Divide and Control: Comparison of Split and Switch Hybridization Sensors

Abstract

Hybridization probes have been intensively used for nucleic acid analysis in medicine, forensics and fundamental research. Instantaneous hybridization probes (IHPs) enable signalling immediately after binding to a targeted DNA or RNA sequences without the need to isolate the probe-target complex (e. g. by gel electrophoresis). The two most common strategies for IHP design are conformational switches and split approach. A conformational switch changes its conformation and produces signal upon hybridization to a target. Split approach uses two (or more) strands that independently or semi independently bind the target and produce an output signal only if all components associate. Here, we compared the performance of split vs switch designs for deoxyribozyme (Dz) hybridization probes under optimal conditions for each of them. The split design was represented by binary Dz (BiDz) probes; while catalytic molecular beacon (CMB) probes represented the switch design. It was found that BiDz were significantly more selective than CMBs in recognition of single base substitution. CMBs produced high background signal when operated at 55°C. An important advantage of BiDz over CMB is more straightforward design and simplicity of assay optimization.

Keywords: Catalytic molecular beacons; Deoxyribozyme sensors; fluorescent sensors; instantaneous hybridization probes; single nucleotide polymorphisms; split hybridization probes.

Figure 1

Predicted structures of the probe-analyte complexes and selectivity data for CMB (A) and BiDZ (B) recognizing TWIST1 gene-related analytes at 30°C. For structures: catalytic core nucleotides are in green; single base substitution site is red underlined; ribonucleotides are in low case. For right panels: all samples contained 200 nM F_ sub and either 5 nM CMB_T1 or 5 nM each DZa_T1 and DZb_T1 in the reaction buffer: 50 mM HEPES, 50 mM MgCl₂, 20 mM KCl, 120 mM NaCl, 0.03% Triton X-100, 1 % DMSO. Samples T1 and T2 contained 5 nM of fully complementary T1–21, T–86 or 5 nM single base mismatched T2–21 or T2–86 analytes, respectively (for full sequences see Table S1). Fluorescence intensity at 517 nm (emission at 485 nm) was measured after 1 hr of incubation. The data is average values of 3 independent experiments.

Figure 2

Predicted structures of probe-analyte complexes and selectivity data for CMB_A2 (A) and BiDZ_A2 (B) recognizing A1–70 analyte at 55°C. For right panels: all samples contained 200 nM F_ sub and either 5 nM CMB or 5 nM DZa_T and 5 nM DZb_T in the reaction buffer. Samples A1 and A2 contained 1 nM complementary A1 and 1 nM mismatched A2 analyte, respectively (for full sequences see Table S1). Fluorescence intensity at 517 nm (emission at 485 nm) was measured after 1 hr of incubation. The data are average values of 3 independent experiments.

Scheme 1

Design of deoxyribozyme (DZ) probes that produce fluorescent signal upon hybridization to specific nucleic acid analytes. A) Parent RNA-cleaving DZ can cleave a fluorophore- and quencher-labelled substrate (F_ sub), thus producing high fluorescence. B) Switch design for DZ hybridization probes: the catalytic core and/or substrate binding arms of DZ are inactivated by binding to the ‘inhibitory fragment’; hybridization of the analyte to the analyte binding domain releases the substrate binding arms of the DZ construct and enables cleavage of F_sub; C) Split design: two DNA strands DZa and DZb hybridize to the analyte sequence and form catalytically active DZ, which cleaves F_sub.