Getting DNA and RNA out of the dark with 2CNqA: a bright adenine analogue and interbase FRET donor

Abstract

With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase Förster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qAnitro and tCnitro, and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.

Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics

“…The 5'-amino-modifier C6 was obtained from GlenResearch. FBA phosphoramidites were prepared as described in the literature (tC 49 , 2CNqA 34 , pA 35 ) and/or acquired from GlenResearch (tC, tC O ). Phosphoramidites were dissolved to a final concentration to 0.1 M (3 equivalents) in DNA-grade acetonitrile (ACN) prior to use.…”

“…from live cell microscopy studies), and study how different FBAs, and positions in the gapmer sequence, affect their RNA target affinities, secondary structures, knockdown activities, and uptake characteristics. The study includes the tricyclic cytosine analogues tC 32 and tC O 33 , as well as the more recently developed quadracyclic adenine analogue 2CNqA 34 , and pentacyclic adenine analogue pA 35 (Fig. 1 a), all of which were incorporated into a 16 nt gapmer sequence (Fig.…”

“…The FBAs included in this work have previously been characterized as monomers 33 , 35 , 37 – 39 , as well as inside RNA 34 , 40 and/or DNA 32 , 33 , 41 , 42 , where they exhibit excellent base-mimicking and emissive properties. MALAT1 is ubiquitously expressed and highly abundant in the nucleus; it is involved in multiple pathological processes, including tumor progression and metastasis, and has consequently been targeted for silencing by several ASO therapies, including gapmers 43 and gapmer conjugates 44 – 46 .…”

Interbase-FRET binding assay for pre-microRNAs

“…ITC was used to explore experimental conditions for the aminoglycoside neomycin binding to unmodified truncated pre-miR-21 ( 2 , Table 1 ), including optimization of the buffer conditions. Ultimately, we employed a sodium cacodylate buffer (20 mM sodium cacodylate, 80 mM NaCl, 100 mM total Na + , pH 7.2) that was previously used in studies of aminoglycosides binding to the HIV-1 RNA dimerization initiation site 41 . This buffer resulted in excellent reproducibility of the ITC binding isotherms and a dissociation constant ( K d ) of 5.2 ± 0.8 µM for the interaction between pre-miR-21 2 and neomycin (Fig.…”

“…We are currently investigating site-selective pre-miR binders using our novel interbase-FRET assay. Additionally, to increase its versatility, we have already expanded the RNA interbase-FRET methodology to include modifications of adenine positions 41 .…”