Discovering the RNA Double Helix and Hybridization

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector Leading Edge Essay

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. That strand that it cleared up many of the issues would come together. Only with time did not increase the radius of the helix, concerning Watson-Crick base pair- and greater understanding of the and they interpreted the added uracil ing and the organization of the double forces stabilizing the double-helical pairing to the adenine in a manner that helix. These studies were carried out structure did these criticisms recede. was seen 2 years later by K. Hoog- several years before synthetic oligo- Ultimately, the above advance was stein in his single-crystal analysis of nucleotides became available for sin- viewed as a paradigm change in the complex of 1-methyl thymine and gle-crystal X-ray diffraction studies. nucleic acid chemistry.

1296 Cell 127, December 29, 2006 ©2006 Elsevier Inc. After 1956, many studies were car- (nonrandom) sequences. In 1960, human genome, and the analysis of ried out on hybridization reactions Paul Doty and the late Julius Mar- DNA for forensic purposes are just a that could make other helical com- mur were studying DNA-DNA inter- few recent examples. plexes. A major problem in the late actions. Their analyses showed that It is remarkable that the discovery 1950s was whether RNA and DNA denatured DNA strands held just of both the first RNA double helix and could actually make a hybrid double below the melting temperature of the the first nucleic acid hybridization helix that might serve as the basis duplex were able to realign and find emerged from a single publication 50 of information transfer from DNA to each other, reforming double helices years ago. RNA. It was commonly believed at and thereby restoring the biological the time that “DNA makes RNA, RNA (transforming) activity of DNA (Doty et AcknowledgmentS makes protein,” but there was no evi- al., 1960). One year later Ben Hall and dence that DNA and RNA could com- the late Sol Spiegelman combined I thank Alexander Rich (MIT) for his helpful comments on the manuscript. bine. Activities in crude biochemi- the polymer DNA-RNA hybridiza- cal preparations that suggested the tion with the Marmur-Doty annealing References existence of DNA-dependent RNA procedure and used this approach polymerases were being studied in to demonstrate the formation of a Day, R.O., Seeman, N.C., Rosenberg, J.M., the late 1950s, but the samples were specific hybrid between DNA of bac- and Rich, A. (1973). Proc. Natl. Acad. Sci. USA not clean enough, as yet, for a direct teriophage T2 and phage-encoded 70, 849–853. demonstration of DNA template- RNA from T2-infected bacterial cells Doty, P., Marmur, J., Eigner, J., and Schild- dependent RNA synthesis. (Hall and Spiegelman, 1961). Thus, kraut, C. (1960). Proc. Natl. Acad. Sci. USA 46, It was clear early on that the by 1961, all of the “first-generation” 461–476. detailed geometry of the RNA duplex hybridization methods were in place Felsenfeld, G., and Rich, A. (1957). Biochim. differed from that of the DNA duplex. for the further development of this Biophys. Acta 26, 457–468. For example, unlike DNA, the confor- approach, which went on to become Felsenfeld, G., Davies, D.R., and Rich, A. mation of RNA in fibers did not seem the technical foundation of modern (1957). J. Am. Chem. Soc. 79, 2023–2024. to change with decreasing humidity. molecular biology. Grunberg-Manago, M., Ortiz, P.J., and Ochoa, In sum, it was not obvious at the time The Southern blot, developed by S. (1955). Science 122, 907–910. that DNA and RNA chains could form Edward Southern in 1975, combined Hall, B.D., and Spiegelman, S. (1961). Proc. a hybrid helix. In 1960, Rich carried hybridization of nucleic acids with Natl. Acad. Sci. USA 47, 137–146. out the first DNA-RNA hybridization, the use of restriction endonucleases, using short (chemically synthesized) gel electrophoresis, and the trans- Rich, A. (1960). Proc. Natl. Acad. Sci. USA 46, 1044–1053. polydeoxythymidylic acid and polyri- fer of separated DNA fragments to boadenylic acid, which he found to a solid support. This method and its Rich, A. (2004). Annu. Rev. Biochem. 73, make a hybrid double helix (Rich, later (massively parallel) incarnations, 1–37. 1960). DNA-RNA hybridization of this termed microarrays, continue to be Rich, A. (2006). J. Biol. Chem. 281, 7693– kind is extensively used today, for of major importance in biological 7696. example in procedures for the isola- research. In the 1980s, hybridization Rich, A., and Davies, D.R. (1956). J. Am. Chem. tion of messenger RNA molecules was at the core of the polymerase Soc. 78, 3548. through their poly A tails, which are chain reaction (PCR), which revolu- Rich, A., and Watson, J.D. (1954a). Nature hybridized to immobilized poly-dT. tionized molecular biological stud- 173, 995–996. The 1960 result reported by Rich ies by making it possible to amplify made it most likely that the mecha- nucleic acid sequences in vitro with Rich, A., and Watson, J.D. (1954b). Proc. Natl. Acad. Sci. USA 40, 759–764. nism of information transfer from DNA great facility and precision. In sum, to RNA involved the (at least transient) many key advances in molecular Rosenberg, J.M., Seeman, N.C., Kim, J.J.P., formation of DNA-RNA duplexes. biology have relied, directly or indi- Suddath, F.L., Nicholas, H.B., and Rich, A. (1973). Nature 243, 150–154. A crucial step in the further devel- rectly, on nucleic acid hybridization opment of hybridization involved as their methodological core. The Warner, R.C. (1956). Fed. Proc. 15, 379. the use of nucleic acids that were in situ hybridization in chromosome Watson, D., and Crick, F.H. (1953). Nature 171, not homopolymers and had specific spreads, the sequencing of the 738–740.