Sickle Cell Research to the Present and the Future

Three-dimensional rendering of sickle cell anemia blood cells. Credit: National Institutes of Health.

By Dr. Marcus Calkins, Part 3 of 3

Forty years after Linus Pauling and his lab demonstrated the molecular basis for Sickle cell disease and James Watson speculated that upregulation of fetal hemoglobin may protect from the disease, methods to control fetal hemoglobin specifically in red blood cells began to be developed. The molecular biology revolution of the late 20^th century had produced extensive knowledge about the molecular systems that drive fetal hemoglobin production, but harnessing that intricate knowledge has taken another thirty years.

Hematopoietic Stem Cells (1990s)

Since the advent of radioactivity research, it has been well-established that red blood cells have a short lifespan of only about 115 days and are continually produced from precursors in the bone marrow. In order to replace defective blood cells in an individual, a protocol for whole-body irradiation and allogenic bone marrow transplant was pioneered by a group of doctors in Seattle in the 1970s, as a treatment for cancer patients.

However, the ability to specifically isolate and identify hematopoietic stem cells from patients was only developed in the 1990s. At that time, nuclear dye exclusion and flow cytometry characteristics were used to isolate the stem cells, but since then, a variety of cell surface markers have been identified, and protocols to isolate, expand and differentiate hematopoietic stem cells have become standardized. In addition, scientists have learned to modify the hematopoietic stem cells at a genetic level, creating the possibility that stem cells may be extracted, genetically modified ex vivo, and then used to reconstitute the bone marrow of patients with blood diseases.

For patients with Sickle cell disease, it may therefore be possible to extract hematopoietic stem cells and inactivate the BCL11A gene, which normally suppresses fetal hemoglobin. The red blood cell progeny of these altered stem cells would then produce fetal hemoglobin that could mask the effects of the disease-causing mutation in the β-globin gene. Afterward, the modified stem cells could be transplanted back into the same patients from which they were isolated, providing the person with a continual supply of red blood cells that expresses fetal hemoglobin and are resistant to sickling.

Gene Therapy and Genome Editors (2000s-2010s)

The final component of a therapy for Sickle cell disease has recently been realized, as it is now feasible to efficiently inactivate BCL11A in isolated hematopoietic stem cells. In the last two decades, several systems of modifying the genome (gene editors) have been developed. Although the first editors to be produced may still find clinical use, CRISPR has quickly overtaken previous technologies to become the most widely applied and well-known gene editing platform.

In the late 1990s, researchers invented two key methods of using proteins to make targeted edits to the genome. These early gene editor proteins are called TALENs and Zinc fingers, both of which are being tested in clinical trials today. Each of these editors is able to target highly specific DNA sequences and make an incision in the DNA helix at a predictable site. Once the DNA strand is incised, error-prone DNA repair processes are activated to fix the incision, often resulting in random base insertions, deletions and changes. In this way, the genetic code is disrupted in some cells, and these random disruptions often serve to inactivate the targeted gene. If those cells with an inactivated gene can be identified and expanded, whole populations of cells with the genetic alteration can be established.

The comparative difficulty of using TALENs and Zinc finger proteins instead of CRISPR is that targeting a particular site in the genome often requires a major technical effort. Since the proteins themselves target the DNA sequence of interest, each target sequence must have its own specialized editor. The introduction of CRISPR/Cas9 in the early 2010s allowed researchers to target various DNA sequences much more easily. This system uses a short guide RNA molecule for DNA targeting, so the same protein can be used to cut any genomic site. Since generating these guide RNAs is a relatively simple procedure, the amount of effort required to design and execute genome edits is greatly reduced.

Theoretically, TALENs, Zinc fingers and CRISPR could all be used to inactivate BCL11A in hematopoietic stem cells. However, design of an appropriate TALEN or Zinc finger might require relatively large investments of money and time.  On the other hand, CRISPR promises to be a more cost-effective and faster approach to editing the genome. In academic studies, CRISPR is already widely used and far more common than the other editing technologies for making genetic modifications to laboratory model organisms. However, with human patients, safety and efficacy greatly outweigh effort and cost. Time will tell which gene-editing platform proves to be most cost-effective, efficient and safest for clinical use.

A New Clinical Reality (2020s)

With these new tools at hand, Watson’s dream of increasing fetal hemoglobin in Sickle cell disease patients is finally within sight. At least two major collaborations to perform ex vivo gene therapy for Sickle cell disease have been initiated since 2018. Both use gene editors to inactivate the BCL11A gene and promote fetal hemoglobin expression in red blood cells.

One collaboration between Bioverativ and Sangamo is testing a protocol for gene editing with a Zinc finger. The estimated completion date for this trial is 2022. Another collaboration that has received a great deal of attention, and was recently published in the New England Journal of Medicine, involves CRISPR Therapeutics and Vertex Pharmaceuticals. This trial is among the first to attempt CRISPR in a clinical setting, and the results are highly anticipated by the research and medical communities, not only for their impact on Sickle cell disease, but also as a bellwether for the use of CRISPR in medical practice. So far the results of the trial are encouraging. As of December 2020, the first two patients to receive therapy were reportedly doing well and were free from symptoms more than one year after receiving the treatment. While this news is exciting, there is still much work to be done before the technique can be applied to a wider population.

It has taken many years and many twists, but the visions of the 1950s are finally beginning to be realized, bringing us to the cusp of an exciting new dawn in medicine. The slow march toward a cure for Sickle cell disease clearly demonstrates that through patience and continued investment in scientific discovery, we can continue to achieve the dreams of our predecessors and plant new seeds for future generations to reap the harvest.  

Sickle Cell Research in the Wake of Pauling and Watson

Harvey Itano, 1954. Image credit: Caltech Archives.

By Dr. Marcus Calkins, Part 2 of 3

The molecular defect in Sickle cell disease was demonstrated by Linus Pauling’s lab in 1949, and one year prior, James Watson had proposed that increasing the level of fetal hemoglobin may provide a means by which the disease could be cured. These two publications provided a direction for what may soon become a real-life cure for the disease. However, many practical questions needed to be answered before a cure could be realized, or even imagined in a realistic sense. The first of these questions had to do with the composition of hemoglobin protein.     

Defining the structure of hemoglobin (1950s-1960s)

The project in the Pauling lab was led by a formidable graduate student of Japanese descent named Harvey Itano. It is noteworthy that despite his birth in Sacramento and his honorific as valedictorian of the University of California, Berkeley class of 1942, Itano was interned at Tule Lake during World War II. He was only released from detention to attend medical school at Washington University in St. Louis. Upon graduating with his M.D., he came to Pauling’s lab to pursue a Ph.D. His widely celebrated doctoral thesis was built on the Sickle cell disease project. After finishing this training in biochemistry and publishing his high-impact paper with Pauling, Itano opened his own lab in which he continued working on hemoglobin.

By applying many of the same methods pioneered in Pauling’s lab, Itano’s small group became central players in identifying aberrant forms of hemoglobin that lead to disease. Because of this careful and detailed work, Itano and other scientists began to suspect that the protein subunits that make up hemoglobin may be derived from multiple, highly similar genes. At the time, this idea represented a major paradigm shift, as it was previously assumed that each enzyme should correspond to a single gene.

This idea also brought up an evolutionary question of how such similar genes might come to exist. In a memorial biography, Itano’s friend and colleague R.F. Doolittle summarized Itano’s forward thinking on the genetic and evolutionary basis of enzyme constituents as follows:

In his thorough review of all the data, Harvey noted that in sheep and cattle, however, there were two kinds of end group (valine and methionine), and concluded that there had to be two each of two kinds of polypeptide in human adult hemoglobins. He went on to discuss how gene duplications could account for all the various hemoglobin chains, including myoglobin and foetal hemoglobin. These were thoughts well ahead of their time.

These major scientific contributions and others led to Itano’s election to the National Academy of Sciences, U.S.A. He was the first of many Japanese Americans to achieve that honor.

The exact change in hemoglobin protein responsible for causing Sickle cell disease was identified at the amino acid level ten years after Itano’s paper with Pauling, when V.M. Ingram used protease digestion to show that the disease came from a glutamine-to-valine mutation in the β-globin subunit of the hemoglobin protein complex. Although the molecular structure of DNA was accurately modeled by Watson and Crick in 1954, revealing tantalizing clues as to how genetic information can be encoded and passed from cell to cell and parent to child, the precise genetic mutation underlying Sickle cell disease would remain unknown for a few more decades, until the molecular biology revolution.

Molecular Switch from Fetal to Adult Hemoglobin (1970s-1990s)

In order to institute the cure dreamed up by Watson, much needed to be learned about hemoglobin composition, genetics, and control in red blood cells. This basic knowledge was largely generated through new technologies developed in the latter part of the 20^th century. The cell and molecular biology revolution began in the mid-1970s and brought with it new techniques for manipulating DNA and other molecules. These advances allowed scientists to define genes and genetic structures, and to begin to understand how gene expression is regulated.

With regard to hemoglobin, it was discovered that two sets of genes (five on Chromosome 11 and three on Chromosome 16) encode the subunit proteins for the hemoglobin molecular complex. Each set contributes one subunit to the complex, and the subunits that are present in the blood change, depending on the developmental stage of the individual. In essence, a switch occurs just after the time of birth, wherein expression of fetal hemoglobin is turned off and adult hemoglobin is turned on. The mechanics of this switch, including the DNA sequences and proteins that make it work, were elucidated during this fruitful period of biological research.

Once the mechanics of the fetal hemoglobin to adult hemoglobin molecular switch were known, it became apparent that one gene, BCL11A, is a dominant factor in shutting down fetal hemoglobin production. If this gene can be silenced or disrupted, fetal hemoglobin expression will continue. Furthermore, there is strong evidence from molecular, cellular and clinical studies that continued expression of fetal hemoglobin will at least partially prevent the harmful accumulation of mutant hemoglobin aggregates and thereby prevent Sickle cell disease.

However, turning off the BCL11A gene is not an easy task. The molecular system evolved over millions of years to function robustly under almost any environmental condition or situation. Like a train hurtling down a track, such developmental programs are extremely hard to redirect, and if they are completely derailed, the individual may experience catastrophic effects. Thus, precise control of only the BCL11A gene, precisely in red blood cells, is necessary to realize the therapy first imagined by Watson. This level of control is possible to achieve in cells outside the body, but inside the body, cells are resistant to change, and our ability to target only one cell type at a time remains relatively primitive. Thus, a cure for Sickle cell disease still needed a method for regulating BCL11A specifically in red blood cells.

The Slow March Toward a Cure for Sickle Cell Disease

Pastel drawing of sickled hemoglobin cells by Roger Hayward, 1964

By Dr. Marcus Calkins

[Ed Note: This is the first of three posts examining the history of sickle cell treatment up to present day. It is authored by Marcus J. Calkins, Ph.D., “a proud OSU alumnus” (Chemical Engineering, B.S., 1999), who now works as a scientific communications service provider and educator in Taipei, Taiwan. In submitting this piece, Calkins emphasized that he has “taken inspiration from Linus Pauling’s research activities, teaching methods and moral character for many years.”]

In 2020, Jennifer Doudna and Emmanuelle Charpentier shared a Nobel Prize for their discovery and development of the CRISPR gene editor. One of the first clinical applications for CRISPR promises to be an ex vivo gene therapy for Sickle cell anemia. If it works, this medical technology will be a major breakthrough in biomedicine, representing the culmination of more than a century of research on Sickle cell disease that encompasses a wide range of topics.

Despite the lifetimes of work that have led to our current exciting position on the precipice of a cure for Sickle cell disease, the basic molecular features of the disease were defined seven decades ago by another Nobel Prize winner, Linus Pauling. The intervening 70 years of work have been required for scientists to learn how we might apply the foundational knowledge to actual patients in a real-life clinical setting. While the pace of progress may seem agonizingly slow to those outside biomedical research, the ground that has been covered is immense, and entire fields of biomedicine needed to be built and optimized before a truly feasible treatment technology could be invented.

Sickle Cell Disease (1910)

Sickle cell disease was first described over the period from 1910 to about 1924. During this time, a series of case reports detailed approximately 80 people of African descent, who had an odd morphology of red blood cells resembling a crescent or a sickle. In many cases, this sickle-like morphology was associated with a devastating condition involving severe anemia and early death. Furthermore, scientists learned that the red blood cell sickling could be exacerbated by depriving the blood of oxygen, either by adding carbon dioxide to cells in a dish or restricting blood flow in the patient. These clinical observations laid the foundation for basic scientists to postulate that the condition was related to hemoglobin, the protein that carries oxygen in red blood cells.

The first person to make this suggestion was Pauling. At some time in 1945, he was chatting with a colleague on the train from Denver to Chicago, when he learned about the difference in sickling between oxygenated and deoxygenated blood. According to the account of his colleague, Pauling was also informed that the sickled red blood cells show birefringence when viewed under a polarizing microscope, which would suggest an alignment of molecules within the cells.

However, by Pauling’s account of the conversation, his immediate guess that Sickle cell disease is caused by a defect in the hemoglobin protein complex was based entirely on the difference in the sickling properties of oxygenated and deoxygenated blood. Notably, Pauling later stated that the idea of Sickle cell disease being singularly caused by the hemoglobin molecule came to him in “two seconds,” but gathering evidence and refinement of the idea took at least three years.

In his public talks, Pauling often emphasized the fact that in the first years of the study, his students performed many experiments but could not identify any obvious biochemical differences between the hemoglobin molecules of patients and control individuals. From his repeated emphasis of this fact, one might speculate that the translation of a two-second idea to a three- or four-year demonstration would have been frustrating for such a quick-minded individual, though Pauling never said as much. Alternatively, he may have simply been emphasizing the challenges and slow, steady nature of rigorous scientific pursuit.

The Molecular Defect and a Potential Cure (1949)

In 1949, prior to the double helix model of DNA and before stem cells were described, the Pauling lab published a paper titled “Sickle Cell Anemia, a Molecular Disease.” In this work, Pauling and his students definitively showed that a slightly abnormal form of hemoglobin is found exclusively in patients with the cell sickling phenotype. Using a 30-foot-long Tiselius apparatus that they had constructed for electrophoresis, a small two-electron difference could be detected in the overall charge of hemoglobin molecules from Sickle cell patients and unaffected individuals. Meanwhile, carriers of the disease had a mixture of the two hemoglobin isoforms.

Importantly, Pauling’s group found that the defect in hemoglobin is not related with its ability to bind oxygen. Instead, it was later shown that the slight change in molecular charge affects the way hemoglobin proteins interact with each other, as would be predicted from the birefringence observation. This aberrant interaction causes the formation of long molecular scaffolds that change the shape of the red blood cell and lead to its dysfunction.

With this publication, Sickle cell disease became the first disorder to be associated with a single molecule. It was also the first with a known genetic basis. In his publication of the same year, J.V. Neel showed that Sickle cell disease follows an autosomal recessive inheritance pattern, meaning that each parent must contribute one copy of the mutated gene for a child to develop the disease. The cell sickling phenotype can occur to some degree in people who only carry one mutant allele, but only those with two copies experience the pernicious effects of the disease. This information, combined with Pauling’s study, established the essential basis for our understanding of Sickle cell disease and serves as a model for many other genetic diseases.

Surprisingly, James Watson (prior to his famed work on the structure of DNA) contributed a prescient idea to Sickle cell disease treatment, when he speculated that cells could be protected by expression of another form of hemoglobin, fetal hemoglobin. Watson made this prediction in 1948, just one year before Pauling’s powerhouse publication. His suspicion was an extension of reports that red blood cell sickling did not happen in the blood of infants who would later develop the condition as children and adults.

The stage was thus set for a Sickle cell disease cure. After the theoretical basis was determined, onlookers might have expected a cure for the disease to be found within a few years. However, extension of the ideas of Pauling and Watson has required incredible efforts by myriad scientists over the course of the next seven decades to create a potential new clinical reality.

Peter Pauling: Epilogue

Peter Pauling with his dad, 1931.

[Part 9 of 9]

Before he passed away in 2003, Peter Pauling saw his daughter Sarah marry, and also witnessed the births of two grandsons, Isaac and Malachi. Over time, he likewise learned to recognize the ebb and flow of his manic and depressive phases, at points struggling to overcome insomnia and drinking too much whiskey or beer, and at others walking the country paths around the mill so giddy with delight, that he felt he could not contain his joy.

In 1992, Jim Watson came to Wales to call on Peter and Alicia. Peter had recently seen a BBC drama depicting the discovery of DNA which, as he explained to his old friend, was not entirely accurate. When Watson asked what they had got wrong, Peter answered firmly that he had appeared only at the very end of the program, and that he showed up on screen driving a white Cadillac convertible. For a car man like Peter, being portrayed in such a vehicle was, apparently, an insult to his sense of personal pride.

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Though thousands of miles apart, Peter remained in regular contact with his father. In 1992, Linus called on his son to ask his advice about what he should do with a collection of secret documents stemming from his years of involvement in the American war effort. As he looked through his files, Linus Pauling had been unable to track down an apparently nonexistent Navy patent for a substance, named “Linusite,” that he helped to develop in secrecy in 1945. Similarly, he noted, his invention of the oxygen meter had presumably remained classified, as was a cone shell windmill that he designed in 1952.

Indeed, Linus had a personal safe full of records relating to such top secret projects, and he had no idea which of them had been declassified. Now, at the age of 91, he wanted to unburden himself of these materials, one way or another. Wishing to help his dad out, Peter called a close friend of his from his undergraduate years at Caltech, Robert Madden, who was then working in the National Security Administration. The elder Pauling’s safe was subsequently inspected, and select material duly vanished into the hands of the federal government.

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Linus and Peter Pauling in England at a model of Bourton-on-the-water, 1948.

A year later, the conversation had turned toward the introspective. As his cancer spread and his health continued to diminish, Pauling lamented to his son that he had never much been there for him during Peter’s childhood; had never thrown the baseball around the yard. His son responded in stark contrast, stating that he looked back on his childhood at Arden Road and Fairpoint Street with great fondness, adding

Well, you did not play much baseball, but then neither did I. You did, however, lie on the side of my bed and taught me how to count in French. Later, when I was old enough to get out and about, you were often out to rescue me, either because I telephoned or Mamma was worried and sent you out to do a general search of the whole of Pasadena and surrounding environs.

In 1994, Alicia sent a letter to Linus on his birthday, saying that she and Peter were thinking of him, and about to toast him, as dinner time was drawing near. The drinking, she hastened to add, would be kept moderate, but the thinking had no limits. She concluded by writing that “Peter hopes to come over shortly – and so do I.”

Less that six months later, Linus Pauling passed away. Peter’s younger brother Crellin wrote to him after their father’s death, and he was heavy with grief. Peter, though, had been experiencing both the depths of depression and the heights of elation for decades. The lesson in all of this, he confided to Crellin, was that when one’s mania had faded, and the depression has set in, one had only to hold on. Be patient and outlast it, for eventually change will come. In this, an entire lifetime of often difficult experience was summed up by Peter Pauling in three simple words:

“Do not despair.”

Peter Pauling and the Discovery of the Double Helix, 1952-1953

Peter Pauling, 1954.

[The life of Peter Pauling: Part 4 of 9]

With Winter break coming fast and Linus Pauling having apparently solved the structure of DNA, Jim Watson and Francis Crick extinguished any hope of modeling their own structure. Eager to take advantage of a few days off, their Cavendish office mate, Peter Pauling, headed for the continent in the company of a friend whom he described as “a mad Rhodes scholar” who had “wooed” him with his “insane plan” for exploring Europe.

On this trip, which was indeed ambitious, Peter visited Munich, Vienna, Linz, Brussels, Frankfurt, and Bavaria, hitchhiking his way from location to location. Crossing Germany, Peter saw neighborhoods still littered with the rubble of the Second World War, alongside industrious people struggling to rebuild. His mode of travel, he confessed to his mother in a letter, had seemed a better idea when its low cost was his only consideration. In person, however, spending several hours standing in or walking through the snow had a way of changing one’s priorities.

Nonetheless, the whole escapade proved a romantic adventure for the young Peter Pauling. He spent Christmas Eve in a gas house belonging to the director of an iron company somewhere in Leoben, Austria. Resting there and watching the snow fall, he wrote again to his mother:

I look out the window to the lovely white mountains. It is grand. Considering the possibilities, Christmas and your birthday [Ava Helen was born on December 24, 1903] could hardly have been spent in a nicer place. Considering impossibilities, I can think of places where I would much prefer to be. Sometimes it is sad to grow up.

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[Triple Helix animation and narration created by Cold Springs Harbor Laboratory]

With the arrival of the new year, the Cavendish researchers put their skis away, shook the snow from their coats, and resumed their work.  It wasn’t long into the term before Peter learned, from two letters received in February, that his father was, in fact, having difficulty with some of the van der Waals distances hypothesized to be near the center of his DNA model. In response – and almost as an afterthought – Peter casually asked his father for a manuscript of the DNA proposal, mentioning that his coworkers in Max Perutz’ unit would like to give it a read. Upon receiving the paper, Peter promptly revealed to Watson and Crick that the Pauling-Corey model was a triple helix, a concept similar to one that Watson and Crick had developed themselves – and rejected – back in 1951.

This moment was a major turning point for Watson and Crick, who only then realized that they still had a chance to discover the structure before Linus Pauling. That said, what followed may not have been quite the race as it was made out to be after the fact. At least, Peter Pauling did not see it that way, and the casual manner in which his father interacted with him (and with others at the Cavendish) seems also to belie such a dramatization.

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[Jim Watson recalls learning of the Pauling-Corey triple helical model. Video created by Cold Springs Harbor Laboratory.]

Near the end of February 1953, while wishing his father a happy birthday, Peter noted that his office still felt that Linus’ structure required sodium to be located somewhere near the oxygens, whose negative charges would have to cancel out to hold the molecule together. “We agree that everything is a little tight,” he said, referring to the small atomic distances between Pauling’s three polynucleotide chains with phosphate groups in the middle.

As communicated in an earlier letter to his son, Linus Pauling had already identified these structural arrangements as a weakness of the model, and he was in the midst of attempting to correct the issue. Peter confided to his father that, at that time, the Cambridge office had nothing better to offer. He added simply that “We were all excited about the nucleic acid structure,” and concluded with his many thanks for the paper.

In response, Linus Pauling asked for updates on any progress that Watson and Crick were making with their own model, and casually requested that Peter also remind Watson that he should arrive for a scheduled protein conference at Caltech by September 20^th. Peter clarified only that the Cavendish group had successfully built the Pauling-Corey model and that Watson and Crick had then discarded it, becoming very involved in their own efforts and “losing objectivity.” It would be up to them, Peter said, to communicate the details of their structure. Shortly thereafter, Watson and Crick sent a letter to Linus Pauling, outlining their structure and including the short article that they had submitted for publication in Nature.

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Crystallographic photo of Sodium Thymonucleate, Type B. “Photo 51.” Taken by Rosalind Franklin, May 1952.

It has been well-established that Pauling and Corey made basic errors in their own modeling  of the structure of DNA. But in March 1953, having no knowledge of the x-ray crystallographic photographs of DNA that had been taken by Rosalind Franklin at Kings College, Pauling felt bewildered by the certainty with which Watson and Crick had rejected his triple helical model. Upon learning its details, Pauling agreed that the double helix model was at least as likely, and he considered it to be a beautiful molecular structure, but he could not understand why his own structure was being ruled out entirely.

At the heart of his confusion lay the fact that he did not believe that any x-ray evidence existed that proved that the phosphate groups might somehow be located on the outside, rather than in the core, of the DNA molecule. Pauling did not believe that this evidence existed because he hadn’t seen it yet; crucially, Watson and Crick had. Indeed, from the point of their realization that Pauling had modeled the structure incorrectly, Watson and Crick worked fervently to once again convince Maurice Wilkins to provide them with Rosalind Franklin’s data.

(On one occasion, they met with Wilkins for lunch at the Crick home, where Peter could often be found for brunch on the weekends. On certain of these earlier brunch occasions, while in the home’s basement dining room, Watson and Crick discussed the feasibility of redoubling their efforts to model DNA while Peter, casually eating biscuits and sipping tea at the table, offered that if they didn’t do it soon, his father would take another shot at it. After the embarrassment of a failed attempt, he assured them, Linus Pauling was a strong bet to get it right the second time around.)

Within a month’s time, and with Rosalind Franklin having left his lab, Wilkins finally consented to providing Watson and Crick with all of the relevant data that he had requested. This proved to be the final piece that the duo needed in building their correct structural model of DNA.

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Pauling en route to Europe, 1953.

While all of this went on, Linus himself was seemingly unconcerned by any “race” for the structure of DNA. In fact, the only racing on his mind was a jaunt across Western Europe in a new sports car.

While Watson and Crick frantically worked to unravel the secrets of DNA before Linus Pauling beat them to it, Linus Pauling himself was debating the virtues of British, German, and Italian motor vehicles. Preparing for multiple trips overseas and in the market for some new wheels, Pauling’s plan was to select a car while in Europe during the Spring for the Solvay Conference, and then to actually pick it up in August, when he and Ava Helen would return to Europe for the International Congress of Pure and Applied Chemistry in Stockholm and Uppsala. The couple would then tour the continent in style before returning to the United States on a Scandinavian freighter and driving across the country from either New York or New Orleans to their California home.

While Peter advised his father that a Jaguar Mark VII was absolutely the best buy of the season, Linus expressed a preference for the slightly more modest convertible Sunbeam-Talbot. Peter countered with the possibility of an Austin A-40 Sports 4-Seater, and Linus finally agreed to have Peter look into purchasing the car on his behalf and scheduling a delivery of sorts. Seeing that his father was finally taking the bait, Peter attempted to spring a trap: “Might you be in need of a chauffeur, mechanic, linguist, travelling companion, navigator, break repairer, tire changer, witty conversationalist etc. on your trip next summer?” he wondered. “I know just the fellow. Good friend of mine.”

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A segment of the original Watson and Crick DNA model. 1953.

As the end of March rolled around and the Solvay Conference approached, Linus Pauling alerted his son to the fact that he had not made hotel reservations or, really, any plans for his visit to Cambridge. This responsibility he delegated wholly to Peter, who was somewhat distracted at the time, writing to his father about the blue sky and sun that had finally begun to break up the English winter gloom, and announcing with pride that he had gone to two balls in one week, getting along quite well with the Scandinavian girls. “As a sensible young American, I stand out in this town of pansy Englishmen,” he declared with impunity.

When Linus finally arrived at Cambridge in April, however, he found his son’s sensibilities to be somewhat lacking. Peter had in fact not made the requested hotel reservations, and while campus accommodations were fine for the son, they were not so wonderful for the elder Pauling. Watson later joked that, “the presence of foreign girls at breakfast did not compensate for the lack of hot water in his room.”

When the moment of truth finally came, Peter and his father strode into the Cavendish offices to see the model that Watson and Crick had constructed. Upon inspection, Linus reiterated the interpretation that he had given to his son earlier: the structure was certainly possible, but to be certain, Pauling would first need to see the quantitative measurements that Wilkins had provided. By way of response, Watson and Crick produced “Photo 51,” Rosalind Franklin’s now-famous image that enabled crucial measurements concerning the structure of the B-form of DNA.

Presented with this evidence, Linus Pauling quickly conceded that Watson and Crick had solved the problem. Later that night, the Paulings, together with Watson, had dinner with the Cricks at their home at Portugal Place to celebrate. To quote Watson, each “drank their share of burgundy.”

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So was it a race? And if so, what was Peter Pauling’s role? Was he a double agent or an informant? Or merely an unwitting accomplice, ignorant of the full implications of his actions?

In trying to answer these questions, it is important to emphasize that, for Peter, the “race for DNA” had never been a race at all. His father, he believed, was only interested in the nucleic acids as an interesting chemical compound. Linus Pauling clearly didn’t attack the structure with the same tenacity as Watson, in particular, who regarded the genetic material as the holy grail of biology, the secret of life. As Peter would write two decades later in New Scientist 

The only person who could conceivably have been racing was Jim Watson. Maurice Wilkins has never raced anyone anywhere. Francis Crick likes to pitch his brains against difficult problems… For Jim, however…the gene was the only thing in life worth bothering about and the structure of DNA was the only real problem worth solving.

In 1966, Jim Watson, then in the process of writing his book on the discovery of DNA, The Double Helix, sent Peter Pauling an early draft. His concern, he explained, was that he accurately portray Peter’s role in the entire affair; that, and he didn’t want Peter to sue him for defamation.

Peter laughed and told his old office mate that he thought it was a very good book; certainly very exciting. However, he pointed out that Watson should ask Linus Pauling for an agreement not to sue him, too. After all, Peter said, “He has more experience than I do.”

Peter Pauling: The Race that Wasn’t, 1952

Peter Pauling with his parents, ca. 1950s.

[The life story of Peter Pauling. Part 3 of 9]

“This tub moves steadily but slowly along.” So wrote Peter Pauling in a letter to his mother, Ava Helen Pauling, riding somewhere in the Atlantic in the hull of a cargo ship that had been built in 1926. “It took us two and a half days to reach the open sea.”

Having said goodbye to the nightlife of Montreal, and having entrusted his brother Crellin with the needle to his old turntable, Peter took to the sea without much to his name save a bottle of duty free Canadian Rye Whiskey; which, he lamented, did not keep him as warm onboard the cold ship as a good overcoat might have done. (Ava Helen, ever concerned for her son’s well-being, would see to it that he would have money to pick up some warmer clothes once he had arrived in Cambridge, paid for in matured war bonds.) Onboard the ship, Peter shared his cramped cabin space with three roommates: a Scot, a “very pleasant and hard-working” Englishman, and an 18 year old “pipsqueak” just out of rugby. Ever the charismatic socialite, Peter must have been excited to spend his days at sea with such an assortment of characters.

Arriving in England in the fall of 1952, Peter began his studies at Cambridge University, working under John Kendrew, a Peterhouse Fellow in Max Perutz’ Molecular Biology Unit at the Cavendish laboratory for physics. Although the Cavendish traditionally had not extended its focus beyond physics and physical chemistry to questions of biology, Sir Lawrence Bragg – director of the Cavendish and chair of the university’s Physics department – had recently supported an expansion of the lab’s scope to include the mapping of biological molecular structures.

This new Molecular Biology Unit would spearhead several important discoveries, among them Kendrew’s and Perutz’ work on the atomic structure of proteins, the program of research that Peter was brought on to support and an accomplishment significant enough to garner the 1962 Nobel Prize in Chemistry. That same year, two other former Cavendish researchers – James Watson and Francis Crick – would receive their shared Nobel Prize in Physiology or Medicine for their discovery of the double helical structure of DNA, a breakthrough that Peter Pauling certainly observed from a front row seat, and even, perhaps, helped to make possible.

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Francis Crick and James Watson, walking along the the Backs, Cambridge, England. 1953. (Image Credit: The James D. Watson Collection, Cold Springs Harbor Laboratory Archives.)

When Peter Pauling first moved into the office that he shared with James Watson, Francis Crick, and Jerry Donahue, Watson noted that Peter was “more interested in the structure of Nina, Perutz’s Danish au pair girl, than in the structure of myoglobin.” Crick, too, felt that the young Pauling was “slightly wild,” but still the office mates hit it off immediately. According to Watson, Peter’s presence meant that, “whenever more science was pointless, the conversation could dwell on the comparative virtues of girls from England, the Continent, and California.” Watson and the young Pauling even made a point of visiting The Rex art house cinema together to watch the 1933 romantic film Ecstasy, which Watson referred to affectionately as, “Hedy Lemarr’s romps in the nude.”

Women aside, Peter was most concerned by the day-to-day troubles that were typical of English life in the early 1950s. He wrote to his mother about the lack of a bathtub in the small, cold, damp room that he now inhabited, and complained about the space’s perpetual lack of sunlight. He did praise his fortune at having scoured London and finding a suitable teapot, and he requested that Ava Helen kindly make him a pair of curtains for his window (which she happily obliged).

In letters to his father, Peter preferred to talk about cars, or his recent dinners with the Braggs and their daughter Margaret, rather than his own research pursuits. Linus, on the other hand, was immediately curious about the intellectual climate at the Cavendish and was especially interested in the work of Francis Crick, who a year earlier had been part of a collaborative effort to develop a theory of mathematical representation for x-ray diffraction that was fast becoming a standard in the field.

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Linus Pauling and Robert Corey examining models of protein structure molecules. approx. 1951. (Image credit: The Archives, California Institute of Technology)

The previous year, 1951, Linus Pauling had bested Bragg and the physical chemists at Cambridge in becoming the first to publish the alpha helical structure of many proteins. Despite the desire prevailing at the Cavendish to eventually beat Linus Pauling at his own game, Watson and Crick had been warned to keep away from the study of DNA by the head of the lab. Bragg knew that Maurice Wilkins and Rosalind Franklin, of King’s College London, were already working on the problem using Franklin’s photos and crystallographic calculations of the A and B forms (low and high hydration levels, respectively) of DNA.

Wilkins’ and Franklin’s work was proceeding slowly, however, and Peter Pauling and Jerry Donahue – another Caltech graduate now stationed overseas as a post-doc – were both in regular communication with Linus Pauling. These contacts provided Watson and Crick with insight into what was going on in Pasadena. In his correspondence, Peter joked about the mounting competition between Caltech and the researchers at the Cavendish and King’s College. “I was told a story today,” he said to his father. “You know how children are threatened ‘You had better be good or the bad ogre will come get you?’ Well, for more than a year, Francis and others have been saying to the nucleic acid people at King’s, ‘You had better work hard or Pauling will get interested in nucleic acids.'”

While Watson and Crick urged Wilkins to provide them with Franklin’s images and calculations so that they might model the structure themselves, Peter stoked the fires of their urgency, assuring them that his father was no doubt only moments away from solving the problem. Donahue was equally convinced: for him, Linus Pauling was the only scientist likely to produce the right structure.

By December, the fate that Jerry Donahue and Peter Pauling had been predicting seemed to come true: a letter from Linus to his son claimed that he had indeed determined the structure of DNA. The letter gave no details, simply confirming for Watson and Crick that Pauling and his Caltech partner Robert Corey had somehow solved the problem. Watson later recounted his colleague’s distress in hearing this news, recalling that Crick “began pacing up and down the room thinking aloud, hoping that in a great intellectual fervor he could reconstruct what Linus might have done.” But it seemed to be too late. Pauling’s DNA paper was set to appear in the February 1953 issue of Proceedings of the National Academy of Sciences. In all likelihood, it would be time to move on to new projects.

Alexander Rich, 1924-2015

Alexander Rich. Photo by Donna Coveney.

Today we remember Dr. Alexander Rich, a student and colleague of Linus Pauling who passed away in April at the age of 90. Rich and Pauling were among the group of scientists who embarked on one of the most exciting scientific quests of the 20th century – the so-called “race for DNA.” Rich’s friends and colleagues also remember him for his endless desire to know more about the processes propelling life, a trait that is evident in his career as a biochemist. According to Pauling, this holistic interest in and understanding of science allowed Rich to make invaluable contributions to multiple disciplines.

Nucleic acids – the carriers of genetic information within a cell’s nucleus – were first identified in 1868 when Friedrich Miescher isolated the DNA compound for the first time. For some eighty-five years, however, the structure of DNA remained undescribed. In the 1940s and 1950s, scientists around the world began to focus more on the problem, working to build an accurate model of the DNA molecule in hopes of fully understanding its role in the process of gene expression.

In 1953, using Rosalind Franklin’s experimental data, James Watson and Francis Crick published their proposal of a double helical structure for the DNA molecule, and quickly became scientific celebrities once their model was deemed correct. Like Rosalind Franklin and, indeed, Linus Pauling, Alexander Rich was among the many researchers whose work and contributions to the understanding of proteins and nucleic acids abetted Watson and Crick’s discovery of the DNA molecule’s structure.

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Born in Hartford, Connecticut in 1924, Alexander Rich served in the U.S. Navy during World War II, then went on to Harvard University, where he received a bachelor’s degree in biochemical sciences in 1947 and graduated from Harvard Medical School in 1949. Soon after receiving his medical degree, he moved to Pasadena, where he worked as a research fellow in Linus Pauling’s lab at the California Institute of Technology, and where he lived with future Nobel laureate Martin Karplus, a fellow student of Pauling’s.

Blessed with a nimble mind, Rich was able to jump back and forth between chemistry and biology as his research interests progressed, all the while paying close attention to the broader implications of his research for the field of medicine. Rich became particularly well-known for his work on the structure and chemistry of fiber compounds, research which quickly became useful to the study of nucleic acids. By isolating strands of nucleic acids within fibrous compounds, Rich was able to produce images of their structure.

Though his pictures were not as clear or impactful as those captured by Rosalind Franklin, many have since posited that his work could have been of equal significance to Franklin’s had Caltech housed more fine-focus x-ray equipment.  Regardless, Rich was held in high esteem by Watson and Crick who, before publishing their DNA structure, asked that Rich review their work and corroborate their ideas.

Collagen model built by Alexander Rich and Francis Crick. September 1955.

In the wake of Watson and Crick’s triumph, the structure of nucleic acids continued to intrigue Rich. This time around however, it was RNA that caught his attention. Like DNA, RNA carries genetic material and is vital to the formation of proteins. It is thus necessary to understand the structure and function of RNA to fully comprehend DNA’s role in protein formation.

Rich began research in this area during James Watson’s brief stay at Caltech, and some now speculate that Rich’s interest in RNA images led Watson to focus entirely on RNA. While in Pasadena, Rich and Watson collected different images of RNA in an attempt to understand its physical structure, but the x-ray crystallographic photographs available at the time were not sufficient enough to discern a conclusive model.

Rich’s stint at Caltech came to an end in 1954 and he subsequently moved into his own laboratory at the National Institute of Mental Health (NIMH). While there he continued to delve into questions regarding the structure and composition of RNA. At the NIMH Rich was, at long last, successful in creating an image of RNA that provided hints about its structure. Rich concluded that RNA consists of a single-stranded nucleic acid that binds with complementary strands of RNA to form a temporary double helix – a process he described as molecular hybridization. Many were skeptical that a single-stranded nucleic acid could temporarily form a double helix, but Rich was able to show that this is made possible by the shedding of water molecules that comes about when the two strands bind.

Not only did this finding contribute enormously to the understanding of RNA’s structure and function, but Rich’s contributions to the understanding of molecular hybridization in nucleic acids has opened up many more possibilities. For example, polymerase chain reaction, a process used to identify genes, is based on the principle of hybridization. Today, methods of this sort are fundamental to all sorts of work in biotechnology and to the analysis of DNA.

Alexander Rich with Linus Pauling, among others, at a scientific meeting in the Soviet Union. Image Source: Alexander Rich Collection.

Following his tenure at the NIMH, Rich became a professor of Biophysics at the Massachusetts Institute of Technology, beginning in 1958 and lasting until his death. His investigations there included the discovery of Z-DNA, which is a type of DNA molecule that takes a zigzag form and follows a left-handed wind rather than the more common right-handed wind. His work at MIT also showed that protein synthesis occurs in a polysome – the name given to a cluster of Ribosomes that work together.

Alexander Rich received high honors for his contributions, including election to the National Academy of Sciences and receipt of the 1995 National Medal of Science – the highest scientific honor bestowed by the U.S. government.  It is no wonder then that Linus Pauling recalled his former pupil with great pride. “Of the several men with MD degrees who have worked with me,” he once noted, “I think that Dr. Rich may well be the one with the broadest grasp of science as a whole.”

Linus Pauling and the Structure of Proteins: A Documentary History

Today is Linus Pauling’s birthday – he would have been 112 years old.  Every year on February 28th we try to do something special and this time around we’re pleased to announce a project about which we’re all very excited: the sixth in our series of Pauling documentary history websites.

Launched today, Linus Pauling and the Structure of Proteins is the both latest in the documentary history series and our first since 2010’s The Scientific War Work of Linus C. Pauling. (we’ve been a little busy these past few years)  Like Pauling’s program of proteins research, the new website is sprawling and multi-faceted.  It features well over 200 letters and manuscripts, as well as the usual array of photographs, papers, audio and video that users of our sites have come to expect.  A total of more than 400 primary source materials illustrate and provide depth to the site’s 45-page Narrative, which was written by Pauling biographer Thomas Hager.

Warren Weaver, 1967.

That narrative tells a remarkable story that was central to many of the twentieth century’s great breakthroughs in molecular biology.  Readers will, for example, learn much of Pauling’s many interactions with Warren Weaver and the Rockefeller Foundation, the organization whose interest in the “science of life” helped prompt Pauling away from his early successes on the structure of crystals in favor of investigations into biological topics.

So too will users learn about Pauling’s sometimes caustic confrontations with Dorothy Wrinch, whose cyclol theory of protein structure was a source of intense objection for Pauling and his colleague, Carl Niemann.  Speaking of colleagues, the website also delves into the fruitful collaboration enjoyed between Pauling and his Caltech co-worker, Robert Corey.  The controversy surrounding Pauling’s interactions with another associate, Herman Branson, are also explored on the proteins website.

Linus Pauling shaking hands with Peter Lehman in front of two models of the alpha-helix. 1950s.

Much is known about Pauling’s famously lost “race for DNA,” contested with Jim Watson, Francis Crick and a handful of others in the UK.  Less storied is the long running competition between Pauling’s laboratory and an array of British proteins researchers, waged several years before Watson and Crick’s breakthrough.  That triumph, the double helix, was inspired by Pauling’s alpha helix, discovered one day when Linus lay sick in bed, bored and restless as he fought off a cold. (This was before the vitamin C days, of course.)

Illustration of the antibody-antigen framework, 1948.

Many more discoveries lie in waiting for those interested in the history of molecular biology: the invention of the ultracentrifuge by The Svedberg; Pauling’s long dalliance with a theory of antibodies; his hugely important concept of biological specificity; and the contested notion of coiled-coils, an episode that once again pit Pauling versus Francis Crick.

Linus Pauling and the Structure of Proteins constitutes a major addition to the Pauling canon. It is an enormously rich resource that will suit the needs of many types of researchers, students and educators. It is, in short, a fitting birthday present for history’s only recipient of two unshared Nobel Prizes.

Happy birthday, Dr. Pauling!

The Triple Helix

“We have seen Paulings paper on Nucleic Acid. Have you? It contains several very bad mistakes. In addition, we suspect he has chosen the wrong type of model.”

-James Watson, letter to Max Delbrück, February 20, 1953.

We were very interested to see that a model of the Pauling-Corey “triple helix” structure of DNA has been built by Farooq Hussain.  As Hussain notes on his website, the model was constructed based on drawings published by Linus Pauling and Robert Corey in their paper detailing the incorrect structure.

The proposed triple helix structure of DNA. Model by Farooq Hussain.

Hussain’s representation of the triple helix is striking; especially so when compared with Watson and Crick’s far more elegant and intuitive double helix, surely the most famous molecule in history.

Double helix model, courtesy of P. Shing Ho.

Indeed, Watson was well within his right to feel confident in his February letter to Delbrück.  As he noted before closing, “Today I am very optimistic since I believe I have a very pretty model, which is so pretty I am surprised no one has thought of it before.”

For more on the triple helix, see our write-up on the subject, published in April 2009.

DNA: The Aftermath

Pastel depiction of the DNA base pairs by Roger Hayward.

The solving of the double helix structure of DNA is now considered to be one of the most important discoveries in modern scientific history. The structure itself suggested a possible mechanism for its own replication, and it also opened up a huge window of opportunity for advances in multiple fields ranging from biology to genetics to biochemistry to medicine. Almost immediately after James Watson and Francis Crick announced their structure, new research began based on the structure’s specifications.

An Early Idea from George Gamow

The Pauling Papers contain an interesting example of research done on the structure of DNA mere months after its discovery. On October 22, 1953, the Russian-born physicist (and founder of the “RNA Tie Club“) George Gamow sent a letter to Linus Pauling that mentioned some work he had been doing with DNA. Gamow explained that he had found a manner by which the twenty amino acids that make up proteins could be related to different combinations of the four nucleotides found in DNA.

At this time, it wasn’t known that the DNA strands unwind during replication, and Gamow assumed that protein synthesis occurred directly on the double helix. He suggested that a “lock and key relationship” might exist between each amino acid and that the “holes” formed between each complementary base pair in the DNA chain. Science is now aware that this is not the case, but Gamow’s letter is nicely demonstrative of the innovative research ushered in by Watson and Crick’s solving of DNA.

Excerpt from Gamow's letter to Pauling, October 22, 1953.

Click here to view Gamow’s entire letter, and here to read Pauling’s response.

RNA

As the buzz around DNA started to die down, scientists began to move toward the next logical step: RNA. By then, Watson and Crick’s structure was widely accepted, and it had been clear for some time that DNA was the site of the gene. So, then, how did DNA transfer its information to RNA, and finally on to proteins?

Gamow’s above suggestion was a possibility, but it didn’t even involve RNA. Watson spent some time playing with the matter, but was not able to equal his luck with DNA. Unfortunately, it would be quite some time before this mechanism was elucidated. Even now, some of the finer details of how this is accomplished are not completely understood.

Four members of the RNA Tie Club, 1955. Clockwise from upper left: Francis Crick, Leslie Orgel, James Watson and Alexander Rich. Founded by George Gamow, the RNA Tie Club met twice a year in pursuit of greater understanding of RNA.

Eventual Honors

Unsurprisingly, as time went on, Watson and Crick began to accumulate awards for their work with DNA. On December 15, 1959, Linus Pauling responded to a previous letter sent to him by Sir William Lawrence Bragg soliciting Pauling’s support of the nomination of Watson and Crick for the Nobel Prize. In this letter, Pauling stated that he would indeed be willing to write the requested letter of support. However, contrary to Bragg’s suggestion that they be nominated for the prize in chemistry, Pauling stated his belief that a prize in physiology or medicine would be much more fitting.

Several months later, on March 15, 1960, Pauling finally sent his letter to the Nobel Committee.  By the time of its authorship, Pauling’s feelings about the importance of Watson and Crick’s work had become even more tepid.

While acknowledging that “the hydrogen-bonded double-helix for DNA proposed by Watson and Crick has had a very great influence on the thinking of geneticists and other biologists,” Pauling notes that their work was, at least to some degree, “stimulated” by his and Robert Corey’s incorrect triple-helix structure, and abetted by Maurice Wilkins‘ x-ray photographs.  Pauling also points out that Wilkins, Corey, Karst Hoogsteen and himself had already tweaked the Watson-Crick model a bit, “which suggests the possibility that a further change in the structure of nucleic acid may be found necessary.”

In the end, Pauling couldn’t bring himself to go through with the promised nomination.

It is my opinion that the present knowledge of the structure of polypeptide chains in proteins is such as to justify the award of a Nobel Prize in this field in the near future, to Robert B. Corey for his fundamental investigations of the detailed molecular structure of amino acids and the polypeptide chains of proteins or possibly divided between him and Kendrew and Perutz. On the other hand, I think that it might well be premature to make an award of a Prize to Watson and Crick, because of existing uncertainty about the detailed structure of nucleic acid. I myself feel that it is likely that the general nature of the Watson-Crick structure is correct, but that there is doubt about details.

Pauling’s hesitations served only to delay their inevitable receipt of a Nobel Prize for a short time. In 1962, Francis Crick, James Watson, and Maurice Wilkins shared the award in Physiology or Medicine “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material.”

The discovery of the structure of DNA was clearly one of the most important discoveries in the modern scientific era. Not only was it a huge breakthrough in itself, but it also opened the door for major advances in numerous other science-related fields. For more information on DNA, check out the rest of the posts in our DNA series or the website on which they are based, “Linus Pauling and the Race for DNA: A Documentary History.” For more information related to Linus Pauling, please visit the Linus Pauling Online portal.