“siRNA traffic lights”: arabino-configured 2′-anchors for fluorescent dyes are key for dual color readout in cell imaging

Abstract

Two fluorescent dyes covalently attached in diagonal interstrand orientation to siRNA undergo energy transfer and thereby enable a dual color fluorescence readout (red/green) for hybridization. Three different structural variations were carried out and compared by their optical properties, including (i) the base surrogate approach with an acyclic linker as a substitute of the 2-deoxyriboside between the phosphodiester bridges, (ii) the 2'-modification of conventional ribofuranosides and (iii) the arabino-configured 2'-modification. The double stranded siRNA with the latter type of modification delivered the best energy transfer efficiency, which was explained by molecular dynamics simulations that showed that the two dyes are more flexible at the arabino-configured sugars compared to the completely stacked situation at the ribo-configured ones. Single molecule fluorescence lifetime measurements indicate their application in fluorescence cell imaging, which reveals a red/green fluorescence contrast in particular for the arabino-configured 2'-modification by the two dyes, which is key for tracking of siRNA transport into HeLa cells.

Broad Applications of Thiazole Orange in Fluorescent Sensing of Biomolecules and Ions

“…Localization of TO and partner dyes opposite one another enables energy transfer or quenching interactions between them, which are interrupted upon target recognition due to separation between the TO-acceptor pair, thus yielding increased TO fluorescence [ 76 ]. Such probes were applied to determine the degree of delivery of siRNAs into cells, along with their inhibitory activity [ 75 , 77 ]. Subsequent works have broadened the library of clickable approaches for TO-labeling of DNA-based probes, combined with other acceptor dyes, through chemical modifications of thymine bases [ 78 , 79 ].…”

Postsynthetic On-Column 2′ Functionalization of RNA by Convenient Versatile Method

“…The cellular uptake of these 2 -cholesterol-conjugated siRNA either in the absence (free uptake) or in the presence of a commercial transfection agent in the HEK293 Phoenix cell line was evaluated (see for details Supplementary Materials, Section 3.3, pages [16][17][18]. The flow cytometry data shows that the efficiency of the cholesterol-conjugated siRNAs carrier-free uptake is rather high (more than 80%, at C siRNA = 1 µM), while lipofectamine 3000-mediated delivery of all the siRNAs provided from approximately 30-65% of transfected cells (Figure 5B).…”

“…To date, a number of pre- and postsynthetic methods for the generation of 2′-modified RNAs have been reported. These methods have been used for chemical synthesis of various conjugates of siRNA [ 1 , 14 , 15 , 16 ], labeled pre-microRNAs [ 17 , 18 , 19 ], peptidyl-conjugates of RNA mimicking the tRNA Ala acceptor arm [ 20 ], branched and lariat RNAs [ 21 , 22 , 23 , 24 , 25 , 26 ], and others. Recently, an original strategy of reversible acylation of 2′-OH groups of native RNA (RNA cloaking) for the controlled blocking of RNA hybridization, folding, and interactions with other biomolecules has been reported [ 27 , 28 , 29 ].…”

Fluorescence Lifetime Imaging Microscopy (FLIM) of Intracellular Transport by Means of Doubly Labelled siRNA Architectures

“…Besides incorporation of the dyes as base surrogates, [6a] we were able to attach the dyes post‐synthetically via copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) to different alkyne moieties, including 2’‐propargylated ribo‐ and arabinofuranosides and acyclic aminopropanediol linkers. This makes the RNA constructs synthetically more accessible and enables the use of a variety of chromophores [6b,d,7] . The photostability of this fluorescent siRNA architecture was further improved by combinations of our photostable cyanine‐styryl‐dyes [8] .…”

“…The photostability of this fluorescent siRNA architecture was further improved by combinations of our photostable cyanine‐styryl‐dyes [8] . These fluorophores, however, show multiple fluorescence lifetimes due to different interactions with RNA, making FLIM‐FRET experiments impossible [6d] . For this purpose, we transferred the “RNA traffic light” concept (Figure 1) to photostable ATTO dyes.…”