Sticky-flares

Prawidłowy przebieg procesów związanych z metabolizmem RNA jest niezwykle istotny z punktu widzenia właściwego funkcjonowania komórki. Stąd tak wiele miejsca w świecie nauki poświęca się badaniom RNA, a w szczególności badaniom rozmieszczenia i ilości RNA w komórce. Najpopularniejsze metody tych badań mają jednak spore ograniczenia. Badanie lokalizacji RNA odbywa się na utrwalonym materiale komórkowym, a najpopularniejsze techniki analityczne skupiają się jedynie na wyznaczaniu ilości transkryptów bez możliwości poznania ich rozmieszczenia w komórce.

Amerykańscy naukowcy z uniwersytetu Northwestern opracowali nowy system umożliwiający jednoczesne wyznaczanie ilości i lokalizacji RNA w żywej komórce. Swój wynalazek nazwali Sticky-flare. Ma on postać sferycznych kwasów nukleinowych (ang. spherical nucleic acids – SNAs), czyli kulistych struktur składających się z oligonukleotydów DNA połączonych z powierzchnią sferycznych nanocząstek złota stanowiących rdzeń. Struktury te są zdolne do transportu przez błony komórkowe i rozpoznawania RNA o konkretnej sekwencji.

sticky flares

Jak to działa? Kowalencyjnie związane ze Sticky-flare oligonukleotydy (zielone) są zhybrydyzowane z oligonukleotydami (ciemnoniebieskie) połączonymi kowalencyjnie z fluoroforem (różowy), którego fluorescencja jest wygaszana ze względu na bliskość nanocząstki złota. Te drugie oligonukleotydy to tzw. flares. Jeśli sticky-flare znajdzie się w pobliżu RNA o sekwencji komplementarnej do oligonukleotydów flare, to dojdzie do hybrydyzacji i uwolnienia oligonukleotydów flare połączonych z RNA. Fluorescencja fluorofora przestanie być wtedy wygaszana, a siła powstającego sygnału będzie wprost proporcjonalna do ilości związanego RNA. Ponadto, hybryda RNA:flare pozwoli na obserwację transportu i lokalizacji wewnątrzkomórkowej RNA.

Naukowcy wykazali funkcjonalność tego nowego układu, badając poziom ekspresji i dystrybucję mRNA kodującego β-aktynę w komórkach HeLa oraz w mysich fibroblastach. Zastosowali go również do śledzenia snRNA wchodzącego w skład kompleksu rybonukleoproteinowego U1.

Źródło:

http://www.pnas.org/content/112/31/9591

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