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Decrypting the in situ three-dimensional spatial configuration of an aptamer is of considerable significance; however, suitable nanoscale resolution tools are lacking. Herein, we show that a new nanometal surface energy transfer (NSET) optical nanoruler has a record resolution, down to single-nucleobase levels. We labeled fluorophores on different T bases of XQ-2d, including 5', 3', 6T, 22T, 38T, and 52T positions. The NSET nanoruler in situ decrypted the base sequence-dependent distance projection on the nanogold surface, demonstrating that 5', 3', stem, and loop structures are symmetrical in three-dimensional spatial configuration. The orientation of the 5' and 3' stem was toward the antiCD71-binding site, whereas the loop was in the opposite direction at a considerable distance. Molecular docking simulation was performed to list all of the possible conformations; however, all base distance parameters projecting on the nanogold surface determined a single conformation of XQ-2d. The specific binding sites of XQ-2d were Lys477, Ser691, and Arg698 on the CD71 receptor.

Developing novel strategies to measure nanoscale distance and molecular interaction on a living cell membrane is of great significance but challenging. Here we develop a model of a linker-free plasmon resonance energy transfer, termed "PRET nanoruler", which is composed of a single-sized nanogold-antibody conjugates donor (G 26 @antiCD71) and a fluorophore-labeled XQ-2d aptamer receptor (XQ-2d-Cy3), that produces a separation distance ( r ) dependent energy transfer (η PRET ). Both the theoretical finite element simulation and experiments evidence the observable PRET between single G 26 NPs and XQ-2d-Cy3. Regardless of the size of η PRET , we could confirm r is less than 5 nm, the separation of two binding sites is in the range of 13.0-18.0 nm. There is a competitive binding of Tf and XQ-2d-Cy3 on CD71 receptors. PRET nanoruler realizes the estimation of the nanoscale separation distance, and determines the molecular interaction and competitive binding. It is an alternative tool for observing nanoscale single molecular events in the future.

3',5'-cyclic adenosine monophosphate (cAMP) is one of the most important and ubiquitous second messengers in cells downstream of G protein-coupled receptors (GPCRs). In a single cell, cAMP can exert innumerous specific cell functions in response to more than one hundred different GPCRs. Cells achieve this extraordinary functional specificity of cAMP signaling by limiting the spread of these signals in space and time. To do so, cells establish nanometer-size cAMP gradients by immobilizing cAMP via cAMP binding proteins and via targeted activity of cAMP-degrading phosphodiesterases (PDEs). As cAMP gradients appear to be essential for cell function, new technologies are needed to accurately measure cAMP gradients in intact cells with nanometer-resolution. Here we describe FRET-based cAMP nanorulers to measure local, nanometer-size cAMP gradients in intact cells in the direct vicinity of PDEs., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The dynamic development of fluorescence microscopy has created a large number of new techniques, many of which are able to overcome the diffraction limit. This chapter describes the use of DNA origami nanostructures as scaffold for quantifying microscope properties such as sensitivity and resolution. The DNA origami technique enables placing of a defined number of fluorescent dyes in programmed geometries. We present a variety of DNA origami nanorulers that include nanorulers with defined labeling density and defined distances between marks. The chapter summarizes the advantages such as practically free choice of dyes and labeling density and presents examples of nanorulers in use. New triangular DNA origami nanorulers that do not require photoinduced switching by imaging transient binding to DNA nanostructures are also reported. Finally, we simulate fluorescence images of DNA origami nanorulers and reveal that the optimal DNA nanoruler for a specific application has an intermark distance that is roughly 1.3-fold the expected optical resolution., (© 2014 Elsevier Inc. All rights reserved.)