Calibration free counting of low molecular copy numbers in single DNA-PAINT localization clusters

Single-Molecule Localization Microscopy (SMLM) has revolutionized light microscopy by enabling optical resolutions down to a few nanometer. Yet, localization precisions commonly not suffice to visually resolve single subunits in molecular assemblies or multimeric complexes. Since each targeted molecule contributes localizations during image acquisition, molecular counting approaches to reveal the target copy numbers within localization clusters have been continuously proposed since the early days of SMLM, most of which rely on preliminary knowledge of the dye photo-physics or on a calibration to a reference. Previously, we developed localization-based Fluorescence Correlation Spectroscopy (lbFCS) as an absolute ensemble counting approach for the SMLM-variant DNA-Points Accumulation for Imaging in Nanoscale Topography (PAINT), for the first time circumventing the necessity for reference calibrations. Here, we present a revised framework termed lbFCS+ which allows absolute counting of copy numbers for individual localization clusters in a single DNA-PAINT image. In lbFCS+, absolute counting in individual clusters is achieved via precise measurement of the local hybridization rates of the fluorescently-labeled oligonucleotides (imagers) employed in DNA-PAINT imaging. In proof-of-principle experiments on DNA origami nanostructures, we demonstrate the ability of lbFCS+ to truthfully determine molecular copy numbers and imager association and dissociation rates in well-separated localization clusters containing up to six docking strands. We show that lbFCS+ allows to resolve heterogeneous binding dynamics enabling the distinction of stochastically generated and a priori indistinguishable DNA assemblies. Beyond advancing quantitative DNA-PAINT imaging, we believe that lbFCS+ could find promising applications ranging from bio-sensing to DNA computing.

Tracking single particles for hours via continuous DNA-mediated fluorophore exchange

Monitoring biomolecules in single-particle tracking experiments is typically achieved by employing fixed organic dyes or fluorescent fusion proteins linked to a target of interest. However, photobleaching typically limits observation times to merely a few seconds, restricting downstream statistical analysis and observation of rare biological events. Here, we overcome this inherent limitation via continuous fluorophore exchange using DNA-PAINT, where fluorescently-labeled oligonucleotides reversibly bind to a single-stranded DNA handle attached to the target molecule. Such versatile and facile labeling allows uninterrupted monitoring of single molecules for extended durations. We demonstrate the power of our approach by observing DNA origami on membranes for tens of minutes, providing perspectives for investigating cellular processes on physiologically relevant timescales.

Adsorption–Desorption Characteristics of ss-DNA on 2D TpTta-COF Studied by Fluorescently Labeled Oligonucleotides

Being the newest member of the 2D materials family, 2D-nanosheet possesses many distinctive physical and chemical properties resulting in a wide range of potential applications. Recently, it was discovered that 2D COF can adsorb single-stranded DNA (ss-DNA) efficiently as well as usefully to quench fluorophores. These properties make it possible to prepare DNA-based optical biosensors using 2D COF. While practical analytical applications are being demonstrated, the fundamental understanding of binding between 2D COF and DNA in solution received relatively less attention. In this work, we carried out a systematic study to understand the adsorption and desorption kinetic, mechanism, and influencing factors of ss-DNA on the surface of 2D COF. We demonstrated that shorter DNAs are adsorbed more rapidly and bind more tightly to the surface of 2D COF. The adsorption is favored by a higher pH. The different buffer types also can affect the adsorption. In Tris-HCl solution, the adsorption reached highest efficiency. By adding the complementary DNA (cDNA), desorption of the absorbed DNA on 2D COF can be achieved. Further, desorption efficiency can also be exchanged by various surfactant in solution. These findings are important for further understanding of the interactions between DNA and COFs and for the optimization of DNA and COF-based devices and sensors.

Tracking Single Particles for Hours via Continuous DNA-mediated Fluorophore Exchange

Fluorophores are commonly used to covalently label biomolecules for monitoring their motion in single particle tracking experiments. However, photobleaching is still a major bottleneck in these experiments, as the fluorophores’ finite photon budget typically limits observation times to merely a few seconds. Here, we overcome this inherent constraint via continuous fluorophore exchange based on DNA-PAINT, whereby fluorescently-labeled oligonucleotides bind to a 54 bp single-stranded DNA handle attached to the molecule of interest. When we assayed our approach in vitro by tracking single DNA origami, first surface-immobilized and subsequently diffusing on supported lipid bilayers, we were able to observe these origami for up to hours without losing their fluorescence signals. Our versatile and easily implemented labeling approach allows monitoring single-molecule motion and interactions over unprecedented observation periods, opening the doors to advanced quantitative studies.

Oligomer based real-time detection of microorganisms producing nuclease enzymes

In this study, we provide a method using fluorescently labeled oligonucleotides for the diagnosis of microorganisms producing nucleases in real time, while growing them in culture media.

Staining of Mitochondria with Cy5-Labeled Oligonucleotides for Long-Term Microscopy Studies

Labeling of organelles for microscopy is achieved generally by specific dyes that either accumulate in a cellular compartment such as cyanine dyes in mitochondria or are only fluorescent under specific conditions such as the low pH in the lysosome. Here we demonstrate that Cy5—a fluorescent molecule that does not enter cells by itself—can be loaded into cells by attaching a short oligonucleotide. This very inexpensive labeling procedure can be done in the presence of serum. Therefore, very sensitive cell types should also be amenable to this procedure, and longer observations can be achieved compared to other commercially available dyes as the labeling reagent does not need to be washed out. This also points to the pitfall of using fluorescently labeled oligonucleotides for live cell imaging where the oligonucleotide is supposed to detect a specific target sequence in its subcellular distribution.