Overview

          This is the story behind the discovery of polynucleotide hybridization. In the early 1950s, the double helix structure of DNA had just been published, however, the structure of RNA was still unknown. Alexander Rich and his colleagues were investigating this question without much success until Rich combined polyadenylic acid and polyuridylic acid and, to his amazement, saw the diffraction pattern of a double helix. He realized that the base pairs had undergone hybridization to form a double stranded RNA structure. In the intervening 60 years, hybridization has become the foundation of much of modern biotechnology.

   

                                                Transcript

00:00:12.16 I'm Alexander Rich. I'm going to tell you about experiments
00:00:18.26 that occurred during the early years of molecular biology.
00:00:26.29 I was a postdoctoral fellow with Linus Pauling from
00:00:34.10 1949-1954, and during that period, at the end of it, he asked me
00:00:46.19 to take x-ray diffraction photographs of DNA. The method for
00:00:57.19 doing this is quite simple. DNA is usually a dry fibrous material,
00:01:08.03 you moisten it with water and put it on a glass slide. The
00:01:16.10 glass slide is put on a microscope bed and attached to the
00:01:24.18 objective lens, you have a small thin glass rod. And using
00:01:31.13 the drive of the microscope, you lower the glass rod
00:01:40.28 till it touches this little gel-like material, and then you slowly
00:01:48.13 withdraw it. And at the end, you're left with a glassy looking
00:01:53.04 fiber. Since DNA is very elongated, you hope to have the molecules
00:02:01.22 parallel to each other. And using x-rays, which are simply
00:02:09.26 short wavelength light waves, but with wavelengths in the range
00:02:17.28 of the distance between atoms. You get a diffraction pattern
00:02:25.08 and from that you can infer something about the structure
00:02:29.21 of the molecule. Rosalind Franklin working in England carried out
00:02:37.20 a very wonderful experiment in which she withdrew a fiber
00:02:43.29 which was highly oriented and played an important role in allowing
00:02:52.08 Watson and Crick to propose their double stranded model
00:02:56.26 of DNA, in which the two strands are coiled around each other
00:03:02.22 with the bases flat in the middle. The bases are what we call
00:03:09.21 complementary, that is adenine pairs with thymine, and guanine
00:03:18.06 pairs with cytosine. These bases stack on each other in the
00:03:23.21 center. When I started working, the Watson and Crick paper was
00:03:34.19 published. And I could immediately see that this was correct.
00:03:39.03 And I then asked myself the following question at a meeting at Caltech,
00:03:51.20 where all the DNA people were there. I asked myself, what about
00:04:00.26 RNA? In RNA, you have the same four bases, except the
00:04:08.15 thymine which pairs with adenine, is usually replaced by uracil.
00:04:15.17 But again, complementary. So after this meeting, I started
00:04:26.14 working with RNA samples. Withdrawing fibers to see if I could get
00:04:33.27 an oriented pattern. Jim Watson, who was at Caltech at the same time,
00:04:40.28 was interested in the problem, so we worked together on this.
00:04:45.09 And during a good part of 1954, we photographed a large number
00:04:56.05 of RNA preparations that we obtained from different people.
00:05:00.26 All of them were frustrating, they did not show evidence
00:05:07.14 of orientation. And so we could not say whether it could form a
00:05:13.25 double helix or not. At the end of 1954, I went to NIH to
00:05:26.27 set up my own lab and continued working on this problem.
00:05:32.16 Around that time, methods were made, enzymes were isolated
00:05:40.24 namely by Severo Ochoa and Marianne Grunberg-Manago, which
00:05:50.09 isolated an enzyme which would take ribonucleotides and polymerize
00:05:56.26 them into chains. So you could make polyriboadenylic acid
00:06:03.24 and polyribouridylic acid. I studied these compounds
00:06:16.08 together with David Davies, who would join me at NIH.
00:06:21.15 And we found they had very little organization within them.
00:06:29.21 But in early 1956, for reasons that were not very clear to me,
00:06:42.20 I decided to mix together polyriboadenylic acid and polyribouridylic
00:06:51.14 acid. Immediately I noted that the gel became rather cloudy
00:07:00.11 and much more viscous. And so I put the glass fiber in and
00:07:08.05 carefully withdrew a fiber of the nucleic acid. And put it in
00:07:20.19 the x-ray beam, and to my astonishment, there was the diffraction
00:07:25.28 pattern of the double helix. Not the DNA double helix, a slightly different
00:07:31.14 form. And we then carried out several analyses of this, and what we
00:07:42.08 concluded was that the molecules had found each other
00:07:50.26 and combined to hybridize. The word "hybridization" was
00:07:57.06 not used at that time. It was used later, but it was meant to
00:08:04.02 describe the binding together of complementary oligonucleotides.
00:08:12.23 Complementary either DNA or RNA. So, all of a sudden,
00:08:25.17 we wrote up a short article or letter to the Journal of the American
00:08:35.24 Chemical Society describing this remarkable event. That there were two
00:08:43.00 issues here, 1. Could RNA form a double helix? The answer
00:08:49.01 was definitively yes. 2. This remarkable phenomenon
00:08:56.25 of the hybridization, or spontaneous formation, of a double helix
00:09:05.15 from individual complementary chains. Many people were very skeptical
00:09:16.00 of that interpretation. Partly they said, look, these chains are very long,
00:09:27.10 they're likely to get entangled. How would they untangle and combine?
00:09:33.24 And these chains are all negatively charged, they have phosphate
00:09:41.25 groups in them. How could those negative charges come together?
00:09:46.21 And finally, 3. An argument concerning thermodynamics. In general,
00:10:02.07 things do not go from disordered to ordered. They go from
00:10:07.25 order to disorder. How could this occur? Well, what was not obvious
00:10:15.10 to everybody, but became obvious to me after studying it,
00:10:21.11 is that the people who made these arguments did not understand
00:10:27.05 the entropic, the randomization, associated with shedding
00:10:36.00 water molecules from these chains when they combined.
00:10:39.12 Now what drives the phenomenon? What drives the phenomenon
00:10:50.11 is the fact that these bases, adenine and uracil, or guanine and
00:11:01.05 cytosine, they are essentially lipid soluble, they like to live in oils
00:11:09.06 not in water, because of their flat ring surface. So what happens
00:11:15.20 is you bring these together, forming a molecule, a double stranded
00:11:22.04 molecule which is essentially oily in the middle. And that's a very strong
00:11:27.15 force for bringing them together. Around that time in 1956,
00:11:37.27 there was a meeting in Baltimore of the McCollum Pratt dealing with genetics.
00:11:50.22 And I presented this material. And at the end of my talk, a
00:11:56.21 middle-aged Englishman in a tweed suit came up to me
00:12:03.13 and said, "Professor Rich, I want to congratulate you. You've
00:12:08.10 discovered molecular sex." This was Julian Huxley, a
00:12:13.16 science writer with a very vivid imagination. And it was his way of
00:12:20.13 describing the specificity of hybridization. The hybridization
00:12:29.11 has become a major tool in the development of molecular
00:12:37.16 biology. For example, a well-known reaction called the polymerase
00:12:43.26 chain reaction, in which you put together a system and essentially
00:12:51.27 increase the amount of DNA starting from very little. This makes
00:12:58.02 possible all kinds of medical and biological experiments. It is
00:13:08.16 based on the combination of a single strand of DNA with a
00:13:16.04 complementary small primer of 20 nucleotides or so. That
00:13:23.06 combination occurs spontaneously because of hybridization.
00:13:28.19 And it allows the whole process to continue. It's very difficult
00:13:38.25 to underestimate the extent to which hybridization dominates
00:13:45.03 a great deal of the developments that have been at the basis
00:13:54.09 of molecular biology. They're used extensively and are used broadly.
00:14:02.03 And are still so today. Thank you.