The MI Team
The HCR™ Platform is an innovative new method for generalized signal amplification borne out of the fields of synthetic biology and molecular programming. The mechanism is based on two DNA monomers (amplifier h1 and amplifier h2) that are kinetically trapped in a hairpin structure and do not interact with one another. The introduction of an initiator strand activates a chain reaction in which the h1 and h2 amplifiers assemble into a long polymer tethered to the initiator strand. Attaching a detectable label to the amplifiers (e.g., fluorophore or hapten, denoted as stars) enables visualization of the amplified signal.
Each HCR™ Probe is designed to be used with a specific HCR™ Amplifier. Because HCR™ Amplifiers are engineered to be orthogonal, each Amplifier will only be activated by its corresponding initiator. For example, a probe set for detecting Transcript 1 that is designed for the B1 Amplifier is unable to activate any other HCR™ Amplifier (e.g., B2-488, B3-546, B4-647... etc.).
For enabling multiplexing, you need to choose a different HCR™ Amplifier (e.g., B1, B2, ...) for each target biomolecule that will be imaged in the same sample (Amplifier B1 for Transcript 1, Amplifier B2 for Transcript 2, ...). It's necessary to choose a different fluorophore label (e.g., 594, 647, ...) for each HCR™ Amplifier that will be imaged in the same sample (e.g., B1-647, B2-594).
All HCR™ Amplifiers (e.g., B1, B2, ...) are engineered to identical performance targets, so there is no reason to choose one system over another. If you are planning to multiplex several targets, then each target should use a different HCR™ Amplifier paired with a spectrally-distinct fluorophore.
The choice of fluorophore will depend first and foremost on the capabilities of your imaging system. There tends to be more autofluorescence in lower wavelengths so we recommend using lower wavelength fluorophores (e.g., 488) for higher expression targets and higher wavelength fluorophores (e.g., 546, 647) for lower expression targets.