Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

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.

Pseudoknot length modulates the folding, conformational dynamics, and robustness of Xrn1 resistance of flaviviral xrRNAs

“…Site-specific internal Cy5 labeling of xrRNAs was achieved using the UBP system containing NaM and TPT3 originally developed by Romesberg’s group 32 , 33 (Fig. 2b ), which were recently utilized to develop strategies for post-transcriptionally site-specific labeling of large RNAs with spin or gold nanoparticle 34 , 35 . Using a similar strategy 35 , DNA templates containing an upstream T7 promoter and dNaM modification at the template strands were prepared by overlapping PCR reactions, the selected residues in the respective xrRNAs were then replaced with alkyne-modified rTPT3 CO by in vitro transcription (Fig.…”

“…Site-specific incorporation of extrinsic fluorophores into large RNAs remains challenging as internal labeling by chemical synthesis is generally limited to RNAs smaller than 80 nts and splint-directed enzymatic ligation is often of low efficiency 41 – 43 . The NaM-TPT3 UBP-based strategy, which can overcome the size constraints in conventional RNA labeling methods, has recently been utilized for a site-specific spin and gold nanoparticle labeling to empower the applications of molecular ruler techniques including pulsed electron–electron double resonance spectroscopy (PLEDOR) and X-ray scattering interferometry in large RNAs 34 , 35 . We expect that the UBP-based site-specific labeling strategy will have broader application in conformational dynamics studies of large RNAs such as Group II introns by smFRET 44 , 45 .…”

Development of a Universal RNA Dual-Terminal Labeling Method for Sensing RNA-Ligand Interactions

“…RNA single-labeling strategies have been successfully developed during the past few years [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] . In general, chemo-enzymatic approaches are used to directly install various labels (such as alkyne or azide) into different ncRNAs for the second-step fast click modifications of various functional groups 17,18 .…”

“…RNA 5'-terminus can be chemically labeled by several methods [14][15][16][17][18][19][20][21][22] . For example, direct 5'-functionalization with diazo reagents was achieved for short RNAs 20 , while the use toward long RNAs needs to be further validated.…”

Recent advances in RNA structurome

“…Recently, an efficient and universally applicable strategy for site-specific nanoparticle labeling of large RNAs has been developed, which is authorized by transcription of an expanded genetic alphabet containing TPT3-NaM UBP. This strategy overcomes the size constraints of conventional RNA labeling methods, thus implementing XSI for large RNA structural studies (Wang et al, 2020a). Because XSI can measure much broader distance distributions (ranging from 50 to 400 Å) than other molecular rulers such as EPR and smFRET, it is expected that XSI will play important roles in structural studies of large RNAs and RNA complexes.…”