How Khorana Synthesized a tRNA Gene and Helped Build the Field of Synthetic Biology
Imagine being able to write the very language of life into existence—not by editing or modifying, but by synthesizing it from scratch using only chemical building blocks. This was the monumental achievement of Har Gobind Khorana, a pioneer who transformed our understanding of the genetic code and then set out to build it with his own hands. His work, which earned him a Nobel Prize in 1968, laid the foundational principles for what would become the field of synthetic biology.
Khorana achieved the first total synthesis of a tyrosine suppressor transfer RNA (tRNA) gene—demonstrating that a fully functional gene could be chemically constructed outside a living cell 1 .
This synthetic gene provided scientists with a powerful tool to probe the deepest mysteries of genetics: How are genes read? How is their information translated into proteins?
At a time when methods for reading DNA sequences were in their infancy, Khorana's approach was to build genes himself, thereby learning their secrets through synthesis. This article explores how this remarkable achievement was accomplished and how its legacy continues to influence modern medicine.
Before attempting to build genes, Khorana first helped crack the cipher that governs all life: the genetic code. The central question of his day was how the four-letter alphabet of DNA—A, T, C, G—could specify the 20-amino acid vocabulary of proteins.
Khorana's breakthrough came through sophisticated chemical synthesis. He developed methods to create specific polyribonucleotides—synthetic RNA chains with repeating sequences 1 . For example, by synthesizing a chain alternating between UC (UCUCUCUC...), he could determine which amino acids were incorporated into the resulting protein. This RNA would produce a protein chain alternating between serine and leucine, revealing that UCU codes for serine and CUC codes for leucine.
Khorana was awarded the 1968 Nobel Prize in Physiology or Medicine, shared with Robert W. Holley and Marshall W. Nirenberg .
As Khorana remarked, having deciphered the code, the next logical step was to synthesize it. This vision would lead to his groundbreaking work on gene synthesis.
By 1970, Khorana had already made history by completing the first total synthesis of a gene, coding for a yeast alanine tRNA 1 . But this initial success revealed new challenges. The synthetic alanine tRNA proved unsuitable for studying certain essential biological processes, such as the mechanisms of transcription initiation and termination.
Khorana embarked on an even more ambitious project: the total synthesis of an Escherichia coli tyrosine suppressor tRNA gene 1 . This 126-base-pair DNA sequence represented a precursor to the tyrosine tRNA molecule, complete with the necessary promoter regions that control its expression 6 .
The project aimed to address fundamental questions about gene expression and protein synthesis, particularly the structure-function relationship in tRNA 1 . By constructing the gene piece by piece, Khorana would create a customizable platform for biological experimentation.
The "suppressor" aspect was particularly valuable—this specific tRNA could bypass genetic "stop" signals, allowing researchers to easily monitor its functionality in living cells. This feature would prove crucial in validating the synthetic gene's biological activity.
Khorana's approach to gene synthesis was as meticulous as it was revolutionary. The process can be broken down into three fundamental stages, each requiring innovative solutions to complex biochemical problems.
The first and most labor-intensive step involved the chemical synthesis of 26 short DNA segments (deoxyribooligonucleotides) that, when combined, would form the complete double-stranded gene 6 . Each segment ranged from 9 to 15 nucleotides in length—painstakingly assembled one nucleotide at a time using sophisticated organic chemistry techniques.
A noteworthy advancement in these syntheses was the pioneering use of reverse phase high-pressure liquid chromatography (HPLC) for the rapid and efficient separation of the synthetic reaction mixtures 8 . This technique represented a significant improvement in purity and yield over existing methods.
With all 26 segments in hand, the next challenge was to link them together in the correct order. Khorana and his team employed two key enzymes to accomplish this:
What made this approach particularly powerful was that each synthetic segment was designed with complementary single-stranded ends 1 . These "sticky ends" allowed the pieces to align perfectly through base-pairing before being permanently sealed by the ligase enzyme.
The final stage involved joining the four larger DNA duplexes, each representing a substantial portion of the gene, to form the entire 126-base-pair DNA duplex 6 . The success of this multi-year project hinged on impeccable precision—a single nucleotide error at any stage would render the final gene non-functional.
| Reagent / Tool | Function in the Synthesis |
|---|---|
| Protected deoxynucleotides | Chemical building blocks for DNA synthesis |
| Polynucleotide kinase | Adds phosphate groups to DNA segments for joining |
| Polynucleotide ligase | Joins DNA segments together by forming phosphodiester bonds |
| Reverse phase HPLC | Purifies synthetic oligonucleotides |
| Restriction endonucleases | Cuts DNA at specific sequences for analysis and cloning |
The synthesis of the promoter region—the genetic "switch" that turns the gene on—exemplifies the precision and careful planning that characterized the entire project. This 62-nucleotide section required the perfect alignment and joining of 10 individual synthetic segments 4 7 .
Khorana's team developed a sophisticated joining strategy that involved creating smaller units first, then assembling them into larger structures. This modular approach allowed them to verify the correctness of each subunit before proceeding to the next assembly stage. The final promoter duplex was not just a chemical achievement; it contained all the necessary information to properly initiate transcription of the synthetic tRNA gene 4 .
The ultimate test of Khorana's synthetic gene came when it was introduced into living bacterial cells. The results, published in the 17th paper of the series, were remarkable 2 :
The gene was successfully joined to both plasmid and bacteriophage vectors after digestion with the EcoRI restriction enzyme
It suppressed amber mutations in both bacterial (trpA, his, lacZ) and bacteriophage genes (Aam32, Bam1)
The cloned gene was transcribed and processed correctly to produce the tyrosine suppressor tRNA precursor
Perhaps most impressively, when the synthetic gene was incorporated into the E. coli chromosome as part of a lambda prophage, it continued to function, suppressing bacterial amber mutations 2 . This demonstrated that the synthetic gene wasn't just active in isolation—it could integrate into the complex network of cellular genetics and function alongside natural genes.
| Validation Method | Result | Significance |
|---|---|---|
| Suppression of amber mutations | Functional compensation for genetic defects | Demonstrated biological activity in living cells |
| In vitro transcription | Production of correct tRNA precursor | Confirmed accurate recognition by cellular machinery |
| RNase P processing | Release of 41-nucleotide fragment | Verified proper folding and processing of tRNA |
| Cloning and re-isolation | Gene size and suppression ability maintained | Proven structural stability and hereditary transmission |
The two-dimensional fingerprinting after T1-RNase digestion provided definitive evidence: the nucleotide sequence of the cloned gene was accurate, and its expression was properly controlled by its synthetic promoter 2 . Khorana had not only built a gene—he had built a functioning genetic component that the cell could read, transcribe, and utilize as if it were its own.
While Khorana's work was fundamental research, it established the principles that underpin today's most advanced genetic therapies. The suppressor tRNA concept he helped pioneer is now being developed to treat devastating genetic diseases.
Approximately 11% of all genetic diseases are caused by nonsense mutations—single nucleotide changes that create premature stop signals in genes, truncating essential proteins 3 . These include conditions like cystic fibrosis, Duchenne muscular dystrophy, and various forms of cancer.
Engineered tRNAs show >20-fold enhanced efficiency in suppressing premature termination codons compared to native tRNAs 3 .
Har Gobind Khorana's journey from a small village in Punjab to the pinnacle of scientific achievement is a story of extraordinary intellect, perseverance, and vision. His total synthesis of a tyrosine suppressor tRNA gene was more than a technical masterpiece—it was a conceptual breakthrough that demonstrated human beings could not only decipher the language of life but could also learn to write it themselves.
The implications of this work continue to unfold decades later. From fundamental insights into gene expression to revolutionary approaches for treating genetic diseases, Khorana's synthesis created a foundation upon which modern molecular biology and medicine are built. As current researchers develop increasingly sophisticated tRNA-based therapies for genetic disorders, they walk a path that Khorana first charted in his laboratory—one nucleotide at a time.
His legacy reminds us that the most fundamental research often yields the most transformative applications, and that understanding life's simplest principles can ultimately empower us to reshape them for human benefit.