Group of molecular tags for optical nanoscopy

Department of Biophotonics

Head: Alexander Mishin, Ph.D.
mishin@ibch.ru+7(495)995-55-57#2518

SMLM; smFRET; nanoscopy; fluorescence labaling

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The Group is working on the creation of reporter systems adapted for live-cell nanoscopy. The group was formed in 2018.

  • Protein-PAINT method: the reversible interaction of a genetically encoded reporter protein and a small-molecule fluorogenic dye freely penetrating the cytoplasmic membrane allows for high photostability and labeling density of target structures. We have developed green, orange, and red fluorogen-protein pairs suitable for localization microscopy (SMLM) as well as STED 

 

  • Protein-PAINT / IRES method: a fully genetically encoded version of protein-PAINT based on the reversible interaction of short peptides (K/E helices). The novel protein labeling system  allows for high-density labeling of target structures in prolonged live-cell nanoscopy experiments. 
  • The use of spontaneous blinking of fluorescent proteins and dyes for nanoscopy
  • Genetically encoded tags for STED and RESOLFT
  • luminescence microscopy and imaging 
NamePositionContacts
Alexander Mishin, Ph.D.headmishin@ibch.ru+7(495)995-55-57#2518
Alexey Gavrikovj. r. f.gavrikovalexey1@gmail.com
Maxim Perfilovj. r. f.sufrep@gmail.com
Irina Shemiakinaj. r. f.

All publications (show selected)

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Alexander Mishin

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 34, office. 632
  • Phone: +7(495)995-55-57#2518
  • E-mail: mishin@ibch.ru

K/E-coils: fluorescent protein labeling and Protein-PAINT nanoscopy

In collaboration with Laboratory of genetically encoded molecular tools

Heterodimerization of K/E-coils was applied for fluorescent labeling of proteins in living cells for the first time.

One of the specially selected α-helices (K or E) serves as a tag for the target protein, and the other is fused with a genetically encoded fluorescent protein. Due to the reversible interaction of K and E-helices, the target protein structure is highlighted, and the continuous exchange of fluorescent proteins in the complex increases the resistance to photobleaching by order of magnitude.

The small size of the labels (only 2-3 kDa) preserves the native dynamics of the studied proteins. Also, this method makes it possible to observe proteins almost immediately after their synthesis.

The most perspective was applying K/E-coils for Protein-PAINT – super-resolution localization microscopy based on reversibly binding labels. The continuous exchange of fluorescent proteins between the target cell structure and the cytoplasmic pool ensures a consistently high labeling density even during prolonged imaging. With the help of K/E-coils, for the first time, it was possible to implement the concept of Protein-PAINT nanoscopy using solely genetically encoded reporters.

Publications

  1. Perfilov MM, Gurskaya NG, Serebrovskaya EO, Melnikov PA, Kharitonov SL, Lewis TR, Arshavsky VY, Baklaushev VP, Mishin AS, Lukyanov KA (2020). Highly photostable fluorescent labeling of proteins in live cells using exchangeable coiled coils heterodimerization. Cell Mol Life Sci 77 (21), 4429–4440

Excimer-FRET cascade in dual DNA probes

In collaboration with Laboratory of molecular design and synthesis

The efficacy of fluorescent hybridization assays is often limited by low signal-to-background ratio of the probes that can be partially overcome by sophisticated signal amplification methods. Deep understanding of the mechanisms of fluorescence quenching and energy transfer in complex DNA probes, choice of optimal donor/acceptor pairs along with rational design can significantly enhance the performance of DNA probes. We propose novel FRET dual DNA probes with the excimer-forming pyrene pair as a donor and sulfo-Cy3 dye as an acceptor. The probes demonstrated remarkable 75-fold enhancement of sulfo-Cy3 fluorescence upon target capturing. Stokes shift up to 220 nm minimizes fluorescence crosstalk. Time-correlated single-photon counting revealed two excited states of pyrene excimer wherein only one is directly involved in the resonance energy transfer to sulfo-Cy3. Optimized DNA probes demonstrated high sensitivity with excellent signal-to-background ratio which were applied for visualization of 18S rRNA by fluorescent in situ hybridization in HEK293T cells.

In Nature Biotechnology, scientists from IBCh RAS have announced the feasibility of creating plants that produce their own visible luminescence. It was revealed that bioluminescence found in some mushrooms is metabolically similar to the natural processes common among plants. By inserting DNA obtained from the mushroom Neonothopanus nambi, the scientists were able to create plants that glow much brighter than previously possible. Plants containing the mushroom DNA glow continuously throughout their lifecycle, from seedling to maturity. This biological light can be used for observing the inner workings of plants. In contrast to other commonly used forms of bioluminescence, such as from fireflies, unique chemical reagents are not necessary for sustaining mushroom bioluminescence.

Publications

  1. Mitiouchkina T, Mishin AS, Somermeyer LG, Markina NM, Chepurnyh TV, Guglya EB, Karataeva TA, Palkina KA, Shakhova ES, Fakhranurova LI, Chekova SV, Tsarkova AS, Golubev YV, Negrebetsky VV, Dolgushin SA, Shalaev PV, Shlykov D, Melnik OA, Shipunova VO, Deyev SM, Bubyrev AI, Pushin AS, Choob VV, Dolgov SV, Kondrashov FA, Yampolsky IV, Sarkisyan KS (2020). Plants with genetically encoded autoluminescence. Nat Biotechnol 38 (8), 944–946

Three-dimensional structure of a new type of photostable biomarker - DiB1

In collaboration with Laboratory of X-ray study

The structure of a photostable non-covalent complex of bacterial lipocalin Blc with a synthetic chromophore M739 (DiB1: M739) has been determined by X-ray method with a high resolution of 1.58 Å. The stereochemical details of the chromophore binding were revealed, and on the basis of the obtained structural data, two new genetically engineered biomarkers (green and yellow) with increased affinity and brightness were designed.

New labels and approaches for low toxic fluorescent labeling of proteins in living cells

In collaboration with Group of chemistry of heterocyclic compounds

Ultraviolet, often used in fluorescence microscopy and nanoscopy, is extremely toxic to cells. Therefore, it is preferable to use labels in the green and red spectral regions.

We found the ability of the fluorescent protein mAvicFP1 to spontaneously blink under the influence of less toxic blue light, and applied this property to nanoscopy and to track single molecules of labeled proteins in living cells.

Fluorogen-activating proteins are new generation labeling systems based on transient interaction of a genetically encoded protein and  externally applied fluorogen. We have created and used  in living cells a new red fluorogen N871b for the FAST reporter protein.