New Method for Producing Significant Amounts of RNA Labeled at Specific Sites
Position-specific labeling of RNA (PLOR) is a newly-developed method to make significant amounts of RNA with one or more labels at specific sites. Initiation involves incubating template DNA coupled to beads with RNA polymerase and a mix of nucleotide triphosphates (NTPs) lacking the nucleotide at the position where the label will be added. After thorough washing, a new NTP mix is added and elongation resumes. Elongation can be paused and restarted repeatedly using different NTP mixes. The process is terminated by lowering the temperature to 4°C and collecting the RNA produced.
Among biomacromolecules, RNA is the most versatile, and it plays indispensable roles in almost all aspects of biology. For example, in addition to serving as mRNAs coding for proteins, RNAs regulate gene expression, such as controlling where, when, and how efficiently a gene gets expressed, participate in RNA processing, encode the genetic information of some viruses, serve as scaffolds, and even possess enzymatic activity. To study these RNAs and their biological functions and to make use of those RNA activities for biomedical applications, researchers first need to make various types of RNA. For structural biologists incorporating modified or labeled nucleotides at specific sites in RNA molecules of interest is critical to gain structural insight into RNA functions. However, placing labeled or modified residue(s) in desired positions in a large RNA has not been possible until now.
Yun-Xing Wang, Ph.D., of CCR’s Structural Biophysics Laboratory, and his colleagues developed a hybrid solid-liquid phase transcription method to synthesize significant amounts of RNAs with labelling at specific sites. The method, named PLOR for position-specific labelling of RNA, involves incubating template DNA molecules that are coupled to agarose beads with T7 RNA polymerase and a mix of nucleotide triphosphates (NTPs) lacking the nucleotide at the position of interest. RNA elongation stops when the polymerase reaches this position. After extensive washing, a new NTP mix containing the labeled nucleotide is added and elongation resumes. Synthesis of the RNA molecules can be paused and restarted repeatedly using different NTP mixes to incorporate labeled NTPs at desired locations. The reaction is quenched by lowering the temperature to 4°C or adding heparin, and the same beads can be used multiple times to increase RNA yield. The process is very specific with an incorrect nucleotide being added about one time in 20,000. Because PLOR requires multiple washes and NTP additions, the researchers also created a fully automated, robotic platform to perform the reactions—this is the first RNA labeling machine in the world.
The investigators decided to test some applications of PLOR using a 71 nucleotide domain of riboA71, a RNA molecule that changes conformation on binding adenine to activate protein production. The crystal structure of riboA71 bound to adenine shows that Loops 1 and 2 interact. To look at the Loop 1- Loop 2 interaction in solution, they synthesized RNAs in which either loop was labeled with isotopes of carbon and nitrogen so they would be distinguishable by nuclear magnetic resonance spectroscopy (NMR) heteronuclear correlation spectra. The spectra showed that these RNA molecules fold the same as the unlabeled versions. Likewise, molecules labeled at both Loop 1 and Loop 2 had a similar structure. The scientists also used a different labelling scheme involving isotopes of carbon, nitrogen, and hydrogen to establish the structure of a region of riboA71 that is otherwise impossible to determine with NMR due to spectral overlaps. In a final demonstration with NMR, using a single labeled residue, they identified four distinct conformations of the riboA71 adenine binding pocket, supporting a currently hypothesized four-state model of its structure.
Turning to single molecule fluorescence studies, the researchers generated riboA71 molecules that included a biotin tag at the 3’ end, one fluorophore-tagged nucleotide at residue 24 in Loop 1, and a second fluorophore-tagged nucleotide at residue 55 in Loop 2. In the absence of adenine, they observed two populations of molecules with distinct interactions between the loops; about 20 percent of the molecules were in a high fluorescence resonance energy transfer (FRET) conformation, meaning the fluorophores are close together. With the addition of adenine, the high FRET population increased dramatically, further supporting the interaction of these loops when riboA71 is bound to adenine. These results also demonstrated that adenine is not required to form the high FRET conformation.
Together, these studies show that PLOR provides efficient synthesis of milligram quantities of labeled RNA. The investigators suggest that future applications of the method may allow for incorporation of nucleotides that resist RNA degrading enzymes for potential in vivo applications. They also propose that PLOR could be used to facilitate crystallographic studies by introducing nucleotides containing heavy atoms.
Summary Posted: Wed, 07/01/2015
Liu Y, Holmstrom E, Zhang J, Yu P, Wang J, Dyba MA, Chen D, Ying J, Lockett S, Nesbitt DJ, Ferré-D’Amaré AR, Sousa R, Stagno JR, Wang YX. Synthesis and applications of RNAs with position-selective labeling and mosaic composition. Nature. June 18, 2015 PubMed Link