Screening of Minimalist Noncanonical Sites in Duplex DNA and RNA Reveals Context and Motif-Selective Binding by Fluorogenic Base Probes

Abstract

We hypothesize that programmable hybridization to noncanonical nucleic acid motifs may be achieved by macromolecular display of binders to individual noncanonical pairs (NCPs). As each recognition element may individually have weak binding to an NCP, we developed a semi-rational approach to detect low affinity interactions between selected nitrogenous bases and noncanonical sites in duplex DNA and RNA. A set of fluorogenic probes was synthesized by coupling abiotic (triazines, pyrimidines) and native RNA bases to thiazole orange (TO) dye. This probe library was screened against duplex nucleic acid substrates bearing single abasic, single NCP, and tandem NCP sites. Probe engagement with NCP sites was reported by 100-1000× fluorescence enhancement over background. Binding is strongly context-dependent, reflective of both molecular recognition and stability: less stable motifs are more likely to bind a synthetic probe. Further, DNA and RNA substrates exhibit entirely different abasic and single NCP binding profiles. While probe binding in the abasic and single NCP screens was monotonous, much richer binding profiles were observed with the screen of tandem NCP sites in RNA, in part due to increased steric accessibility. In addition to known binding interactions between the triazine melamine (M) and T/U sites, the NCP screens identified new targeting elements for pyrimidine-rich motifs in single NCPs and 2×2 internal bulges. We anticipate that semi-rational approaches of this type will lead to programmable noncanonical hybridization strategies at the macromolecular level.

Keywords: RNA recognition; fluorescent probes; noncanonical pairs; nucleobases; synthetic bases.

Figure 1.

Structures of the fluorogenic probes used in this study. All probes made based on literature procedures to install the R₁ and R₂ linkers indicated. Ammeline was synthesized with both R₁ and R₂ linkers to produce N1 and N2, respectively.

Figure 2.

Heat map of fluorogenic binding for the abasic (Left) DNA and (Right) RNA) libraries. Fluorescence is reported as average intensity from triplicate measurement relative to unbound probe. Samples were prepared in HEPES buffer (pH 7.5, 50 mM, 100 mM NaCl) with 2 μM probe, 2 μM duplex. See histogram analysis with error bars in Supporting Information.

Figure 3.

(Top) Fluorescence heat maps of NCP-probe binding. Average emission intensities from triplicate measurement are shown according to the scales at right, indicating fold enhancement over unbound probes for DNA (Left) and RNA (Right). Probes and NCPs in both maps are ordered with the highest integrated relative fluorescence signal in DNA at the top and right, respectively. (Bottom) Possible interactions of synthetic bases melamine (M), ammeline (N) and W1 with NCPs approached from the (above) major groove and (below) minor groove. Samples were prepared in HEPES buffer (pH 7.5, 50 mM, 100 mM NaCl) with 2 μM probe, 2 μM duplex.

Figure 4.

(Top) Heat map of tandem NCP RNA library screen. Tandem NCPs are ordered by calculated relative stability, with the most stable on the left. Probes are ordered with the highest integrated fluorescence (most reactive) in the top row and lowest in the bottom row. Fluorescence indicated is relative enhancement over background TO emission. (Bottom) Probe emission data grouped by XQ/WZ size ratio in the 2×2 internal bulge. Size ratio is calculated using purine or pyrimidine molecular weight as an approximation for each base in the tandem NCP. Schematic illustration of the size ratio is indicated above the green bars highlighting the most reactive probe in each grouping. The most reactive probes in each group are labeled accordingly. Samples were prepared in HEPES buffer (pH 7.5, 50 mM, 100 mM NaCl) with 4 μM probe, 2 μM RNA duplex. Data is the average of triplicate measurement, with histograms and error bars available in Supporting Information.

Figure 5.

Binding isotherms of M, W1 and N2 probes binding to the indicated tandem NCPs, obtained by fluorescence (RFU) as a function of RNA (duplex) concentration, showing fit to a 1:1 binding model and standard deviation error bars from triplicate measurements. RNA substrates and probes were the same as in Figure 4.

Scheme 1.

Illustration of the three nucleic acid library designs targeted by the fluorogenic probe library. (Left) The abasic library in DNA and RNA pairs a purine (R) or pyrimidine (Y) with an abasic site (Ap) wherein the anomeric carbon is replaced with CH₂; the single noncanonical pair (NCP) library contains all non WC pairings in DNA and RNA; the tandem NCP library in RNA only contains 43 unique motifs of two non WC pairs in tandem. (Right) Illustration of fluorogenic probe binding to the noncanonical sites.