Arnav's Blog: Testing Synthetic DNA Nucleotides Using Cleavable Fluorophores at Columbia University

Introduction:

Hello All! My name is Arnav Rai and I am rising Junior this year. This summer I have the opportunity to work in Dr. Jingyue Ju's Genetics Laboratory at Columbia University. As of now Dr. Ju and his colleagues are working on ways to make DNA sequencing more efficient. In addition, Dr. Ju's lab develops synthetic nucleotides and then tests them to see how well they incorporate into a DNA sequence. As research interns, we are not privy to the exact chemical structures of the nucleotides as that is highly sensitive information. This is because if the synthetic nucleotides end up being successful in DNA sequencing the University can then license them to different companies. But as interns, we are able to conduct the Single Base Extension experiments to test the Nucleotides.

Week 1: 7/17/17 - 7/21/17

Over the course of the first week, I spent time getting used to the lab and lab equipment. In the Genetics lab, we are often forced to use very small sample sizes. so it is of paramount importance that all member of the lab is proficient in using pipettes and droppers. On the first day, we measured volumes of liquid and transferred them using pipettes.

After we practiced using basic lab equipment, my supervising scientist Dr. James Russo sat down with me and we fleshed out the details of the project and the procedure we would be using. The premise of our project is to make DNA sequencing more accurate and efficient by using light and fluorescence. DNA sequencing was originally done using a method called Pyrosequencing. Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle, it relies on the detection of pyrophosphate released during nucleotide incorporation. There are major issues when it comes to pyrosequencing, which we will discuss later on. Dr. Russo and I discussed how we would improve upon this method. For a while, Dr. Russo's and his colleagues have been developing fluorophores that can be attached to a nucleotide and based on the fluorescence detected the DNA incorporation can occur. Essentially we will be attaching a unique fluorophore (something that gives off fluorescence) to the four types of nucleotides: dATP, dCTP, dGTP, dUTP. And since we know that DNA will only combine in certain pairs, in a DNA sequence only one nucleotide will incorporate at a time, and based on the color of the fluorescence given off we can determine which nucleotide was incorporated and how successful it was. Having only taken Chemistry to this point I spent the rest of my first week learning the basic of DNA and how DNA extends. A nucleotide is composed of a carbon-sugar, a nitrogenous base, and a phosphate group.

In order for DNA to extend an OH group has to be present on the 3 prime position of the 5-carbon sugar that makes up the nucleotide. As seen in this diagram there is an OH group on the 3 prime position, meaning that DNA incorporation can further occur. If you notice, there is a Phosphorus group also at the end of the DNA strand. This is because when a DNA polymerase reaction occurs an O-P-O chain is added in order to extend the backbone of the DNA. The polymerase adds 3 O-P-O groups as seen in the diagram below:

After the new nucleotide is incorporated the DNA strand only has need for one of the O-P-O groups, and as a result the two extra O-P-O groups detach. And this phenomena is the basis of the outdated pyrosequencing method. Pyrosequencing relies on the detection of the released phosphate groups. So if a Genetics scientists were trying to incorporate a nucleotide he/she would have to set up four test tubes each with a different nucleotide. Conduct the incoroporation and use light to try and pick up the phosphate signals.

So for the below DNA chain the Pyrosequencing data would look like so:

The blue writing is what we added in order to clarify the data. But as you can see the height of the peaks is an estimate. The height of the peaks is meant to represent how many nucleotides in a row there are. So the height of the 10A peak represents the 10 Adenine nucleotides that incorporated and paired with the 10 Thymines in the Template strand.

We were only able to get up to covering the basics and conducting the control of our experiment. But I am looking forward to next week when we actually begin to test the Fluorophores!

Week 2: 7/24/17 - 7/28/17

After learning the fundamentals of sequencing and the basis of our research we begun our work on attaching cleavable fluorophores to regular DNA nucleotides in order to test sequencing. One thing I forgot to mention last time is that the fluorophores we are attaching act as reversible terminators. This means that the Fluorophore is attached to the nucleotide using some sort of linker (in our case we used an allyl group). The linker attaches the Fluorophore to the 3I prime group of the nucleotide. This caps the nucleotide and limits the DNA strand to add only one base. This process is called Single Base Extension (SBE). In order for us to record our results accuracy in it is very important we observe each nucleotides incorporation individually so we can calculate exactly what percent of the nucleotide was incorporated.

For this portion of the research I worked alongside Dr. Shundi Shi and Dr. Stefan Rover in making the Synthetic nucleotides. Both Dr. Rover and Dr. Shi should us how to attach the fluorophore group using an allyl group as the linker. The allyl group is an especially good linker because after we extend the DNA Template by one base, we can terminate the allyl group and continue to sequence the DNA. With the attached fluorophores this is what the four main Nucleotides will look like.

Once we made the fluorophores we used Mass Spectrometry to make sure no additional salts or non nucleotide activity occurred during the linking process. In our lab we use MALDI-TOF Mass Spectrometry. MALDI-TOF stands for Matrix Assisted Laser Desorption/Ionization – Time Of Flight. It describes the way a sample is converted into charged particles, or ionized, and what method has been used to separate the different mass particles after charging. In order to use MALDI-TOF we take one microliter of sample and mix it with one microliter of matrix. We then put the matrix-sample

mixture onto a metal chip which we then place into the MALDI-TOF Each of the dots on the chip represent different experiments that are done. The Matrix helps crystallize the sample which is why the dots appear white. After taking the MS reading the graphs we got looked like so for each of the nucleotides. On the left is the Mass reading of the nucleotide with the fluorophore attachment and right is the reading without the fluorophore attachment. Since we know the mass of the fluorophore we can check to see if any additional salts formed.