A Spinach molecular beacon triggered by strand displacement

Abstract

We have re-engineered the fluorescent RNA aptamer Spinach to be activated in a sequence-dependent manner. The original Spinach aptamer was extended at its 5'- and 3'-ends to create Spinach.ST, which is predicted to fold into an inactive conformation and thus prevent association with the small molecule fluorophore DFHBI. Hybridization of a specific trigger oligonucleotide to a designed toehold leads to toehold-initiated strand displacement and refolds Spinach into the active, fluorophore-binding conformation. Spinach.ST not only specifically detects its target oligonucleotide but can discriminate readily against single-nucleotide mismatches. RNA amplicons produced during nucleic acid sequence-based amplification (NASBA) of DNA or RNA targets could be specifically detected and reported in real-time by conformational activation of Spinach.ST generated by in vitro transcription. In order to adapt any target sequence to detection by a Spinach reporter we used a primer design technique that brings together otherwise distal toehold sequences via hairpin formation. The same techniques could potentially be used to adapt common Spinach reporters to non-nucleic acid analytes, rather than by making fusions between aptamers and Spinach.

Keywords: aptamer; aptamer beacon; fluorescent RNA; molecular beacon; nucleic acid diagnostics; nucleic acid engineering; strand displacement.

FIGURE 1.

Re-engineering the RNA aptamer Spinach to be a sequence-dependent molecular beacon. All sequence blocks are designated as numbered domains (* indicates complementary domains). (A) The 9-bp stem (highlighted in brown) of the folded, 80-nt minimized Spinach aptamer (24-2-min) was designated domain 6* at its 5′-end and domain 6.1 at its 3′-end. (B) The 5′-end was extended by adding an 8-nt domain 5* (highlighted in blue) while the 3′-end was extended by adding a duplicate of domain 6.1 (termed 6.2) and two 8-nt domains, designated 5 and 2 (highlighted in blue), respectively. Sequences derived from Saccharomyces cerevisiae tRNA^trp (highlighted in light purple) flanked the engineered Spinach. RNA folding should result in domains 5 and 6.2 being contiguously paired, trapping the aptamer in a nonfluorescent conformation in which domain 2 remains unpaired. (C,D) Domain 2 serves as a toehold for strand displacement by a trigger sequence containing domains 2*–5*–6* (highlighted in orange), leading to refolding of the aptamer into its active conformation. Associations between trigger and Spinach.ST are indicated with a double-sided arrow, while the conformational change that Spinach.ST undergoes is indicated by a double-tipped arrow. All nucleic acid structures were generated using NUPACK (Dirks and Pierce 2003, 2004; Dirks et al. 2007; Zadeh et al. 2011).

FIGURE 2.

Sequence-dependent activation of Spinach.ST molecular beacons. (A) The molecular beacons Spinach.ST1, Spinach.ST2, and Spinach.ST3 were designed to be specific for three different target nucleic acid sequences. They are activated by only their complementary trigger oligonucleotides Trigger 1, Trigger 2, and Trigger 3, respectively. Spinach.ST RNAs were synthesized by T7 RNA polymerase-mediated transcription of 500 ng of PCR-generated duplex DNA transcription templates. Spinach.ST transcripts were incubated with 0.5 μM trigger DNA oligonucleotides and Spinach.ST activation was measured as fluorescence accumulation over time at 37°C. Raw fluorescence values are shown in arbitrary units (a.u.) and data representative of replicate experiments are depicted. (B) Spinach.ST molecular beacons can detect single-nucleotide mismatches in their target oligonucleotides. Spinach.ST1 transcripts were incubated with 0.7 μM cognate trigger oligonucleotides that were either fully complementary (1T) or contained a single-base mismatch at various positions within the toehold domain 2* (1T-1M to 1T-5M). Trigger sequences with 2*–5*–6* domain organization are depicted in the 5′–3′ direction with mismatches highlighted in red. Spinach.ST1 activation was measured in real-time at 37°C. Incubation with a noncognate trigger oligonucleotide (2T) served as the negative control for Spinach.ST1 activation. Raw fluorescence values are shown in arbitrary units (a.u.) and data representative of replicate experiments are depicted.

FIGURE 3.

Application of Spinach.ST to the detection of NASBA amplicons. (A) Schematic of primer design for real-time, sequence-specific NASBA detection using Spinach.ST2. Sequence blocks are depicted as numbered or lettered domains (* indicates complementarity). NV4FP and NV4RP are the forward and reverse primers used for NASBA. PBS-F and PBS-R* domains hybridize with the target nucleic acid and initiate polymerization. Hybridization is depicted using vertical slashes, while the potential hybridization of the PBS-F domain within the NV4FP primer to a complementary region is indicated by colons. Dashed regions in the target, double-stranded DNA transcription template, and RNA transcript denote target sequences not involved in priming or in Spinach.ST activation. The Spinach.ST trigger in the NASBA RNA amplicons is highlighted in gray. Spinach.ST structures were generated using NUPACK. (B,C) Sequence-specific fluorescent detection of single-stranded DNA (B) or RNA (C) templates by real-time Spinach.ST-NASBA. Varying concentrations of NV4 template DNA or RNA were amplified at 37°C by NASBA using T7 RNA polymerase and MMLV RT in the presence of cotranscribed Spinach.ST2 and 40 µM DFHBI. Raw fluorescence values are shown in arbitrary units (a.u.) and data representative of replicate experiments are depicted. (D) Denaturing 10% polyacrylamide gel analysis of NASBA amplification reactions of single-stranded DNA (lanes 1–5) or RNA (lanes 6–10) templates; cotranscription of Spinach.ST2 is also shown. Template concentrations are in nanomolar (nM) amounts. Two microliters of each NASBA reaction were analyzed. Single-stranded DNA oligonucleotides were used as size markers (lane 11).

FIGURE 4.

Discrimination of alleles in real-time by NASBA and sequence-modulated Spinach. (A) Schematic depicting the target alleles NV4 and the SNP-containing NV4.4M. Only the domain 2 and 2* sequences are shown for clarity. The single-nucleotide difference between the two alleles (highlighted in a large bold font) is located within the target region termed domain 2. Both NV4 and NV4.4M alleles were amplified using the common primers NV4FP and NV4RP. NASBA-generated NV4 and NV4.4M RNA amplicons form associative Spinach.ST triggers that have a single base difference between them (highlighted in bold font) in their 2* domains. The sequence-modulated molecular beacons Spinach.ST2 and the SNP-complementary Spinach.ST2.4M have corresponding single-nucleotide differences (highlighted in a large bold font) in their toehold domain 2. RNA amplicons bearing fully complementary associative triggers should activate Spinach.ST molecular beacons by facilitating toehold-mediated strand displacement. Activated Spinach.ST molecules increase the fluorescence of DFHBI (depicted as a green hexagon). Activation of Spinach.ST molecules by RNA amplicons bearing a single-nucleotide mismatch in their toehold domain should be significantly impaired (denoted as dashed double-tipped arrows interrupted with an X). (B,C) Detection of NASBA amplicons by allele-specific, sequence-modulated Spinach.ST. NASBA reactions were used to amplify either no template (black line) or 20 nM of ssDNA targets NV4 (red line) or NV4.4M (purple line) in the presence of 40 µM DFHBI and 138 ng of dsDNA transcription templates for either Spinach.ST2 (B) or Spinach.ST2.4M (C). Raw fluorescence values are shown in arbitrary units (a.u.) and data representative of replicate experiments are depicted.