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. 2020 May 12;13:75. doi: 10.3389/fnmol.2020.00075

Figure 1.

Figure 1

Principle of in situ hybridization chain reaction (HCR) and short hairpin design. (A) Each hairpin DNA (H1, H2) has toehold, stem and loop domains and is conjugated to a fluorophore. Whereas the sequence of the toehold domain of H1 (a) is complementary to that of the loop domain of H2 (a’), the sequence of the loop domain of H1 (c) is complementary to that of the toehold domain of H2 (c’). In the presence of an initiator that is composed of a’ and b’, the initiator hybridizes with the toehold and stem domains (a,b) of H1. Then, the remaining part of H1 (c,b’) hybridizes with the toehold and stem domains (c’,b) of H2. Thereafter, H1 and H2 continue to hybridizes with each other (lower). (B) Split-initiator probe set. One probe has sequence a’, first 3 nucleotide (nt) of sequence b’, 2 nt spacer sequence, and 25 nt sequence complementary to the target mRNA. Another probe has 25 nt sequence complementary to the target mRNA, 2 nt spacer sequence and the last 9 nt of sequence b’. (C) In situ HCR using split-initiator probes. Three sets of split-initiator probes hybridize with the target mRNA and lead to HCR. (D) Design principles of short hairpin DNA. A set of short hairpin DNAs and split-initiator probes with actual nucleotide sequences are shown in Supplementary Figure S1.