Overlooked enzyme turns out to be master builder

Can be used to build synthetic biomaterials that could find use in applications ranging from drug delivery to nanowires

Original source: Materials Today

Materials scientists have written the recipe for how to use an oddball enzyme to build new biomaterials out of DNA. Their work provides instructions for researchers the world over to build self-assembling molecules for applications ranging from drug delivery to nanowires.

The molecular machinery of the human body typically relies on genetic templates to carry out construction. For example, enzymes known as DNA polymerases read DNA strands base-by-base to build accurate copies.

There are, however, a few black sheep in the world of molecular biology that do not require a template. One such outlier enzyme, called terminal deoxynucleotidyl transferase (TdT), works in the immune system and catalyzes the template-free addition of nucleotides – the building blocks of DNA – to single-stranded DNA.

Adding seemingly random nucleotide sequences to a single DNA strand wouldn’t seem to have much biological use – but materials scientists have now figured out how to take advantage of it.

In a new paper in Angewandte Chemie International Edition, researchers at Duke University build on their previous work and describe in detail how the TdT enzyme can produce precise, high molecular weight, synthetic biomolecular structures much more easily than current methods. Researchers can tailor the synthesis process to create single-stranded DNA that self-assembles into ball-like containers for drug delivery or incorporate unnatural nucleotides to provide access to a wide range of medically-useful abilities.

“We’re the first to show how TdT can build highly controlled single strands of DNA that can self-assemble into larger structures,” said Stefan Zauscher, professor of mechanical engineering and materials science at Duke University. “Similar materials can already be made, but the process is long and complicated, requiring multiple reactions. We can do it in a fraction of the time in a single pot.”

TdT has an important advantage over typical, synthetic chain-building reactions: it continues to add nucleotides to the end of the growing chain as long as they are available. This opens up a vast design space to materials scientists.

Because all TdT enzymes work at the same pace and never stop, the resulting strands of DNA are all very close in size to each other – an important trait for controlling their mechanical properties. The never-ending process also means that researchers can force-feed TdT any nucleotide they want – even unnatural ones – simply by providing no other options.

“Your body makes strands of DNA out of only four nucleotides – adenine, guanine, cytosine and uracil,” explained Chilkoti, professor and chair of the department of biomedical engineering at Duke. “But we can create synthetic nucleotides and force the enzyme to incorporate them. This opens many doors in making DNA-based polymers for different applications.”

For example, unnatural nucleotides can incorporate molecules designed to facilitate ‘click chemistry’ – allowing the attachment of a whole suite of biomolecules. Researchers could also start the building process using a specific DNA sequence, called an aptamer, that can target specific proteins and cells.

“This enzyme has been around for decades, but this is the first time somebody has mapped these concepts into a blueprint for synthesizing a whole new family of polynucleotides,” said Zauscher. “In the past, biochemists have largely been interested in what TdT does in the human immunological system and how it does it. We don’t care about all of that, we’re just interested in what material building blocks we can make with it. And the precision with which we can make polymers with this enzyme is actually quite exceptional.”