2′‐Bispyrene‐Modified 2′‐O‐Methyl RNA Probes as Useful Tools for the Detection of RNA: Synthesis, Fluorescent Properties, and Duplex Stability

Abstract

The synthesis and properties two series of new 2'-O-methyl RNA probes, each containing a single insertion of a 2'-bispyrenylmethylphosphorodiamidate derivative of a nucleotide (U, C, A, and G), are described. As demonstrated by UV melting studies, the probes form stable complexes with model RNAs and DNAs. Significant increases (up to 21-fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the probes with complementary RNAs, but not with DNAs. The fluorescence spectra are independent of the nature of the modified nucleotides. The nucleotides on the 5'-side of the modified nucleotide have no effect on the fluorescence spectra, whereas the natures of the two nucleotides on the 3'-side are important: CC, CG, and UC dinucleotide units on the 3'-side of the modified nucleotide provide the maximum increases in excimer fluorescence intensity. This study suggests that these 2'-bispyrene-labeled 2'-O-methyl RNA probes might be useful tools for detection of RNAs.

Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides

“…Krasheninina et al developed and studied three variants of pyrene excimer-forming 2′- O -methyl RNA probes, namely linear probes, dual probes, and MBs for the detection of RNA [ 82 , 83 , 84 , 85 ]. The probes were obtained via simple and efficient postsynthetic labeling method as described in [ 86 , 87 ].…”

“…The first variant, linear 2′- O -methyl RNA probes containing one to three 2′-bispyrene-modified monomers ( Figure 6 ) were found to be RNA-specific fluorescent probes [ 82 , 83 ]. In general, the probes, being much more synthetically accessible, demonstrated similar properties that were observed for other variants of probes comprising monomers with pyrene introduced to the 2′-position of ribose through a short linker, indicating the disposition of pyrenes in the minor groove of the RNA duplex.…”

“…Namely, introduction of the 2′Nbpyr monomers ( Figure 6 a) decreased the thermal stability of duplex probes with RNA targets and, depending on the nucleotide context, had different effects on duplexes with DNA, whereas significant increases (up to 21-fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the 2′-bispyrene-modified probes with complementary RNA ( Figure 6 b), but not with DNA. We found that the fluorescent properties of the probes upon hybridization with RNA depended on the nucleotide context of the modified ribonucleoside and were optimal in the case of 3′-flanking CC, CG, or UC dinucleotide units [ 83 ].…”

“… ( a ) 2′-Bispyrene-modified nucleotide monomers studied in [ 82 , 83 ] ( 2′Nbpyr ) and the disposition of pyrene in the hybrid probe-target duplex; ( b ) Fluorescence emission spectra of linear probes containing one 2′Nbpyr monomer (B: cytidin-1-yl). A: adenin-9-yl, U: uracil-1-yl, C: cytidin-1-yl, and G: guanin-9-yl.…”

Design of a synthetic luminescent probe from a biomolecule binding domain: selective detection of AU-rich mRNA sequences

“…Even though synthetic chemists have developed a large number of low-molecular-weight fluorescent probes that can detect various analytes (reactive oxygen species, amino acids, anions or metal cations) or that can respond to their microenvironment (pH, redox potential), 2–8 selective sensing of larger biomolecules such as proteins, DNA or RNA is more challenging with small molecular probes. 4,9–16 Compared to low-molecular-weight analytes, the selective recognition of large biomolecules requires a high number of interactions between the probe and its target, which are difficult to implement and control with small synthetic molecular probes.…”