Site-Specific Fluorescent Labeling of RNA Interior Positions

Abstract

The introduction of fluorophores into RNA for both in vitro and in cellulo studies of RNA function and cellular distribution is a subject of great current interest. Here I briefly review methods, some well-established and others newly developed, which have been successfully exploited to site-specifically fluorescently label interior positions of RNAs, as a guide to investigators seeking to apply this approach to their studies. Most of these methods can be applied directly to intact RNAs, including (1) the exploitation of natural posttranslational modifications, (2) the repurposing of enzymatic transferase reactions, and (3) the nucleic acid-assisted labeling of intact RNAs. In addition, several methods are described in which specifically labeled RNAs are prepared de novo.

Keywords: RNA interior positions; site-specific fluorescent labeling.

Figure 1

Wybutosines, guanosine derivatives.

Figure 2

Covalent modification of DHU via THU formation.

Figure 3

Current minimal substrate for ThiI. Reprinted with permission from reference [58].

Figure 4

Site- specific TGT labeling of RNA. Reprinted with permission from reference [66].

Figure 5

Site-specific modification of RNA loops and gaps induced by DNA. Reprinted with permission from reference [67].

Figure 6

(A). (i) DNA Helper Sequences (light gray) annealing, (ii) hybridization of DNA Reactive Sequence (dRS, dark grey) precursor, (iii) activation by sodium periodate, (iv) transfer of the reactive group and modification of an internal adenine. (B). Mechanism of reactive group transfer. Adapted with permission from Reference [68].

Figure 7

(A). RNA catalysts for labeling of a specific adenosine (starred) within a model sequence in cis were identified from a partially structured random RNA pool (N40). The constant region of the RNA pool contained the hypothetical substrate sequence and a pair of recognition arms complementary to the substrate sequence. (B). The labeling reaction involving N⁶-substituted ATP adenylylating the 2’ hydroxyl of the targeted adenosine. (C). The product of reaction shown in B showing substitutions used for fluorophore labeling. Adapted with permission from reference [72].

Figure 8

Formation of RNAs site-specifically labeled with ^thG. Reprinted with permission from reference [70].

Figure 9

(A). The scheme for site-specific incorporation of the unnatural base pair X(Pa)-Y(Ds) into first DNA and then RNA. (B). An azide derivative of Pa was used to site-specifically incorporate an Alexa dibenzocyclooctyne fluorophor into several different RNAs by click chemistry. Adapted with permission from reference [83].