N6^6-Isopentenyladenosine in RNA Determines the Cleavage Site of Endonuclease Deoxyribozymes

RNA-cleaving deoxyribozymes can serve as selective sensors and catalysts to examine the modification state of RNA. However, site-specific endonuclease deoxyribozymes that selectively cleave posttranscriptionally modified RNA are extremely rare and their specificity over unmodified RNA is low. In this study, we report that the native tRNA modification N$^6$-isopentenyladenosine (i$^6$A) strongly enhances the specificity and has the power to reconfigure the active site of an RNA-cleaving deoxyribozyme. Using in vitro selection, we identified a DNA enzyme that cleaves i$^6$A-modified RNA at least 2500-fold faster than unmodified RNA. Another deoxyribozyme shows unique and unprecedented behaviour by shifting its cleavage site in the presence of the i$^6$A RNA modification. Together with deoxyribozymes that are strongly inhibited by i$^6$A, these results highlight intricate ways of modulating the catalytic activity of DNA by posttranscriptional RNA modifications

High-Throughput Activity Profiling of RNA-Cleaving DNA Catalysts by Deoxyribozyme Sequencing (DZ-seq)

RNA-cleaving deoxyribozymes have found broad application as useful tools for RNA biochemistry. However, tedious in vitro selection procedures combined with laborious characterization of individual candidate catalysts hinder the discovery of novel catalytic motifs. Here, we present a new high-throughput sequencing method, DZ-seq, which directly measures activity and localizes cleavage sites of thousands of deoxyribozymes. DZ-seq exploits A-tailing followed by reverse transcription with an oligo-dT primer to capture the cleavage status and sequences of both deoxyribozyme and RNA substrate. We validated DZ-seq by conventional analytical methods and demonstrated its utility by discovery of novel deoxyribozymes that allow for cleaving challenging RNA targets or the analysis of RNA modification states

In Vitro Selection of Deoxyribozymes for the Detection of RNA Modifications

Deoxyribozymes are artificially evolved DNA molecules with catalytic abilities. RNA-cleaving deoxyribozymes have been recognized as an efficient tool for detection of modifications in target RNAs and provide an alternative to traditional and modern methods for detection of ribose or nucleobase methylation. However, there are only few examples of DNA enzymes that specifically reveal the presence of a certain type of modification, including N6-methyladenosine, and the knowledge about how DNA enzymes recognize modified RNAs is still extremely limited. Therefore, DNA enzymes cannot be easily engineered for the analysis of desired RNA modifications, but are instead identified by in vitro selection from random DNA libraries using synthetic modified RNA substrates. This protocol describes a general in vitro selection stagtegy to evolve new RNA-cleaving DNA enzymes that can efficiently differentiate modified RNA substrates from their unmodified counterpart

RNA-Cleaving Deoxyribozymes Differentiate Methylated Cytidine Isomers in RNA

Deoxyribozymes are emerging as modification-specific endonucleases for the analysis of epigenetic RNA modifications. Here, we report RNA-cleaving deoxyribozymes that differentially respond to the presence of natural methylated cytidines, 3-methylcytidine (m$^3$C), N$^4$-methylcytidine (m$^4$C), and 5-methylcytidine (m$^5$C), respectively. Using in vitro selection, we found several DNA catalysts, which are selectively activated by only one of the three cytidine isomers, and display 10- to 30-fold accelerated cleavage of their target m$^3$C-, m$^4$C- or m$^5$C-modified RNA. An additional deoxyribozyme is strongly inhibited by any of the three methylcytidines, but effectively cleaves unmodified RNA. The m$^X$C-detecting deoxyribozymes are programmable for the interrogation of natural RNAs of interest, as demonstrated for human mitochondrial tRNAs containing known m$^3$C and m$^5$C sites. The results underline the potential of synthetic functional DNA to shape highly selective active sites

Masked red-emitting carbopyronine dyes with photosensitive 2-diazo-1-indanone caging group

Caged near-IR emitting fluorescent dyes are in high demand in optical microscopy but up to now were unavailable. We discovered that the combination of a carbopyronine dye core and a photosensitive 2-diazo-1-indanone residue leads to masked near-IR emitting fluorescent dyes. Illumination of these caged dyes with either UV or visible light (λ < 420 nm) efficiently generates fluorescent compounds with absorption and emission at 635 nm and 660 nm, respectively. A high-yielding synthetic route with attractive possibilities for further dye design is described in detail. Good photostability, high contrast, and a large fluorescence quantum yield after uncaging are the most important features of the new compounds for non-invasive imaging in high-resolution optical microscopy. For use in immunolabelling the caged dyes were decorated with a (hydrophilic) linker and an (activated) carboxyl group.Fil: Kolmakov, Kirill. Max Planck Institute for Biophysical Chemistry; AlemaniaFil: Wurm, Christian. Max Planck Institute for Biophysical Chemistry; AlemaniaFil: Sednev, Maksim V.. Max Planck Institute for Biophysical Chemistry; AlemaniaFil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Max Planck Institute for Biophysical Chemistry; AlemaniaFil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry; AlemaniaFil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemani

N6^6-methyladenosine-sensitive RNA-cleaving deoxyribozymes

Deoxyribozymes are synthetic enzymes made of DNA that can catalyze the cleavage or formation of phosphodiester bonds and are useful tools for RNA biochemistry. Here we report new RNA-cleaving deoxyribozymes to interrogate the methylation status of target RNAs, thereby providing an alternative method for the biochemical validation of RNA methylation sites containing N$^6$-methyladenosine, which is the most wide-spread and extensively investigated natural RNA modification. Using in vitro selection from random DNA, we developed deoxyribozymes that are sensitive to the presence of N$^6$-methyladenosine in RNA near the cleavage site. One class of these DNA enzymes shows faster cleavage of methylated RNA, while others are strongly inhibited by the modified nucleotide. The general applicability of the new deoxyribozymes is demonstrated for several examples of natural RNA sequences, including a lncRNA and a set of C/D box snoRNAs, which have been suggested to contain m$^6$A as a regulatory element that influences RNA folding and protein binding

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RNA-cleaving deoxyribozymes can serve as selective sensors and catalysts to examine the modification state of RNA. However, site-specific endonuclease deoxyribozymes that selectively cleave posttranscriptionally modified RNA are extremely rare and their specificity over unmodified RNA is low. In this study, we report that the native tRNA modification N$^6$-isopentenyladenosine (i$^6$A) strongly enhances the specificity and has the power to reconfigure the active site of an RNA-cleaving deoxyribozyme. Using in vitro selection, we identified a DNA enzyme that cleaves i$^6$A-modified RNA at least 2500-fold faster than unmodified RNA. Another deoxyribozyme shows unique and unprecedented behaviour by shifting its cleavage site in the presence of the i$^6$A RNA modification. Together with deoxyribozymes that are strongly inhibited by i$^6$A, these results highlight intricate ways of modulating the catalytic activity of DNA by posttranscriptional RNA modifications

Carborhodol: A New Hybrid Fluorophore Obtained by Combination of Fluorescein and Carbopyronine Dye Cores

Asymmetric hybrid fluorophores are built from the structural elements of two (or even more) symmetric dyes and can develop valuable new features which their parents do not possess. A new hybrid carborhodol dye was obtained by the combination of fluorescein and carbopyronine fluorophores. The brightly fluorescent hybrid dye with a linker and reactive group was prepared in 12 steps with overall yield of 1.6%. In aqueous solutions, it has absorption and emission maxima at 586 and 613 nm, respectively. Antibodies labeled with a carborhodol dye possess broad absorption and emission bands so that the effective Stokes shift is increased (compared with small Stokes shifts of the parent dyes) and the fluorescence quantum yield of 39% at a degree of labeling of 5.2. Two samples of secondary antibodies labeled with carborhodol and the benchmark red-emitting rhodamine dye (KK114) were used in two-color imaging experiments with excitation at 514–532 (carborhodol dye) and 633–640 nm (KK114). When emitted light was detected above 650 nm, the novel carborhodol dye provided a lower crosstalk than spectrally similar emitters (e. g., Atto594; crosstalk 40–60% with KK114 under the same conditions). The optical resolution of ca. 80 nm was attained using the new dye in stimulated emission depleted (STED) microscopy. The relatively short fluorescence lifetime in conjugates with antibodies (τ = 1.2–1.6 ns) suggests the possibility of dual FLIM with numerous dyes having τ values in the range of 3–5 ns. All of these features make the carborhodol fluorophore a valuable addition to the family of the red-emitting fluorescent dyes