Discovering the RNA Double Helix and Hybridization
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Leading Edge Essay Discovering the RNA Double Helix and Hybridization Alexander Varshavsky1,* 1Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA *Contact: [email protected] DOI 10.1016/j.cell.2006.12.008 The discovery, 50 years ago, that RNA could form a double helix made possible a number of advances, including an understanding that led, decades later, to the discovery of micro- RNAs and RNA interference. Remarkably, the first nucleic acid hybridization reaction was also described in the same report. It is difficult to imagine today how little two papers in which they analyzed naturally occurring RNA. These syn- was known about the molecular basis X-ray diffraction photographs of thetic RNA chains were apparently of living cells 50 years ago. The DNA RNA fibers (Rich and Watson, 1954a, linear, suggesting that the natural double helix, described in the 1953 1954b). The diffraction patterns they RNA molecules would not contain Watson and Crick Nature paper (Wat- saw were too diffuse for a definitive significant branching. son and Crick, 1953), was a uniquely statement about the underlying RNA important insight into a molecular structure. It was puzzling that the The Key Discovery structure that could both contain same diffraction pattern was pro- In the spring of 1956, Rich and Dav- genetic information and replicate it. duced by RNA molecules that had ies found that, on mixing sodium However, the role of the other nucleic vastly different base ratios, unlike the salts of polyadenylic acid (poly A) and acid, RNA, was clothed in uncertainty. 1:1 ratios of adenine to thymine and polyuridylic acid (poly U), there was It was believed by many that RNA was guanine to cytosine that had been “a very rapid increase in viscosity, as involved in protein synthesis, but that found in DNA samples. A number of well as a drop in the optical density was still a conjecture based on indi- RNA viruses that were characterized at 260 nm” (Rich and Davies, 1956). rect arguments. Given that RNA had by the early 1950s seemed to lack The drop in optical density was also an extra hydroxyl group, the molecule any specific ratios of this kind. The reported at the same time by Rob- could be, in principle, branched, RNA fibers were negatively birefrin- ert Warner in Ochoa’s laboratory at unlike the linear DNA molecule. The gent, a characteristic of DNA fibers New York University (Warner, 1956). 1953 Watson-Crick paper about the as well, suggesting that the bases in The report by Rich and Davies stated DNA double helix mentioned that “it RNA often could be oriented perpen- that tough, glassy, negatively bire- is probably impossible to build this dicular to the fiber axis. Beyond that, fringent fibers were drawn from the structure with ribose, instead of the very little could be inferred. viscous solution of poly A plus poly deoxyribose, as the extra oxygen In 1954, Rich moved to the National U. The fibers produced “a well-ori- atom would make too close a Van der Institutes of Health (NIH) to set up a ented X-ray diffraction photograph Waals contact.” Did that mean that Section on Physical Chemistry, and with a distribution of intensity which RNA could not form any double helix? soon David Davies joined him (Rich, is characteristically helical.” The dif- If so, how could RNA viruses (which 2004). A significant event in 1955 was fraction pattern had many similarities were studied at the time) replicate if the discovery of the enzyme poly- to that seen with DNA, but there were their RNA could not form a double nucleotide phosphorylase in Severo major differences in the first-layer helix in the same manner as DNA? Ochoa’s laboratory (Grunberg-Man- line, which was strong for RNA but In late 1953, Alexander Rich was a ago, et al., 1955). This template-inde- quite weak for DNA. The calculated postdoctoral fellow with Linus Paul- pendent enzyme could convert ribo- diameter of RNA molecules was 26 Å, ing at Caltech, and James Watson nucleoside diphosphates into RNA significantly larger than the 20 Å seen was a postdoctoral fellow with Max polymers. Rich and Davies employed with DNA fibers. A short report by Delbrück. Both Rich and Watson this technique to synthesize RNA, Rich and Davies (1956), published in were interested in whether RNA could began studying diffraction patterns the Journal of the American Chemi- form a double helix. They proceeded from synthetic RNA fibers, and dis- cal Society (JACS), stated: “These to carry out X-ray investigations of covered that a random-sequence results show for the first time that it RNA fibers, using the technique that copolymer containing adenine and is possible for the RNA backbone had proven so successful in the study uracil residues produced a diffraction to assume a configuration not unlike of DNA structure. This work yielded pattern very similar to that found in that found in DNA, using the same Cell 127, December 29, 2006 ©2006 Elsevier Inc. 1295 complementarity in the base pairs. 9-methyl adenine. Formation of a tri- We know today that the double- This implies that there may exist a ple-stranded RNA molecule was the helical RNA plays a number of crucial form of the RNA molecule similar to first indication that RNA was capable roles in biology. It serves as a struc- that of DNA and that this would be of significant structural complexity, a tural framework for many molecules, the form in which RNA carries out its continuing theme in modern analyses including tRNA and ribozymes. Dou- implied molecular duplication in the of RNA structures. ble-helical RNA is also the basis for plant and smaller animal viruses.” By 1962, improvements in analyz- nucleic acid replication in many sys- The RNA double helix was formed ing RNA duplex diffraction patterns tems, directly analogous to DNA rep- by a remarkable reaction, in solu- led to the realization that the RNA lication. More recently, the RNA dou- tion, between poly A and poly U. Rich duplex was very similar to the (dehy- ble helix has become a key actor in and Davies added, “We would like to drated) A-form of DNA. However, the remarkable phenomenon of RNA point out that this method for forming there were still uncertainties about interference (RNAi) and the underly- a two-stranded helical molecule by the structure, a consequence of the ing RNA-based circuits. The RNA simply mixing two substances can be fact that fiber diffraction is nowhere double helix pervades all of modern used for a variety of studies directed close to producing enough experi- biology. It is medically relevant as toward an understanding of the for- mental data to fix the position of every well, given the possibility of RNAi- mation of helical molecules utilizing atom, a feat that could be achieved based drugs. specific interactions.” The astonish- only by single-crystal X-ray crystal- ment at this discovery can be seen lography. Rich and coworkers contin- The Nucleic Acid Hybridization today in a letter that Rich wrote to ued to pursue RNA structure, and in In the 1950s, the possibility that long Linus Pauling 2 weeks after sending 1973 they published the first single- polymeric molecules could spon- off the note to JACS (Rich, 2006). The crystal structure of the RNA double taneously form a double helix in letter conveyed an amazement that helix. The structures of GpC (Day et solution was perceived by many as this reaction could happen sponta- al., 1973) and ApU (Rosenberg et al., far-fetched. Hence the surprise and neously and that it was “com- (initial) skepticism that greeted pletely reproducible.” The first the discovery of nucleic acid hybridization reaction was hybridization. Many felt it was thus discovered, and it elicited unlikely that hybridization of a great deal of skepticism. long chains of nucleic acids Shortly afterwards, Gary would happen without the aid Felsenfeld joined the NIH of an enzyme. (The doubters, laboratory and proceeded to while incorrect about in vitro carry out a systematic study settings, were right about of the reaction involving a mix- the in vivo situation: in the ture of these two RNA poly- crowded intracellular milieu, mers (Felsenfeld and Rich, where nonspecific aggrega- 1957). By carefully measuring tion is a major problem, both the optical density in the ultra- formation and disruption of violet, it was possible to show RNA and DNA duplexes are unequivocally that the two indeed orchestrated by spe- strands formed a 1:1 structure cific enzymes, as was discov- Figure 1. The RNA Double Helix Revealed with equal molar contents of Optical density (at 260 nm) of mixtures of poly U and poly A ered years later.) One group adenine and uracil (Figure 1). showing a 1:1 complex that indicated the formation of an RNA of chemists felt that entropic double helix. (Adapted from Felsenfeld and Rich, 1957.) effects made it unlikely that The RNA Double Helix large, tangled molecules with Felsenfeld, Davies, and Rich (Felsen- 1973) were solved at 0.8 Å resolution thousands of residues would untan- feld et al., 1957) reported that the addi- and revealed the RNA double helix in gle themselves to form linear double tion of small amounts of magnesium full detail. The accompanying News helical arrays in solution. Theorists ions would convert the two-stranded & Views article in Nature commented pointed out that the two polynucle- RNA molecule into a three-stranded that the ApU structure was the “miss- otide chains were highly negatively molecule, which contained a third ing link” of nucleic acid structure in charged, making it unlikely that they strand of polyuridylic acid.