I have really been enjoying the Society for Biomaterials conference! There is a lot of great work here going on at the nano / bio interface. A lot more people are working with hydrogels and wound healing than I would have assumed too. It’s amazing! I got to talk to companies who deal with hydrogels and the measuring of hydrogel and tissue stiffness. It’s been quite fun as well to talk to people during the poster sessions. I learned a lot. I got to hear Dr. Jordan Green from Johns Hopkins give a talk yesterday. I also heard several interesting talks on gene and mRNA delivery techniques using nanoparticles. It was all in all a great conference. I highly recommend this one if you are at the nano / bio interface!
I was able to give a 10 min talk, last day last slot, Saturday April 14th. In spite of the unlucky placement, it’s such an honor. I’d almost rather be last, because that way the sleep audience will go easier on me for questions. I’ll try to highlight some of the people and companies that I met at the conference later.
A lot has happened in this new year of 2018. One of them is that I went to the Biophysical Society Meeting in San Francisco! I met a lot of great people, and my friend Gokul dragged me out to see the Golden Gate Bridge, for which I am especially grateful. I never would have done it without him. My research has focused on building on my published work, moving splicing of RNA using my nanozyme into cells. I also have a second project looking at the mechanism of how DNAzyme functionalized particles work inside cells.
Family in 2018
Meanwhile, family has been busy too. Isaiah has continued to grow. He started walking on his own as of Superbowl weekend, when he took his first tentative steps. My grandmother has moved in with my parents since she can no longer live on her own, due to some mild dementia. So, my mom is watching both her and my son Isaiah while I go to work. It’s a lot for her to handle, but so far we are making it.
Today: This week, I’m giving group meeting, so I am preparing for that. I had the pleasure today of meeting and speaking with former Mirkin graduate Dr. KiBum Lee. We students took him to lunch to Saba, a popular Italian joint at Emory Point. I really enjoyed meeting him and hearing about his research, as there is a lot of overlap between us. I really hope we get to collaborate one day.
Tomorrow: Tomorrow, I am running a gel to verify some of Kevin Yehl’s work, and prepare for some of my own. I’m salting gold nanoparticles (AuNPs) – which means – I am making more DNAzyme-AuNP conjugates. I am also changing the media on my cells, in preparation for my attempts at validating my splicing reporter, once I can get the plasmid from Oskar, Emory’s cloning expert. I am also discussing a piece of literature with a member of the Heemstra lab. We may collaborate on a new idea that Khalid and I have been cooking up. Finally, there is subgroup meeting at 4 p.m., where I am pitching this idea, and showing latest data. It will be a busy day.
Isaiah is also having a check-up and more shots. On April 14th, I give a 10 min talk at the Biomaterials Society Conference in Atlanta. More soon!
Kevin Yehl, who graduated from Khalid Salaita’s lab about 2 years ago, came back for biophysics week (Monday, March 12, 2018) to give a talk about his post-doc work in the Lu Lab at MIT. It was amazing! He summarized his graduate studies, then gave us a sneak peek at what he’d been up to the last two years. It won’t disappoint. Can’t talk about it right now though as it’s still under review. My goal that week was to spend as much time with Kevin as possible. He used to be my mentor or a sorts, in the lab. I’d discuss data with him and my project was an idea of his that he never finished, and that I took under my wing.
I met him in the morning and helped him get setup with his computer for his talk. I went to one of the subgroups he attend – the rolling motors group – which I don’t usually go to. After the talk, I went out with him and Khalid and a collaborator. Then, finally, I had dinner with him along with other students from around Emory. All in all, it was a fantastically fun day because I got to spend most of it with Kevin! I’m going to try better to keep in touch with him.
The answer to the above question is: for fun! This article is at once quite silly. Those who need to run nap-25 columns already know how to do it, or they can find out in a much more handy way by reading GE Healthcare’s awesome protocol here. The only ones who might not know how to run a nap-25 column are those who are not scientists, and most of them won’t care to know! Nevertheless, in case you fall into the category of non-scientist who would like to know about some aspects of esoteric science, I write this note to you!
What is a nap-25 column? Why use one?
Nap-25 columns are used to purify DNA. In our case, we buy thiolated DNA from Integrated DNA Technologies (IDT) – DNA that has a disulfide in it – then reduce this DNA with DTT. A small molecule linker comes off, and this linker and the DTT must be removed from the DNA before we attach the DNA to gold nanoparticles. Thus, we use the awesome nap-25 column. (There is also a nap-5 column. Nap-25 columns are used to purify larger amounts of DNA, up to but not exceeding 70 nmol of a ~33 nucleotide sequence.)
Using a nap-25 is as easy as 1-2-3!
First, to use a nap-25 column, you…
Connect said column to a ring stand with a clamp.
Cut off the tip of the column at the end so the buffer can begin to drain.
Take off the cap.
Then, let the buffer drain from the column and flush it three times with the appropriate buffer with which you want to elute the column – in our case – Nanopure water. I mark the column with a permanent marker with tick marks to indicate which wash I am on so I don’t forget. To run a sample through the column, you…
Add 1 ml of DNA sample and allow it to enter the column completely.
Add 1.5 ml of Nanopure water and allow it to enter the column and drip through completely.
Elute the DNA with 2.5 ml Nanopure water and collect the liquid.
You can then measure the absorbance at 260 of the DNA on a Nanodrop instrument to find the concentration. Done!
Adding the sample to the column: two fluorescent DNAs as examples
When the DNA has run half-way through the column, you can still see it due to the DNA’s color
I had such a fun time at the Cuneo’s today for Thanksgiving! The Cuneo’s are my sister-in-law’s family. She is the eldest of 12 and has a four year old brother! It was so neat to be able to share a table with that many people all at once. How special! The turkey was smoked and extremely tasty. One was plain and one was wrapped in bacon. There was every kind of food imaginable there, even home-cooked pumpkin pie! Isaiah did so well and didn’t cry at all. He slept on the way to and from also. I was extremely impressed and so glad he could enjoy the company!
On Thanksgiving, it seems the perfect time to list the things for which one is thankful. I feel I have too many to count, but I shall try.
Thanksgiving: What Am I Thankful For?
Jesus: I have real reason to be thankful for my Lord. Not only does He provide for me all the things I have and am thankful, but He allowed me, a mere nobody, to know I am loved. That is my identity – that I am loved by Him. Without that, my life would be a dry husk that doesn’t make any sense. I never expected to know that. “Jesus loves me,” were just words on a page to me until he emblazoned them onto my heart in 2015. I found He loves not just me but every person uniquely. I am so grateful to be His handiwork, his co-laborer and His daughter. I didn’t think I was important enough for Him to really care about or notice me. But, I found out, He is SO great, that He notices us who are so small. Because He lives in my life, I have hope, excitement for the future, and a hunger to know Him more. I wonder at the great adventure He has in store for John and I and am excited to live it!
John: John daily loves me through watching Isaiah, his warm hugs, doing things for me and our lovely chats about books. His love and support are a constant I can depend upon. He supports me in everything I do. When I’m worried or scared, he prays with me and lends me strength through just being there. He gave up his career for me. Without John, I would be lost. I love him to the depth of my soul and I wouldn’t be the same without him.
Isaiah: Isaiah has taught me so much, even in his short 11 months of life. He taught me how selfish I really am, and made me trust God more. He’s also given me a window into the world of childhood and allowed me to be a kid again. Being a mom is not glamorous. It is hard work, and not very fun most of the time. But I wouldn’t trade it. I want this. I want Isaiah in my life and I can’t wait to see who he will grow into.
Mom: Mom watches Isaiah for me every day while I am at work. It is a labor of love. It is a lot of hard work! Everyone with a baby knows it. Because she watches him, and not a daycare, I can rest in peace, knowing he is being watched by someone I trust and who loves him. It also gives me more flexible hours with which I can work. Mom also comes over to my house and help me clean it on a semi-regular basis. Mom does dishes and cleans the cat litter pans. Mom also makes dinner every night and lets me eat some. I would be lost without my mom. She is a pillar in my life for good, growth and strength.
Dad: Dad mows our lawn sometimes during the summer, helps my mom with Isaiah before I come over, and is a great entertainer and granddad. He will be Isaiah’s only living granddad. I am so thankful that Isaiah still has a granddad to love and he does love him. Isaiah finds dad or “Opa” as we call him, infinitely entertaining and funny.
Khalid: Khalid makes this list in a big way. I consult him about my experiments and he is always brimming with ideas. I am constantly amazed at his knowledge base – it’s very inspiring. I hope that I can build one as extensive. Khalid is to me at work what my parents are to me at home. I am so honored that I get to collaborate with someone so ingenious. Talking about science and batting ideas back and forth with him is one of my favorite pastimes. It’s so mentally stimulating, engaging and fun! Khalid is also extremely supportive of me, of John, of Isaiah and my career. I appreciate that he understands when family matters have to trump work, and doesn’t get upset. With him at the helm, I can know that my future in science is in good hands, the lab is in good hands, and I can focus on my experiments in peace knowing all is right with the world.
Brenda: My friend and mentor Brenda Harmon helped shape me into the person I am today. She was my organic chemistry lab teacher, but taught us how to think by making our own protocols and how to be true scientists. She is the reason I fell in love with the chemistry behind the biology. And she is the reason I didn’t quit science when it got down near impossible to keep going. Without her, I wouldn’t be in grad school now. She continues to be my best friend, one of my biggest supporters, and I love her to death.
Pam: There is no person I know like Pam Martin. She has the most beautiful spirit in the world and I am so lucky to call her friend. She prays with me about anything and everything in life, and I share and talk about with her all my deep ponderings on life and hear hers in turn. We share our dreams, our fears our joys. With her prayers behind me, I feel protected and loved. She is such a support!
Graduate school: I cannot tell you what graduate school means to me. I am so thankful to be in it, getting the opportunity to pursue my dream of being on the cutting edge of knowledge and even getting paid for it! That is all I have ever wanted: not a specific position (like being a PI), not a specific place (like Berkeley or MIT), not even the degree itself. I just want to be on the cutting edge of knowledge as long as I can, and I am doing that. My most intense moment of euphoria in my whole life was the Emory visitation weekend, when I was listening to Khalid describe the project I am working on now and have helped make a reality. It was in that moment that I realized that what I had been working toward so long – doing research – I would finally be able to do! I could finally study and experiment whatever I wanted! I had freedom! I could do real research! I was a valuable, recruitable resource! My dreams were becoming reality! I already have my dreams, even if I never get anything else. Graduate school has also given me friends. I didn’t have a lot of friends growing up, being shy, and none in college, because I studied too much – but now – in grad school, I actually have friends in my classmates, and it has been a real joy to share life with them.
ARCS: The ARCS scholarship is an incredible blessing. It has allowed me to stay afloat, with a baby in tow. It was extremely validating. I felt such relief, that I could be among such an elite crew of people honored with the award. My sponsors Sara Jean and Fred Burke are wonderful and amazing people. I am so blessed to know them. Their support has meant the world to John and I. It has also been awesome to get to go to events where I can network with some truly awesome scientists, feel honored and valued, for who I am and what I do. Those moments can be rare in science. And I value every one.
My first paper: My first paper was recently published as a “just accepted” manuscript in ACS Chemical Biology, as I am sure everyone knows, as I cannot stop talking about it. Having a first author paper has been a dream of mine ever since I was a freshman in college. Now, thanks to everyone’s help and support, it has become a reality. I cannot describe my joy at seeing my work for the last four years in print, not to mention that, at least 60% of the paper is completely my original words! Khalid helped shape the words, and he changed some things, but much of the writing stayed true. He did not completely re-write it. It remains my own. That makes me so proud.
My house: I love our house. John and I waited a long time before buying one, and we bought one large enough to support multiple kids in the future. I am so grateful to have a place to call my own and to raise a family in. I never dreamed I would own a place like our home that is so fancy-looking and great! I love it. It’s perfect for us.
My computer(s): I have a desktop and a laptop. Both these computers were purchased to be top-of-the-line and fast. I love fast computers. Most everything I love doing is on a computer: writing, blogging, reading science articles, building websites, some minor gaming when I can – it’s my connection to knowledge and to social media and the rest of the world. Therefore, I love my computers and am so thankful for them every day.
My cats: I have always lived with cats but never had my own until recently, when we purchased three orange kittens in 2016. Having an orange kitten in particular was a childhood dream of mine. Our cats also taught me responsibility, similar to having a baby, but less work. Still work though – having my own cats is a lot different than growing up with them. They are more special and also more work! And I do love them dearly. I can tell them apart by their meow’s and they each have their own unique personalities.
Books: I love books. I’m glad they exist. I love reading them, especially about God, and getting transported into other worlds not my own. I love trying to write books. I love collecting them and putting them on shelves. Books are all around just awesome.
This list is just a sprinkling of the things that are dearest to my heart, that I think about all the time and for which I am most grateful. I wouldn’t be who I am today without these people and things. Thank you from the bottom of my heart for being a part of my life – every one of you – whether I know you in passing or not. I am so grateful for each and every one of you. Life is so much more exciting when it is shared.
Site-SelectiveRNASplicingNanozyme: DNAzyme and RtcBConjugates on a GoldNanoparticle
(In this post, I use blue notes to describe details of my science to an educated non-science audience.)
I am overjoyed to announce the going live of my very first scientific article in the journal ACS Chemical Biology! It can be found here in PubMed or here at the ACS Chemical Biology website (1).
I have waited SO LONG to tell you all about my work! I have been working diligently on this system (with some side projects as well) for about four years.
In it, I describe the construction and activity of my RNA splicing nanozyme! It is built using 10-23 DNAzymes and the E.coli ligase RtcB attached to a gold nanoparticle scaffold. Note that this nanozyme only removes introns. It cannot add exogenous RNA into a strand. The complete nanozyme construct functions currently at 10% efficiency. However, we have some plans to boost that yield in the future. When used in excess and not attached to a particle, the enzymes themselves can achieve up to 66% yield or higher in some cases (data not shown). RtcB is the limiting reagent, as it is likely to be a single-turnover enzyme in E.coli. The amount of RtcB on the nanozyme is also far less than the attached DNAzymes: only between 1-5 RtcB per particle and between ~50-100 DNAzymes per particle.
What is a 10-23 DNAzyme?
DNAzymes are short pieces of DNA about 33 nucleotides long that can act like enzymes. The particular DNAzyme I am using in my system works enzymatically by cutting RNA strands. It can be targeted to cut anywhere along a particular RNA target. An RNA ligase is a protein enzyme that joins RNA strands together. By coupling these two enzymes with each other on a gold nanoparticle, RNA “scissors” and RNA “glue,” this nanoparticle machine, nanomachine or “nanozyme” can effectively cut up RNA and paste it back together. In my paper, I use its activity to remove a short segment from an RNA strand.
Two 10-23 DNAzymes (DZ) are shown at left. These are the ones I used. The blue section is the catalytic core, responsible for the enzymatic activity of the DNAzyme. The core chelates (or latches onto) a manganese ion, which assists in shaping and activating the core to perform its catalytic function of cutting RNA.
What is RtcB?
RtcB is a protein – more specifically – an enzyme called a ligase that ligates (or joins) RNA molecules together. It is the “molecular glue” of my nanozyme. It is thought to be involved in RNA repair in E.coli. In mammalian cells, it acts differently than the E.coli protein (and also has another name – HSPC117). It associates with other proteins such as archease (2,3) and DDX1, is multi-turnover (2) and is involved in tRNA splicing in mammalian cells (3). It is also involved in upregulating the unfolded protein response (4). We chose RtcB as the ligase for our nanozyme because it can specifically act on DNAzyme cleaved ends (2’3′-cyclic phosphates and 3′-OHs), ligating them directly, rather than the longer “healing and sealing” mechanism preferred by other ligases.
What is the end game? Why does this nanozyme matter?
The home run goal for this nanozyme would be for it to be used to correct genetic defects in RNA inside cells – though that has still to be realized. It is also the first example of DNAzymes collaborating with a ligase for RNA splicing, and a completely new method of manipulating RNA in vitro and potentially one day, inside cells.
With much thanks!!
I cannot mention this project without thanking the people involved who helped make it happen. At the top of the list is my PI, adviser and boss Khalid Salaita. He is the most amazing mentor I could hope for and has been a cheerleader behind me all the way, through the long process of trouble shooting and getting this nanozyme working. Oh the stories I have to tell! Also high on the list is Kevin Yehl, who originally thought of the idea of combining DNAzymes and RtcB, but didn’t have the time to work on it. So, I took up the torch and carried it forward. I often turned to Kevin, during the years that followed, for advice and help interpreting data. He’s known as a wizard for good reason. Then, there’s Kornelia, who helped make a batch of cysteine labeled RtcB for me, Brendan, who helped me with flow cytometry showing cell uptake of the nanozyme, and Roxy, who did all the FLIM experiments needed to show that RtcB is bound to the gold surface. Then, there are the unsung heroes like Yun, who I love to death. She was my special “stress friend” my first three years as a graduate student, meaning that when I was stressed, I would find her and cry on her shoulder. She also taught me loads about how to carry myself and present better. She now lives in China with her husband. Yuan was also another special friend who supported me undyingly during my work and I so appreciate her kind words, encouragement and random cookies she would give me. Thank you to all you amazing guys!! Without you, I wouldn’t have been able to endure to see this project through.
If you have comments or questions please email me! I absolutely love talking about this work.
1. Petree, Yehl, Galior, Glazier, Deal, Salaita. DOI: Site-Selective RNA Splicing Nanozyme: DNAzyme and RtcB Conjugates on a Gold Nanoparticle. ACS Chemical Biology. DOI: 10.1021/acschembio.7b00437
2. Desai, Beltrame, Raines. (2015). Coevolution of RtcB and Archease created a multiple-turnover RNA ligase. RNA. http://www.rnajournal.org/cgi/doi/10.1261/rna.052639.115.
3. Popow, J., Jurkin, J., Schleiffer, A., & Martinez, J. (2014). Analysis of orthologous groups reveals archease and DDX1 as tRNA splicing factors. Nature, 511(7507), 104–7. http://doi.org/10.1038/nature13284
4. Kosmaczewski, S. G., Edwards, T. J., Han, S. M., Eckwahl, M. J., Meyer, B. I., Peach, S., … Hammarlund, M. (2014). The RtcB RNA ligase is an essential component of the metazoan unfolded protein response. The EMBO Journal, 15(12), 1278–1285.