Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability

Abstract

Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability.

Figure 1

In situ amplification via hybridization chain reaction (HCR). (a) HCR mechanism. Metastable fluorescent hairpins self-assemble into fluorescent amplification polymers upon detection of a cognate initiator. Initiator I1 nucleates with hairpin H1 via base-pairing to single-stranded toehold “a”, mediating a branch migration^, that opens the hairpin to form complex I1·H1 containing single-stranded segment “c*-b*”. This complex nucleates with hairpin H2 by means of base-pairing to toehold “c”, mediating a branch migration that opens the hairpin to form complex I1·H1·H2 containing single-stranded segment “b*-a*”. Thus, the initiator sequence is regenerated, providing the basis for a chain reaction of alternating H1 and H2 polymerization steps. Red stars denote fluorophores. (b) In situ hybridization protocol. Detection stage: probe sets are hybridized to mRNA targets, and unused probes are washed from the sample. Amplification stage: initiators trigger self-assembly of tethered fluorescent amplification polymers, and unused hairpins are washed from the sample. (c) Experimental timeline. The same two-stage protocol is used independent of the number of target mRNAs. For multiplexed experiments (three-color example depicted), probe sets for different target mRNAs (five probes depicted per set) carry orthogonal initiators that trigger orthogonal HCR amplification cascades labeled by spectrally distinct fluorophores.

Figure 2

Comparing in vitro amplification performance for (a) published RNA HCR in stringent amplification conditions (40% formamide, 45 °C) and (b) next-generation DNA HCR in permissive amplification conditions (0% formamide, room temperature). For each system, reactions were run with 200 nM of each hairpin for 1.5 h (to challenge polymer growth) and with 1 μM of each hairpin overnight (to challenge hairpin metastability). Agarose gels demonstrating hairpin metastability in the absence of initiator and increasing polymer length with decreasing initiator concentration (1×, 0.1×, 0.01× I1). Green channel: HCR-Alexa647. Red channel: dsDNA ladder prestained with SYBR Gold.

Figure 3

Multiplexed signal amplification using four orthogonal DNA HCR amplifiers (B1, B2, B3, B4). Agarose gel demonstrating minimal leakage in the absence of initiators and strong activation of the cognate amplifier by each of four initiators (I1^B1, I1^B2, I1^B3, I1^B4). Reaction conditions: 4 HCR amplifiers in all reactions, 400 nM for each hairpin, 0.01× initiator, 5× SSCT buffer, 4 h reaction at room temperature. See Section S3 in the SI for additional data.

Figure 4

Comparing in situ amplification performance for published RNA HCR in stringent amplification conditions (40% formamide, 45 °C) and next-generation DNA HCR in permissive amplification conditions (0% formamide, room temperature). (a) mRNA expression imaged by confocal microscopy with the microscope gain adjusted to avoid saturating pixels using DNA HCR. Sample: whole-mount zebrafish embryo. Target: transgenic mRNA Tg(flk1:egfp). Probe sets: one RNA or DNA probe. Green channel (excitation 633 nm): HCR-Alexa647 staining plus autofluorescence. Gray channel (excitation 488 nm): autofluorescence to depict sample morphology. Embryos fixed: 27 hpf. Scale bar: 50 μm. (b) Pixel intensity histograms for background (in WT embryos lacking the target; depicted rectangles in Figures S13 and S14 in the SI) and signal plus background (in transgenic embryos containing the target; depicted rectangles in panel (a)). (c) Characterizing signal and background contributions for representative rectangles (mean ± standard deviation, N = 3 embryos). See Section S4.1 in the SI for additional data.

Figure 5

Comparing signal strength using direct-labeled DNA probes without and with DNA HCR in situ amplification. (a) mRNA expression imaged by confocal microscopy with the microscope gain adjusted to avoid saturating pixels using DNA HCR. Sample: whole-mount zebrafish embryo. Target: transgenic mRNA Tg(flk1:egfp). Probe set: five Alexa647-labeled one-initiator DNA probes. Green channel (excitation 633 nm): probe-Alexa647 staining plus autofluorescence without or with HCR-Alexa647 staining. Gray channel (excitation 488 nm): autofluorescence to depict sample morphology. Embryos fixed: 27 hpf. Scale bar: 50 μm. (b) Pixel intensity histograms for background (in WT embryos lacking the target; depicted rectangles in Figure S17 in the SI) and signal plus background (in transgenic embryos containing the target; depicted rectangles in panel (a)). (c) Characterizing signal and background contributions for representative rectangles (mean ± standard deviation, N = 3 embryos). See Section S4.2 in the SI for additional data.

Figure 6

Comparing signal strength using DNA HCR in situ amplification with one-initiator and two-initiator DNA probes. (a) mRNA expression imaged by confocal microscopy with the microscope gain adjusted to avoid saturating pixels using the two-initiator DNA probe. Sample: whole-mount zebrafish embryo. Target: transgenic mRNA Tg(flk1:egfp). Probe sets: one-initiator DNA probe (I1 or I2) or two-initiator DNA probe (I1 + I2). Green channel (excitation 633 nm): HCR-Alexa647 staining plus autofluorescence. Gray channel (excitation 488 nm): autofluorescence to depict sample morphology. Embryos fixed: 27 hpf. Scale bar: 50 μm. (b) Pixel intensity histograms for background (in WT embryos lacking the target; depicted rectangles in Figures S20–S22 in the SI) and signal plus background (in transgenic embryos containing the target; depicted rectangles in panel (a)). (c) Characterizing signal and background contributions for representative rectangles (mean ± standard deviation, N = 3 embryos). See Section S4.3 in the SI for additional data.

Figure 7

Multiplexed mapping of mRNA expression in a fixed whole-mount zebrafish embryo. (a) Expression atlas for four target mRNAs: Tg(flk1:egfp), tpm3, elavl3, ntla. (b) mRNA expression imaged via confocal microscopy at four planes within an embryo. Probe sets: five two-initiator DNA probes per target. Amplifiers: four orthogonal DNA HCR amplifiers carrying spectrally distinct fluorophores. Embryos fixed: 27 hpf. Scale bar: 50 μm. See Section S4.4 and Movie S1 in the SI for additional data.

Figure 8

Subcellular signal localization and co-localization in a fixed whole-mount zebrafish embryo. Redundant two-color mapping of a target mRNA expressed predominantly in the interstices between somites (Tg(flk1:egfp); two probe sets, two amplifiers, channels 1 and 2) simultaneous with mapping of a target mRNA expressed predominantly in the somites (desm; channel 3) and nuclear staining with DAPI. (a) Subcellular co-localization of Tg(flk1:egfp) signal (each pixel is 129 nm × 129 nm) with highly correlated pixel intensities (Pearson correlation coefficient: r = 0.97). To avoid inflating the correlation coefficient, we exclude pixels that fall below background thresholds in both channels (excluded pixels fall in the black box at the lower left corner of the correlation plot). For each channel, the background threshold is defined as the mean plus two standard deviations for the pixels in the depicted white square. (b) Localization of signal within the cell cytoplasm for targets with interleaved expression patterns. Probe sets: three and five two-initiator DNA probes for Tg(flk1:egfp), three two-initiator DNA probes for desm. Amplifiers: three orthogonal DNA HCR amplifiers carrying spectrally distinct fluorophores. Embryos fixed: 27 hpf. Scale bar: 10 μm. See Section S4.5 in the SI for additional data.