Cyclic Caged Morpholinos: Conformationally Gated Probes of Embryonic Gene Function

Abstract

Deciphering the molecular mechanisms that underlie embryogenesis requires an ability to alter gene function with spatial and temporal precision. Synthetic reagents can be valuable tools in this discovery process, especially in model organisms currently intractable to targeted genomic changes. In particular, morpholino-based antisense oligonucleotides (MOs) have been widely used to inhibit gene expression in metazoans that develop ex utero (Figure 1a), [1][2][3][4] and caged versions of these reagents (cMOs) can enable conditional gene silencing through targeted illumination. [5][6][7][8][9] Composed of morpholino-based nucleosides and a phosphorodiamidate backbone, these nuclease-resistant probes are typically injected into zygotes as 25-base oligomers, after which they hybridize to complementary RNAs and disrupt splicing or translation. While cMOs can provide important new insights into embryonic gene function, their utility has been hindered by their empirical design, synthetic complexity, in vivo instability, reliance on complementary inhibitors, and/or use of multiple ** We thank A. Puri and I. Hinkson for their assistance. This work was supported by the NIH (R01 GM087292 and DP1 OD003792 to J. K. C.; R01 DK061215 to S. D. L.)

Sequential Gene Silencing Using Wavelength‐Selective Caged Morpholino Oligonucleotides

“…[17,18] We microinjected the DMNB, NB, DEACM, and DEACM-MN cyclic ntla cMOs individually into zebrafish zygotes (115 fmol/embryo for all reagents except for the DEACM-MN cMO; see below) and either briefly illuminated the embryos at 3.5 h post fertilization (hpf) with 365, 405, or 470 nm light or maintained them in the dark. [3,7] As expected, the DMNB and NB cyclic ntla cMOs were efficiently uncaged by 365 nm light, thereby inducing strong ntla-morphant phenotypes in 89 % and 86 % of the embryos injected with these respective reagents (Figure 2 c). [3,7] As expected, the DMNB and NB cyclic ntla cMOs were efficiently uncaged by 365 nm light, thereby inducing strong ntla-morphant phenotypes in 89 % and 86 % of the embryos injected with these respective reagents (Figure 2 c).…”

“…[1] We and others have developed caged morpholino oligonucleotides (cMOs) that can perturb targeted RNAs in vivo, [2][3][4][5][6][7][8] and these optochemical tools have been used to interrogate the functions of individual genes, such as the zebrafish transcription factors no taila (ntla) and ets variant gene 2 (etv2). The efficacy of these reverse-genetic chemical probes has been demonstrated in zebrafish embryos, and these reagents have been employed to examine the mechanisms of mesoderm patterning.…”

“…[1] We and others have developed caged morpholino oligonucleotides (cMOs) that can perturb targeted RNAs in vivo, [2][3][4][5][6][7][8] and these optochemical tools have been used to interrogate the functions of individual genes, such as the zebrafish transcription factors no taila (ntla) and ets variant gene 2 (etv2). [1] We and others have developed caged morpholino oligonucleotides (cMOs) that can perturb targeted RNAs in vivo, [2][3][4][5][6][7][8] and these optochemical tools have been used to interrogate the functions of individual genes, such as the zebrafish transcription factors no taila (ntla) and ets variant gene 2 (etv2).…”

“…[7] Antisense MOs are typically 25 bases in length and designed to complement splicing or translational start sites in targeted RNAs. [7] Antisense MOs are typically 25 bases in length and designed to complement splicing or translational start sites in targeted RNAs.…”

Hydrogel formation by the ‘topological conversion’ of cyclic PLA–PEO block copolymers

“…As noted above, the terminal structure plays an important role in the gel formation; therefore, we expected the cyclic polymers, which lack a terminal structure, to have less ability to gel (Scheme 1b). We also synthesised cyclic PLLA-PEO and cyclic PDLA-PEO, which have o-nitrobenzyl (NB) groups as photocleavable units, and devised a plan to use light to perform a 'topological conversion' to transform cyclic polymers, 19,20 which are expected to have low ability to gel, to form linear polymers with the ability to gel (Scheme 1c).…”