNA-based enzymes, also known as DNAzymes, have opened new opportunities for signal generation and amplification in several fields including biosensing. However, biosensor performance can be hampered by heterogeneity in the catalytic activity of such DNAzymes, especially when relying on a limited number of molecules to generate signal. In this regard, single-molecule studies are essential to discern the behavior among such heterogeneous molecules otherwise masked by ensemble measurements. This work presents a novel methodology to study the 10–23 RNA-cleaving DNAzyme at the single-molecule level. By means of measuring the distance-sensitive efficiency of Förster Resonance Energy Transfer using alternating-laser excitation on a superresolution microscope, we determined the kinetics of individual DNAzymes in terms of substrate turnover, rates of different reaction steps, and changes in performance over time. Our results revealed that, despite high concentrations of the reaction cofactor (i.e., Mg²⁺), a maximum of only 70% of the DNAzymes are actively cleaving multiple substrate sequences; the DNAzyme molecules also showed a wide range of substrate turnover rates. Our findings shed new light on the functional diversity of DNAzymes and the importance of exploring sequence modifications to improve their catalytic performance. Ultimately, this work presents a technique to obtain time-dependent information, which could be easily implemented to study other types of enzymes or biomolecular interactions.

Abstract:

Biosensors play key roles in medical research and diagnostics. However, the development of biosensors for new biomolecular targets of interest often involves tedious optimization steps to ensure a high signal response at the analyte concentration of interest. Here we show a modular nanosensor platform that facilitates these steps by offering ways to decouple and independently tune the signal output as well as the response window. Our approach utilizes a dynamic DNA origami nanostructure to engineer a high optical signal response based on fluorescence resonance energy transfer. We demonstrate mechanisms to tune the sensor's response window, specificity and cooperativity as well as highlight the modularity of the proposed platform by extending it to different biomolecular targets including more complex sensing schemes. This versatile nanosensor platform offers a promising starting point for the rapid development of biosensors with tailored properties.