Caged oligonucleotides for bidirectional photomodulation of let-7 miRNA in zebrafish embryos

Abstract

Many biological functions of microRNA (miRNA) have been identified in the past decade. However, a single miRNA can regulate multiple gene targets, thus it has been a challenge to elucidate the specific functions of each miRNA in different locations and times. New chemical tools make it possible to modulate miRNA activity with higher spatiotemporal resolution. Here, we describe light-activated (caged) constructs for switching let-7 miRNA 'on' or 'off' with 365 nm light in developing zebrafish embryos.

Keywords: Antagomir; Let-7; MicroRNA (miRNA); Zebrafish.

Figure 1. let-7 early induction phenotype

Zebrafish embryos injected at the 1-cell stage with 10 μM let-7 miRNA show the let-7 early induction phenotype when imaged at 24 and 50 hpf (right images), compared to un-injected wildtype (Tu × Tu) embryos (left images). The early induction let-7 miRNA phenotype is characterized by decreased head formation, shortened tail, and yolk that fails to extend along the tail.

Figure 2. Structures of caged hairpin antagomirs CHANT1 and CHANT2

A) CHANT1: A 22-mer miRNA antagomir targeting let-7 miRNA was covalently attached to a complementary 12-mer blocking sequence via a nitrobenzyl photocleavable linker, PL (in red). Upon photolysis, the 12-mer more readily dissociated, restoring antagomir function. B) CHANT2: A 22-mer miRNA antagomir targeting let-7 miRNA was covalently attached via PL (in red) to a 5′-9-mer-PL-10-mer blocking sequence. Upon PL photolysis, antagomir function was restored. All sequences consisted of 2′-OMe RNA bases.

Figure 3. CHANT1 in vivo experiments

A) Embryos injected at the 1-cell stage with 10 μM let-7 miRNA showed let-7 early induction phenotype. B) Embryos injected with 10 μM let-7 miRNA + 50 μM CHANT1 showed similar let-7 early induction phenotype prior to 365 nm irradiation. C) Embryos injected with 10 μM let-7 miRNA + 50 μM CHANT1 developed normally after 365 nm irradiation for 10 min at 1 hpf. All embryos were imaged at 24 hpf.

Figure 4. CHANT2 in vivo experiments

A) Embryos injected at the 1-cell stage with 10 μM let-7 miRNA showed let-7 early induction phenotype. B) Embryos injected with 10 μM let-7 miRNA + 50 μM CHANT2 antagomir showed let-7 early induction phenotype prior to 365 nm irradiation. C) Embryos injected with 10 μM let-7 miRNA + 50 μM CHANT2 developed normally after 365 nm irradiation for 10 min at 1 hpf. All embryos were imaged at 24 hpf.

Figure 5

CHANT1 vs. CHANT2 in vivo efficiency.

Figure 6. CIRClet7 in vivo experiments

A) Zebrafish embryos injected with 20 μM CIRClet7 and incubated in the dark developed normally. B) Embryos irradiated with 365 nm light developed with the early induction let-7 miRNA phenotype. All embryos were imaged at 24 hpf.

Scheme 1. Synthesis and photocleavage of circular caged miRNA (CIRClet7)

A photocleavable oligonucleotide duplex with 3′-amine and 5′-thiol was circularized with a heterobifunctional crosslinker. The crosslinker was first reacted with the 3′-amine, and circularization proceeded after 5′disulfide reduction with TCEP. Upon photolysis, the active miRNA strand was released with a 5′-phosphate for processing by RISC.