Measure Twice, Cut Once: How Measuring Molecular ‘Scissors’ Can Further Groundbreaking Research

My grandfather was a carpenter, so growing up, I often heard the adage “Be sure to measure twice, so you only have to cut once.” The saying is also attributed to tailors — you have to make sure your measurements are correct before you cut the fabric.

Little did I know that years later the saying would take on new meaning as I studied a different type of scissors, made of the genetic material ribonucleic acid (RNA) that affects how our genes work.

RNA is sometimes called the “messenger” because it regulates how the genes in our DNA express themselves. Part of my research involves studying a type of scissors that cut RNA into two pieces. These RNA scissors are called self-cleaving ribozymes.

These RNA scissors play crucial roles in gene expression across all walks of life — from simple viruses to complex genes in the human brain — and have broad implications for understanding and treating infection and disease.

Self-cleaving ribozymes naturally occur in viruses and many lifeforms. But researchers have learned we can repurpose them to help engineer RNA vaccines, medical treatments or even supplements you may take, such as probiotics. This is called cellular engineering, which is what I do at NIST. Self-cleaving ribozymes are indispensable for producing the RNA elements I use to program cells.

Researchers must ensure that ribozymes work as needed for their intended use. This can be tricky when we apply self-cleaving ribozymes to areas they did not evolve to be in, such as engineering bacteria to produce biofuels or altering gene expression to treat cancer. We need accurate measurements of ribozymes in these contexts to succeed in this research.

Going back to the parallels to carpentry, we can think of different contexts for a ribozyme as different legs on a table. If we cannot accurately measure each leg, we will ultimately build a wobbly table. Similarly, if we want to use a ribozyme sequence identified in a bacterium as part of an RNA therapeutic for humans, we need to be able to accurately measure how well the ribozyme cuts in the new therapeutic context.

Accurate Measurements of RNA Scissors

Carpenters and tailors use standardized length measurements and have established methods for calibrating their instruments for accuracy. But calibrating measurements at the molecular level is not as straightforward, particularly when trying to study how these molecules behave inside living cells, like the bacteria that I work with.

Directly measuring how well a ribozyme cuts inside a cell is challenging, so often researchers remove the RNA from the cell and use this extracted RNA for their measurements. But these additional manipulations have the potential to influence the result of the final measurement.  

Most ribozymes are produced inside cells, but some are produced outside cells. If these ribozymes are produced outside the cell, we call that “in vitro.” 

So, in my lab, before attempting to extract RNA from cells to conduct measurements, we first tested how in vitro ribozymes could change through all the sample manipulations required for extraction. To do this, we selected ribozymes from other NIST-led work that we knew barely cut in vitro. We mimicked taking these ribozymes through all the sample manipulations we use to prepare for measurement.

We then redid our measurements. Lo and behold, our results after mimicking RNA sample preparation from cells indicated substantially more cutting than we measured before preparation.

So, the sample preparation was affecting the measurement in a way that made the measurements inaccurate.

This is a problem because medical researchers, for example, may think they’re getting these measurements just right, but then they wouldn’t be able to figure out why the treatment isn’t working as well as it should. Their inaccurate measurements indicate the ribozyme cuts very well, so they may try to fix the wrong problem in the drug development process. Our team wanted to solve this measurement challenge so researchers don’t waste time and effort trying to troubleshoot the wrong issues.

Looking into the problem further, we can think of the ribozymes that do not cut in a specific context as being similar to scissors with a string tied around the blades. The process of preparing the RNA from cells caused the string to loosen, allowing the scissors to cut before the measurement took place. We don’t want this to happen because it negatively affected our research.

To work around this, we placed an extremely strong “knot” in the form of a DNA strand around the scissors so they couldn’t cut during sample preparation. Only by using this modified protocol could we get the same measurements before and after the sample was prepared, both for ribozymes inside the cell and for in vitro ones.

The measurement mystery was solved! We’ve published this research so others in the field can apply this protocol to their own measurements, helping further important work in medicine and cellular engineering. These results highlight the importance of conducting multiple measurements on the same sample.

My grandfather was right that if you want to get the correct cut, it’s best to always measure at least twice!

The Bigger Picture

Studying ribozymes in detail is a side gig for me; my main research is in repurposing biomolecules like DNA and RNA to serve as software for programming biology, known as molecular programming. Self-cleaving ribozymes are indispensable for producing the RNA elements I use to program cells. Preparing these elements is similar to making a paper snowflake, in which the paper must be folded a certain way before it is cut. The RNAs I design must fold into a specific structure before the ribozymes cut to function properly.

While designing these RNAs for use outside of cells, I found many contexts in which the ribozymes didn’t cut, causing my designs to fail. I realized measurements of ribozyme activity were going to be crucial for successfully moving my designs to cells, so I began exploring new methods to conduct these measurements.

My NIST training taught me to calibrate any new measurements against known reference samples and techniques — always measure a known sample with at least two different measurements! And that’s how this side project was born.

Our accurate ribozyme measurement technique is now an integral part of my design cycle and will help NIST researchers engineer cells to tackle a wide array of problems, from engineered bacteria that diagnose and treat disease in the gut or skin to engineered yeast that produces medicines or biofuels.

Beyond NIST, these measurements will support further development of the growing bioeconomy, and that’s rewarding for me as a measurement researcher who always remembers to measure twice.