Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription.
Ontology highlight
ABSTRACT: Conjugation of RNAs with nanoparticles (NPs) is of significant importance because of numerous applications in biology and medicine, which, however, remains challenging especially for large ones. So far, the majority of RNA labeling relies on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nucleotides (nts). We, here, present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with a gold nanoparticle (Nanogold) empowered by transcription of an expanded genetic alphabet containing the A-T/U and G-C natural base pairs (bps) and the TPT3-NaM unnatural base pair (UBP). We synthesize an amine-derivatized TPT3 (TPT3A), which is site specifically incorporated into a 97-nt 3'SL RNA and a 719-nt minigenomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by in vitro T7 transcription. The TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-N-hydroxysuccinimidyl (NHS)-Nanogold NPs via an amine and NHS ester reaction and further purified under nondenaturing conditions. TPT3 modification and Nanogold labeling cause minimal structural perturbations to the RNAs by circular dichroism, small angle X-ray scattering (SAXS), and binding activity assay. We demonstrate the application of the Nanogold-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The internanoparticle distance distributions in the 3'SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus, validate the applicability of this labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.
SUBMITTER: Wang Y
PROVIDER: S-EPMC7502756 | biostudies-literature | 2020 Sep
REPOSITORIES: biostudies-literature
ACCESS DATA
