Abstract
Visualizing the functional interactions of biomolecules such as proteins and nucleic acids is key to understanding cellular life on the molecular scale. Spatial proximity is often used as a proxy for the direct interaction of biomolecules. However, current techniques to visualize spatial proximity are either limited by spatial resolution, dynamic range, or lack of single‐molecule sensitivity. Here, we introduce Proximity‐PAINT (pPAINT), a variation of the super‐resolution microscopy technique DNA‐PAINT. pPAINT uses a split‐docking‐site configuration to detect spatial proximity with high sensitivity, low false‐positive rates, and tunable detection distances. We benchmark and optimize pPAINT using designer DNA nanostructures and demonstrate its cellular applicability by visualizing the spatial proximity of alpha‐ and beta‐tubulin in microtubules using super‐resolution detection.
Highlights
Florian Schueder+, Juanita Lara-GutiØrrez+, Daniel Haas, Kai Sandvold Beckwith, Peng Yin, Jan Ellenberg, and Ralf Jungmann*
If the target pair is in close proximity, the two split docking sites will spatially co-localize, yielding a full, detectable DNA-PAINT docking strand by the transient hybridization of a complementary stem
We detected a positive pPAINT signal in 91 % of all cases. To ensure that this high detection efficiency is not an artifact of potential false positive signals, which might originate from solitary split docking strands, we performed experiments where only the 3’or 5’-extension was incorporated in our DNA origami platform, and detected negligible pPAINT signals in 3 % and 1 % of all cases for the 5’- and 3’-extension, respectively (Figure 1 b and Table S2)
Summary
Florian Schueder+, Juanita Lara-GutiØrrez+, Daniel Haas, Kai Sandvold Beckwith, Peng Yin, Jan Ellenberg, and Ralf Jungmann*. If the split DNA-PAINT docking sites co-localize, a binding signal would again be detectable, highlighting spatial proximity of two molecular targets. If the target pair is in close proximity, the two split docking sites will spatially co-localize, yielding a full, detectable DNA-PAINT docking strand by the transient hybridization of a complementary stem (black sequence section in Figure 1 a).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
