RNA structural studies have been primarily conducted in vitro. Despite the remarkably detailed views of RNA folding obtained, a true understanding of structure-function relationships will require probing RNA within living cells. Therein, RNA is subject to influences such as the rate of transcription and interactions with proteins and small molecules. In a brief communication published in Nature Chemical Biology, Spitale et al. introduce an RNA SHAPE procedure geared for use in living cells. SHAPE, a method for mapping RNA structure, stands for selective 2′-hydroxyl acylation analyzed by primer extension. It is based on the premise that 2′ hydroxyl groups in RNA nucleotides are differentially reactive depending upon their flexibility, with fewer constraints tolerating conformations in which the adjacent 3′ phosphodiester anion can help boost reactivity. In a typical SHAPE assay, an in vitro–transcribed RNA is incubated with the acylating agent N-methylisatoic anhydride (NMIA), which adds a bulky 2′-O-adduct in regions that are solvent-exposed or less protected by base pairing or binding partners. When the modified RNA is reverse-transcribed, cDNA synthesis yields prematurely truncated products that can be mapped by gel electrophoresis. Unfortunately, with so many other possible substrates available, NMIA does not measurably modify RNAs in living cells. So, Spitale et al. designed a new acylation elecrophile, NAI (2-methylnicotinic acid imidazolide). In vitro, NAI gives very similar 2′-hydroxyl acylation footprints to NMIA, but NAI's higher effective half life and better solubility in aqueous solutions let it modify RNA within cells. The authors treated E. coli; yeast; and cultured human, murine, and Drosophila cells with NIA, then reverse-transcribed the 5S rRNA, comparing the footprint with the RNA's known structure. As expected, bases that were modified were in loops that participated in noncanonical Watson-Crick pairing, or were solvent-accessible. As opposed to structural studies of in vitro–transcribed 5S rRNA, intracellular SHAPE picked up interactions with other ribosomal RNAs and proteins. When the new method's results diverged from traditional SHAPE, the affected bases corresponded almost exactly with those detected as functionally important through mutagenesis studies. Thus, in vivo SHAPE builds upon the in vitro version by revealing reactivity patterns indicative of tertiary structure and protein interactions. The authors suggest that NAI, which also appears to modify lower-abundance nuclear RNAs, will help elucidate structure-function relationships of both coding and noncoding RNAs.
Spitale, R.C. et al. 2013. RNA SHAPE analysis in living cells. Nat. Chem. Biol. 9(1):18-20.Connect the dots
The photostability and intrinsic brightness of quantum dots (QDs) have made them highly favored for detecting proteins on living cells. As an alternative to antibody-conjugated QDs, which depend on the availability of good-quality antibodies to one's protein of interest, Alice Ting's group at MIT in Cambridge, Massachusetts previously described the use of biotin ligase and streptavidin to target QDs to proteins. In that method, the protein target is expressed as a fusion with a 15–amino acid acceptor peptide, which is biotinylated by a coexpressed biotin ligase; the modified protein can then be bound by streptavidin-conjugated QDs. Despite its elegance, the biotin ligase method can't be used for targeting two proteins in the same cell, and then there's the problem of QD cross-reactivity with endogenous biotinylated proteins. Previous reports have used protein fusions with HaloTag, cutinase, or acyl carrier protein, together with a suitable QD conjugate. However, these fusion tags are relatively large and may interfere with protein trafficking. Ting and colleagues investigated the use of lipoic acid ligase (LplA), a protein structurally homologous to the biotin ligase from the original method. In the new scheme, the target protein is produced as a fusion with a 13–amino acid ligase acceptor peptide (LAP). A single amino acid change in LplA allows it to accept an adenylate ester of 10-bromodecanoic acid (10-Br-AMP) as a substrate, ligating it to LAP. The newly bromoalkylated protein then becomes a substrate for a HaloTag-conjugated QD, resulting in a covalently labeled protein. After a 5-minute ligation step plus an equally brief incubation with HaloTag-QD, the authors observed a 15:1 signal-to-noise ratio in imaging. In other experiments, they verified the labeling was stable, persisting for at least 48 hours. And as hoped, the new labeling method can be used side-by-side with the previous biotin ligase technique, as shown by simultaneous targeting of LAP-LDL receptor and biotin acceptor peptide–EGF receptor by HaloTag and streptavidin QDs, respectively. This new orthogonal labeling technique should be useful for single-molecule detection, particularly in cases where long-term tracking is desired. Beyond imaging applications, bromoalkylated LAP-containing proteins can be covalently linked to any HaloTag conjugate, including other proteins.
Liu, D.S. et al. 2012. Quantum dot targeting with lipoic acid ligase and HaloTag for single-molecule imaging on living cells. ACS Nano. 6(12):11080-11087.