What was the biggest challenge in proving that the codon was a triplet?

Step-by-step solution

Providing proof that the codon was a triplet, was a more daunting task. The chemical method developed by Har Gobind Khorana was instrumental in synthesizing RNA molecules with defined combinations of bases (homopolymers and copolymers). Marshall Nirenberg’s cell-free system for protein synthesis finally helped the code to be deciphered. Severo Ochoa enzyme (polynucleotide phosphorylase) was also helpful in polymerising RNA with defined sequences in a template independent manner (enzymatic synthesis of RNA).