Fluorescent probing for RNA molecules by an unnatural base-pair system

Abstract

Fluorescent labeling of nucleic acids is widely used in basic research and medical applications. We describe the efficient site-specific incorporation of a fluorescent base analog, 2-amino-6-(2-thienyl)purine (s), into RNA by transcription mediated by an unnatural base pair between s and pyrrole-2-carbaldehyde (Pa). The ribonucleoside 5′-triphosphate of s was site-specifically incorporated into RNA, by T7 RNA polymerase, opposite Pa in DNA templates. The fluorescent intensity of s in RNA molecules changes according to the structural environment. The site-specific s labeling of RNA hairpins and tRNA molecules provided characteristic fluorescent profiles, depending on the labeling sites, temperature and Mg2+ concentration. The Pa-containing DNA templates can be amplified by PCR using 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), another pairing partner of Pa. This site-specific fluorescent probing by the unnatural pair system including the s-Pa and Ds-Pa pairs provides a powerful tool for studying the dynamics of the local structural features of 3D RNA molecules and their intra- and intermolecular interactions.

2‐Phenanthrenyl–DNA: Synthesis, Pairing, and Fluorescence Properties

“…Most of these investigations were driven by the search for additional base pairs to be used for the extension of the genetic alphabet, [1][2][3][4][5][6][7][8] as tools in biotechnology, [9][10][11] for probing recognition, fidelity, and nucleotide processing by DNA polymerases, [12][13][14][15] or for designing novel genetic systems. [16,17] Of special interest amongst these artificial constructs are aromatic base replacements that interact with each other, specifically without the formation of hydrogen bonds, merely on the basis of edge-on or face-on hydrophobic or stacking interactions.…”

Stacking with the unnatural DNA base 6-ethynylpyridone

“…This increases the genetic information that can be encoded by DNA. A plethora of studies have explored extending the genetic lexicon by incorporating unnatural base pairs into DNA or RNA [1][2][3][4][5][6][7][8][9][10]. Most unnatural base pairs successfully incorporated into DNA or RNA contain different hydrogen-bond patterns than the canonical base pairs, though also base pairs with no hydrogen bonds at all have been employed [11][12][13][14].…”

Synthesis and characterization of antibacterial 2-(pyridin-3-yl)-1H-benzo[d]imidazoles and 2-(pyridin-3-yl)-3H-imidazo[4,5-b]pyridine derivatives

“…Benzmidazole and imidazo [4,5-b]pyridine groups readily interact with the biopolymers of living organisms [18]. Because this type of structure is known to have a wide range of biological activity, for example antibacterial [19], antiviral [20], antimicrobial [21], antiulcer proton-pump inhibiting [22,23], and anticancer [24] activity, and because, in the pharmaceutical sciences, new drugs are usually discovered on the basis of molecular modification of lead compounds or already established pharmacophores, we synthesized fourteen novel derivatives of 2-(pyridin-3-yl)-1H-benzo[d]imidazole and 2-(pyridin-3-yl)-3H-imidazo [4,5-b]pyridine.…”