An Interview with Manuel Varela & Ann Varela: Transfer RNA and Antisense Medicines—Paul Zamecnik—They inspire them well at Harvard!

Jul 11, 2020 by

Paul Zamecnik

Michael F. Shaughnessy

1) Paul Zamecnik, in a sense, lived a scholar’s dream, so to speak—educated at Harvard, taught at Harvard—practiced medicine at Harvard Med, but where and when was he born?

Dr. Paul Zamecnik was a prominent biochemist who helped to discover the then elusive transfer RNA and who later helped to invent antisense DNA medicine. Cleveland, Ohio, is the birthplace of Paul Charles Zamecnik, who was born in November 1912. Zamecnik is the son of John Charles, a banker, and Mary Gertrude McCarthy. Zamecnik was interested in science as a child. Those that knew him as an adult, report that he was a good storyteller, good-natured, open-minded, and a gentleman. Zamecnik graduated from high school at the age of 16.Zamecnik majored in Chemistry and zoology, graduated in 1933 from Dartmouth College, and obtained his B.A. degree. During his tenure at Harvard Medical School, he took his M.D. degree in 1936.

Mary Connor wed Zamecnik in 1936. The Zamecnik’s had three children, seven grandchildren, and five great-grandchildren.

Between the years of 1936 and 1939, Zamecnik had internships at Collis P. Huntington Memorial Hospital located in Boston, Harvard Medical School, and Lakeside Hospital in Cleveland, Ohio. While at Lakeside Hospital, Zamecnik developed an interest in how cells regulate growth, thus leading him to the field of protein chemistry. Zamecnik acquired a fellowship at the Carlsberg Laboratory in Copenhagen, Denmark.

He worked in Kaj Linderstrøm-Langs laboratory and studied protein chemistry until he returned to the United States in 1940 to work at the Rockefeller Institute.

After a year at Rockefeller, Zamecnik relocated to accept a Professorship of medicine at the Huntington Memorial Hospital, which was associated with Harvard University and at the Massachusetts General Hospital. While there, Zamecnik collaborated with Fritz Lipmann. Their research centered on using radioactive isotopes to prove that proteins were built from amino acids in a process requiring ATP. Zamecnik became an Associate Professor of Medicine in 1942 at Harvard Medical School. Zamecnik was a Senior Scientist at Massachusetts General Hospital. Zamecnik died of cancer on October 27, 2009, at his Boston residence.

2) Transfer RNA—why is this important, and how does it fit into the big picture?

Transfer RNA (tRNA) is a piece of the molecular machinery that helps a living cell to make protein. The production of proteins is a cellular process called translation, and tRNA is one of the players engaged in that process. During translation, amino acids are connected to make longer strings of amino acid chains called peptides. The translational machinery then releases the completed full-length polypeptides. The tRNA molecules deliver specific amino acids to the ribosome so that it can attach them to form long strings.

Figure Transfer RNA molecule.

Transfer RNA is an L- or T-shaped molecule of RNA. In the figure, on the left, the tRNA molecule, shown in two-dimensions, has a binding site for attachment of amino acid. The tRNA molecule has some parts of it as a single strand and other parts attached to form a double-stranded molecule, the latter of which is referred to as intramolecular base-pairing regions. Thus, tRNA is both a single- and a double-stranded RNA molecule.

A typical tRNA molecule has three loop structures, the bottom loop of which forms a binding site for attachment to an mRNA message molecule. The attachment site on the tRNA is called an anti-codon (e.g., CUC in the figure, bottom left), and the mRNA has the codon (e.g., GAG). The codon message on mRNA specifies a given amino acid. In the figure example, the GAG codon (left, bottom) specifies a glutamate amino acid (left, top).

In the figure on the right, a three-dimensional format for a typical tRNA molecule is depicted. At the bottom right is shown the anti-codon ribonucleotides, CUC. At the top right, the amino acid attachment site is shown without the amino acid. Still, it shows the 5′ (pronounced 5-prime) and 3′ (pronounced 3-prime) ends of the tRNA molecule coming together, where the 3′ end of the tRNA molecule binds its dedicated amino acid.

For each one of the individual 20 amino acids, there are dedicated tRNA molecules for them. In many cases, more than one tRNA molecule can specify a given amino acid. Such a coding system is known as degeneracy, i.e., more than one codon, and, thus, anti-codon, can code for an amino acid.

Paul Zamecnik was involved in the discovery of tRNA. At the time, the concept of its existence was theoretical, and the evidence for it elusive. Francis Crick had predicted its presence in cells as a so-called adaptor molecule when he and others formulated the Central Dogma, the belief without evidence that the code of life was centered around DNA but carried as a message in RNA and translated into the production of protein.

The trail to the discovery of tRNA started with Robert Loftfield. In the late 1940s, he became the first graduate student in the laboratory of Professor Robert Woodward, a professor of chemistry at Harvard. Using cyanide as a starting point at Harvard, Loftfield had prepared amino acids alanine and glycine that had been radioactively labeled with carbon-14. Zamecnik worked with Loftfield, Ivan Frantz, and Mary Stephenson to study rat liver slices to measure carbon-14-alanine incorporation into protein. They found good protein-making activity.

Next, they discovered that the inhibitor of ATP synthesis, called dinitrophenol, blocked the carbon-14-alanine inclusion into protein. The result was intriguing because it suggested that the phosphorylation of amino acids was an essential step in making protein. They followed up on this notion by searching for specific proteolytic enzymes involved in protein synthesis but were thwarted by the new finding that reversing proteolytic enzyme activity did not seem to be necessary for translation.

Using Loftfield’s carbon-14-labeled amino acids, Zamecnik’s laboratory developed a method to purify these amino acids in rat liver slices and measure protein. They often kept finding, however, that the labeled carbons ended up in breakdown products or in building blocks of other amino acids, instead of within intact proteins.

Thus, Zamecnik invoked a so-called cell-free system to sort out the confounding degraded protein results. The cell-free system consisted of lysing a collection of cells by mincing rat liver, passing the tissue through the slim crevices of round-bottomed flasks, and adding a buffer to make a homogenate cell lysate without the cell membrane in their laboratory preparation. The method produced a cell-free extract that could be used to measure protein synthesis—it also eliminated the confounding proteolysis from their experimental process.

Then, Dr. Mahlon Hoagland entered the picture, and he was asked to search for the elusive phosphate activation molecule that was postulated to occur after the dinitrophenol experiments. Presuming that phosphate activation of amino acids occurred in ribosomes, microsomes were prepared.

Microsomes consist of endoplasmic reticulum membranes that contain ribosomes. Hoagland also prepared microsomes and enzymes that were not attached to the endoplasmic reticulum, a so-called soluble enzyme preparation. Working with Elizabeth Keller, Zamecnik, and Hoagland observed the production of ATP-activated amino acid and phosphate incorporation into ATP within their microsome preparation.

Next, the Zamecnik laboratory reconstituted the known components back together to make protein in the test tube. They added ribosomes, ATP, carbon-14-labeled amino acids, rat liver lysate, and even their soluble enzyme mixture, but they obtained no new protein! Something was missing from their translational machinery!

They then added a solution that had been previously thrown away as so-called “junk RNA.” This junk RNA solution was thought not to be necessary because it separated itself from the microsomes, the protein-making machinery, during the lab preparation. Thus, this so-called junk RNA, which was initially called soluble RNA (sRNA), was incorrectly considered unnecessary.

Nevertheless, they added the sRNA to radioactively labeled ATP and found that the label went to AMP. However, they were not sure whether the ATP association was specific to RNA or merely an artifact. So, they repeated the experiment and invoked a control group consisting of Loftfield’s carbon-14-valine, instead of carbon-14-ATP.

The results were staggering. They found that the radioactive valine firmly attached to the 5′ end of the sRNA! Furthermore, in the test tube with radioactive ATP, they found it firmly attached to the 3′ end of the same sRNA!

Figure Transfer RNA two-dimensional structure.

The next experiment turned out to be the one in which the now-famous tRNA was discovered. Hoagland, Stephenson, Jesse Scott, and Zamecnik purified the sRNA and renamed it transfer RNA to distinguish it from ribosomal RNA (rRNA). They added their new tRNA to a mixture of amino acids, ATP, a mixture of soluble enzymes, ribosomes (then called microsomes), and a pure preparation of radioactive amino acid labeled by Loftfield. They found a series of radioactive amino acids connected into long chains of protein!

Thus, the highly critical tRNA molecule had been discovered. The pioneering work was duly published in the prestigious Journal of Biological Chemistry in 1958.

3) Denying a virus or cancer—what it seems to need to survive was one of Zamecnik’s endeavors—and it seems logical—to deny the virus or cancer—whatever it needs to continue—seems logical—how did he hypothesize this and go about it?

For over a century, investigators have known of the association between certain viruses and cancer. The importance of these microbes and tumorogenesis stems from initial work in the early 1900s, with the studies of Dr. Peyton Rous. He had shown that infection of laboratory birds, such as chickens, resulted in the induction of tumor masses. The genome of the Rous sarcoma viruses is made up of RNA, rather than DNA. David Baltimore had later demonstrated that these Rous sarcoma viruses packed intact enzymes for making DNA on the insides of the virions themselves.

Zamecnik’s interest in the Rous sarcoma virus began in the 1930s when he studied cell transformation using the cancer-causing microbe. At that time, he did not focus intensely on the microbe as a prime means of investigation. Zamecnik returned to the Rous sarcoma virus in the early 1970s when he followed up on previous studies involving the activation of amino acids with RNA.

At some point, it became clear that one needed to understand the mode of amino acid activation. Thus, it would be necessary to know the precise sequences of the tRNA molecules that bound to each of the amino acids. Hence, sequencing RNA was the main focus of research in the 1970s.

At first, they spent five years determining the sequence of tRNA, starting from its 3′ end. They learned that this 3′ end of tRNA had a short stretch of adenosines in a row, called poly-A, for short. Then, they learned about the next 21 ribonucleotides adjacent to the poly-A tail. To their dismay, however, they found out that another laboratory, this one headed by Dr. Walter Gilbert, who was at Harvard biological sciences department, worked with Allan Maxam, in which they had already sequenced over 100 residues, starting from the 5′ end of tRNA.

At about that time, after publishing their tRNA sequences together, it was discovered that DNA could be made from the RNA using reverse transcriptase. Still, tRNA was surprisingly needed to initiate DNA synthesis. That is, tRNA was the primer for the reverse of transcription! It was also discovered that the reverse transcriptase enzyme needed another enzyme activity, called RNase H, to remove the tRNA primer after it performed its initiating primer function. Lastly, Zamecnik became aware that during this viral RNA genome copying mechanism into DNA that the RNA template could be complementary to the newly made DNA and that the ends of the two molecules (DNA and RNA) could come together and form a circle during reverse transcription.

Taking all of these facts into consideration, Zamecnik hypothesized that DNA and RNA could bind with each other forming a so-called RNA-DNA duplex hybrid.

He further rationalized that if indeed viral RNA and DNA could form hybrids, then it might be possible to design a synthetic DNA which might bind to Rous sarcoma virus RNA genomes.

If true, then the viral RNA would be made unavailable as a template for reverse transcription to make DNA or even stopped in RNA genome replication. It was a tremendously insightful deduction. It would change the history of biochemistry and molecular biology.

If the possibility existed that the RNA genome could be blocked, then Rous sarcoma virus multiplication inside host cells might be stopped. Thus, the virus would be prevented from causing cancer. Such was the basis for Zamecnik’s discovery of the antisense DNA medicines. Cancer-causing viruses could be stopped in their tracks!

4) His ideas even led to the blocking of the HIV (AIDS) virus via antisense oligonucleotide. Now, first, what exactly is an oligonucleotide, and how would it work against HIV, and then this word “antisense”—can you describe it and how it fits into the big picture?

The idea was that short stretches of DNA, called antisense oligonucleotides, could be fashioned in the laboratory, and bind complementarily to the RNA genomes of dangerous viruses. Zamecnik’s notion, formulated above, about the antisense oligonucleotides, arose out of the need to prevent pathogenic viruses from wreaking their havoc upon healthy cells and, thus, prevent cancers, and in the case of HIV, prevent the severe illness known as AIDS. The HIV (human immunodeficiency virus) destroys specific immune cells that are needed for full immune responses to a great many microbial pathogens and cancer. Thus, HIV depletes the body’s ability to mount an immune response to foreign and potentially detrimental antigens.

Disease-causing viruses must be able to make more copies of their mature viral forms to mediate their dastardly, perhaps lethal, effects upon healthy cells. This need to multiply viruses requires that genomes of these pathogenic viruses be faithfully copied into large numbers within cells. Similarly, in the case of RNA viral pathogens, the RNA genomes must be copied, just as well.

Therefore, if a short stretch of a complementary DNA molecule, i.e., an antisense oligonucleotide, is added to a mixture of host cells and their viral disease-causing pathogenic microbes, then the RNA would be unable to undergo genome replication. The idea of the antisense oligonucleotide as medicines for viral diseases is brilliant because these drugs could be made in highly specific ways, thus, targeting precise species and even highly specific strains of viruses.

5) His work seems to be the precursor of an entirely new realm of work—”biotechnology, and antisense therapeutics.” How is his work impacting the field today?

Working closely with Mary Stephenson in 1978, Zamecnik had the keen insight to formulate a basis for antisense oligonucleotide technology for Rous sarcoma viruses. The method was initially geared towards the inhibition of viral replication using complementary pieces of DNA, which would form hybrid RNA-DNA duplexes and prevent viral infection. Stephenson and Zamecnik referred to these complexes as “hybridons.” The field of cancer biology was fraught with serious medical issues. Therapeutically speaking, Zamecnik’s discovery provided promising avenues in dealing with cancer-causing (oncogenic) viruses. Investigators would later refer to these agents as antisense DNA molecules. The work would lead to the development of antisense RNA therapeutic agents, as well.

Zamecnik had the insight to combine disparate fields of biochemistry and molecular biology to develop the antisense oligonucleotide therapy. He considered the emergence of restriction endonuclease enzymes, initially found in bacteria, and their ability to defend themselves from viral infection. Bacteria cleaved specific DNA sequences and in their ability to rearrange certain stretches of DNA by cutting and reassembling them elsewhere, perhaps in another location in the genome or even in a plasmid molecule. The knack for restricting an enzyme’s DNA-cutting ability to only a specific DNA sequence would be, in Zamecnik’s mind, useful for targeting a synthetic oligonucleotide to a specific DNA sequence, too.

Zamecnik was well aware, as most biochemists and molecular biologists were, that DNA sequencing technology relied upon the role of primers for sequencing purposes. These sequencing primers, i.e., oligonucleotides, consisted of short pieces of DNA molecules that were highly specific to certain regions of DNA near regions of unknown to be sequenced. The sequencing technology would then use the sequencing primers to make new DNA into the territories of unknown base sequences, and investigators would soon learn these new sequences. Nevertheless, it all required, from the start, the employment of oligonucleotide DNA.

Zamecnik also reasoned that if DNA sequencing and viral replication required knowledge of the molecular and cellular mechanism of DNA synthesis, then oligonucleotides could be exploited to enhance sequencing and inhibit replication, respectively. Thus, investigations of the inner workings of how organisms made DNA would be necessary to understand how these systems worked and how they could be manipulated.

Zamecnik needed to understand how pieces of DNA or RNA interacted with each other to form hybrids, e.g., DNA-DNA, RNA-DNA, and RNA-RNA. He needed to know the strand orientation (i.e., 5′ → 3′ or 3′ → 5′) and whether nucleic acids of similar or distinctive strand senses, i.e., plus (sense), or minus (antisense), would interact, and if so, how? We know today that DNA strands of complementary sequences but opposite senses with their proper strand directions could interact and stably bind. Such knowledge would be necessary to develop hybridization technology and, thus, antisense oligonucleotide therapy.

6) This issue goes out to all administrators who want to put all classes and labs online—Zamecnik once said, “The lab is where the action is!” Why was he so correct in this assertion?

Zamecnik was reported to have said the above quotation in 2000 during an event sponsored by the prestigious Massachusetts Institute of Technology (M.I.T.) in Cambridge, MA, in which he was included in a collection of innovative inventors, a list put together by the Lemelson-M.I.T. program. The program highlights exceptional inventors who make outstanding contributions to science and society. Zamecnik was probably referring to the fact that in the laboratory, new technologies are continually being developed, which can be used positively for the benefit of disease treatment. He was speaking directly about antisense biotechnology, of course.

One can undoubtedly extend the meaning of the famous quotation in terms of effective teaching. University and college faculty know the importance of a “learning by doing” approach to education. Students learn effectively by conducting “hands-on” experiments in the lab and seeing the results of experimental exercises for themselves. Spontaneous conversations started in the lab may also promote invaluable insight to deepen understanding of the laboratory experiment’s objectives.

The students seem to have a vested interest in analyzing the results of their experiments. In a Microbiology Laboratory taught by one of us (M.F.V.), the students formulate their hypothesis about the specific nature of a microbe that they find on an inanimate object of their choosing. The students then clone the microbe and study its biochemical behavior to identify their bacterial clone. Since the individual microbes that the students isolate are directly tied to their individual everyday lives, such students have a keen sense of wanting to know and understand the complexities of the course concepts. For instance, the students are eager to know whether their microbe produces bubbles when hydrogen peroxide is poured on their clones. Such students then focus more clearly on the mechanism of the catalase enzyme that produces the bubbles. The students perform their experiments and productively learn from them.

7) What were some of his awards and acknowledgments?

Zamecnik published over 210 peer-reviewed scientific articles and made several highly significant discoveries, most notably the discovery of tRNA and the invention of antisense oligonucleotide therapy. Thus, it is surprising that the Nobel was not forthcoming for Zamecnik. He did win, however, many distinguished awards, including the National Cancer Society National Award in 1968. He was the recipient of the James Ewing Award, Borden Award, Passano Award, and a three-time recipient of the John Collins Warren Triennial Prize of Massachusetts General Hospital. He won the National Medal of Science in 1991. He was awarded the first-ever Albert Lasker Lifetime Achievement Award for Special Achievement in Medical Science in 1996. Zamecnik had been elected to the National Academy of Sciences in 1968. He was also a member of the American Academy of Arts and Sciences and the Institute of Medicine.

Zamecnik became a full-fledged member of numerous scientific organizations, including the American Society of Biological Chemistry, the American Association for Cancer Research (President 1964-65), the Association of American Physicians, the American Philosophical Society and the Danish Academy of Sciences and Letters.

8) He lived a long and fruitful life—what was he doing in his later years?

After retirement, Zamecnik continued to work actively in the laboratory, at Mass General, in Boston, MA. His chief focus was on the development of antisense therapeutics.

The oligonucleotide DNA technology would be useful for molecular biology and biotechnology. For instance, improvements in the efficiency of DNA sequencing technology made possible the genetic mapping and nucleotide sequence determination for entire genomes, creating the new field of genomics. The work would lead to the sequencing of collections of whole genomes in the environment or, for instance, in the guts of animals, like humans, thus, creating a new field called metagenomics.

The work would lead to new developments in editing genomes by younger investigators. In more recent times, the genome-editing technology was made possible by the early work of Zamecnik. An organism’s genome can be edited readily using CRISPR. The abbreviation is short for clustered regularly interspaced short palindromic repeats. The relatively new CRISPR technology makes use of oligonucleotides such as guide-RNAs (gRNA), now known as crRNAs (CRISPR RNA molecules), which are used to target the genome region to be edited. In modern times, biomedical investigators are focusing on tweaking CRISPR to find and repair defective human genes to address untreatable genetic diseases.

9) What have I neglected to ask about this Harvard trained scholar and researcher?

Zamecnik wrote in a memoir that the amino acid chemistry work, in 1946, of a young Harvard Ph.D. student by the name of Robert B. Loftfield started the process that led to their discovery of transfer RNA. At the time, Loftfield had successfully synthesized alanine with radioactive carbon-14 using the so-called Strecker chemistry method. The carbon-14-labeled amino acids would be used by the Zamecnik laboratory to measure protein synthesis and discover tRNA.

Nearly 40 years later, Dr. Loftfield would be become one of the most rigorous professors ever of a two-semester course in biochemistry for one of us (M.F.V.). Professor Loftfield taught an Intensive Biochemistry course at the University of New Mexico in Albuquerque, NM. He actively taught structures, reactions, and mechanisms in substantial detail. He took us, students, through every reaction in each of the essential biochemical pathways.

Loftfield held weekly recitations in which we undergraduate students were chosen at random to go to the board and provide an answer to a randomly chosen biochemistry question from the back of the textbook’s chapters. We soon learned, some of us more quickly than others, to hide if we knew answer and volunteer to go to the board if we did not know the answer! If he somehow sensed that we didn’t know something, it was guaranteed that we’d be at the board.

We marveled one day during a lecture as we witnessed Loftfield draw on the board (without notes), taking the entire chalkboard, the intact chemical structure of acetyl coenzyme A. The next week, we suffered when the acetyl-coenzyme A structure appeared as a question on the exam.

File:Acetyl CoA.png

Figure Acetyl Coenzyme A Structure.

Despite the tremendous amount of time and effort we students spent learning biochemistry in Loftfield’s courses, we eventually learned to admire our dear professor, who, sadly, died at the age of 94 in 2014. As a faculty, he inspired me to invoke many of his quite effective teaching methods when I became a university professor.

One day, when I was a Ph.D. graduate student at the UNM Health Sciences Center, we attended a seminar given by Loftfield titled “How I helped discover transfer RNA.” His presentation that day was inspiring. The seminar that day had been memorable as it was captivating because it was the first time that I had ever heard of Zamecnik and his colleagues on how they went about discovering tRNA.

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