Peering inside protein complexes with AFM

Abstract

A new method uses click chemistry to covalently modify any RNA of interest. RNAs have crucial roles in diverse cellular processes, and studying these RNAs in cells is important for revealing their functions. However, methods for site-specific, covalent tagging of RNAs have lagged behind those for proteins. For these reasons, Jiangyun Wang at Beijing National Laboratory for Molecular Sciences and a team of researchers worked to develop a new method for covalent modification of any RNA of interest in mammalian cells. The method employs an archaeal tRNA synthetase (Tias) that modifies its cognate tRNA (tRNA Ile2) by adding a molecule called agmatine. The authors found that Tias could also be used to covalently add agmatine analogs that contain a click-chemistry handle. To label an RNA of interest, researchers first genetically tag it by incorporating the tRNA Ile2 sequence into its gene. Then this tagged gene is transcribed in cells that contain Tias and the agmatine analog. Tias then specifically adds the agmatine analog to the tagged RNA. Finally, click chemistry can be used to add functional groups to the RNA for a range of applications from fluorescence imaging to nuclear magnetic resonance spectroscopy. According to Wang, this work was inspired by the team's previous research on the mechanism of recognition between codons in mRNA and anticodons in tRNA. In its normal cellular context, chemical modification by Tias ensures that tRNA Ile2 decodes the correct codon. Wang says that he and his colleagues were " fascinated by the chemical basis for this novel codon-anticodon interaction and decided to investigate further, " which ultimately led to the development of this new labeling strategy. The work led to many new insights regarding the structure-function relationship of Tias, as the team solved the crystal structure of Tias in complex with one of the structural BiologY Peering inside protein complexes with Afm Atomic force microscopy is applied to image the location of chemical groups inside single protein complexes. Atomic force microscopy (AFM) is a versatile tool that, among many applications, allows researchers to image single molecules, all the way down to the atomic level. This technique produces a topographic map of molecular structure. However, imaging details of the internal structure of a large molecule such as a protein complex is typically not possible with AFM. In recent work, Ozgur Sahin of Columbia University and his postdoc Duckhoe Kim devel-oped a method that can be applied to peer inside a protein complex by targeting specific chemical groups and tugging on them with an AFM cantilever. They created short, single-stranded DNA probes that are functionalized with a binding moiety on one end. These DNA probes are designed to hybridize with complementary DNA probes tethered to an AFM cantilever. Sahin and Kim designed this cantilever-tethered probe sequence to contain two different regions that hybridize to two different complementary DNA molecules used as the binding probes. By doing this, they could use the different AFM force-time waveforms gen-erated as a result of hybridization to one complementary sequence or another to distinguish distinct binding interactions. Sahin and Kim initially experimented with the cantilever-tethered probe sequence by test-ing its interaction with two different complementary DNA probe molecules attached to a surface. " When the AFM detects an interaction, the computer highlights the corresponding pixel in the image with a specific color, " explains Sahin. " We noticed that the highlighted pixels were clustered into regions less than 2 nanometers and more typically less than 1 nanometer. " A careful statistical analysis of the data revealed that their AFM setup had subnanometer resolution. Because they achieved such high resolution, the researchers were encouraged to apply the approach to locate specific chemical moieties within a protein complex. npg