Winter quarter has gotten off to a busy start.  I am taking more units than I was the previous quarter which was also hectic for me; however since I was  able to discover the miraculous ways of time management with a daily planner I have been able to jump into this quarter with the ability to handle a busy schedule.  Luckily I was able to sort out my problems with procrastination and this quarter I have been able to dedicate a steadily increasing amount of time to my research work in the lab.  My graduate mentor had a new baby girl at the beginning of January so I was on my own with synthesizing twenty-one new RNA molecules that I will be working with this quarter.  I ran into a few speed bumps along the way but I knew that I would have some troubles making some of the molecules due to their sequence nature.  When synthesizing RNA we use synthetic DNA that we have purchased and amplify it via PCR.  The cleaner the PCR product of DNA the more easy it is to transcribe the RNA from the complementary DNA.  However many of my molecules were very rich in guanine and cytosine bases which can cause difficulties when performing PCR.  Yet I was able to maneuver around these problems by adjusting my annealing temperatures during PCR incubation.  Now that my molecules have been synthesized I only have to label them radioactively in order to analyze their assembly through gel electrophoresis experiments. For some of the new molecules I have synthesized I will be studying the strength of their ability to fold into a right angle.  This will be done by mixing assembled right angle motifs with a probe molecule that binds most strongly to molecules that are in less rigid right angle structures.  Hence, I can measure the association constant for the probe to various right angle molecules.  We will be testing different molecules that have mutations within the right angle region to test which one folds into a more rigid right angle.  The molecules that fold into a right angle the most strongly will have the smallest association towards the probe.

This fall quarter has gone by in a flash.  Experiencing my first upper division class load was initially mind boggling.  Last year it was fairly manageable to put in a lot of hours in the lab outside of my class schedule.  I was taking sixteen units last quarter and this quarter I took sixteen units as well however the distances between lower and upper division classes seemed to be worlds apart for me.  At the beginning of the quarter when the work load was already piled high I had the amazing opportunity to attend the SACNAS conference, which meant that I would have to finish double homework assignments in advance for physical chemistry and do a make-up lab for biochemistry.  So initially I was flustered with lab write-ups and homework assignments and my flustered state basically continued to nearly the end of the quarter when I finally figured out how I could balance my schedule and allot more of my time to research.  Although I wish I could have reached this conclusion before because it would have definitely minimized my stress levels, I am glad that I can jump into the new year with a new strategy on time management.  The turning point in my quarter happened when I purchased a daily planner.  This little UCSB standard planner helped me visualize my weeks in advance so that I could plan out what needed to be done instead of reaching the day before a lab report is due and then realizing “OH NO!”, I also have p. chem homework due the next day!  And I was able to have stress-free fun in my dwindling spare time because I knew when everything had to be done and I could relax without worrying that I had forgotten about an assignment.  All in all I recommend that everyone gets a planner if you sometimes struggle with procrastination or time management like myself.

Although I just went on about my time management strategies of the quarter which seems to have nothing to do with my research work, I actually believe that there are parts of your life-style that can have a great impact on your research work.  I learned this quarter that managing your time can play a big role in your effectiveness as a researcher.  It was difficult for me to understand this before because my class schedule allowed a substantial amount of extra time that I could spend in the lab working on my research project.  However this past quarter my class and life schedule were more hectic and it cut into the time I could spend doing research, and in some cases I was rushed and didn’t fully document all of my experiments in an organized fashion.  Hence at the end of the quarter I had to go back through my notebook and the pile of papers I had printed out and figure out what electrophoresis gels went with what experiment spreadsheets and what assembly protocols I had used for each experiment.  I managed to link everything together, yet it took a good deal of time, something that could have been avoided if I had simply organized my experiments in my lab notebook as I went along through the quarter.  Now that I have made these mistakes and worked out the kinks in my time management strategies, I am excited to enter the new year with the ability to work more efficiently on my research project especially since I designed a plethora of new RNA molecules that I will start working with next quarter.

In my last post I spoke about the new mutant RNA corner pieces that I was sequencing.  The results I was able to get from the gel electrophoresis experiments were interesting and some of the molecules assembled into a tertiary structure we were expecting while others seemed to behave slightly mysteriously.  One of the mutations at the crux of the corner piece seems to play the largest role in interacting with adjacent bases in order to allow the RNA to fold into a corner piece where all three strands are perpendicular.  This point mutation is a guanine base that has been changed to a uracil.  Another point mutation that it interacts with is an adenine base that buldges out of the helical base pairing structure on the adjacent strand.  These mutations allow the uracil and the adenine to interact with each other to stabilize the corner motif’s tertiary structure.  According to the interactions between these two mutations it should seem that they would be the most critical sequence changes in making the corner piece rigid.  There is also a third mutation that separates the fiber motif from the corner motif, which is a guanine buldge, however it does not seem play as large of a role in causing the molecule to fold into a corner piece.  With the previous thoughts in mind I looked at the first couple of gel electrophoresis experiments I had run and found that the two mutations which seemed to form the most rigid strong species bands on the gel were caused by the A buldge and the G buldge.  My P.I. wondered if I had possibly mixed the tubes, and at the time I was fairly certain that I did not mix up two molecules because I had run the experiment several times and I didn’t think that I would make the same mistake that many times but who knows sometimes.  Later that day however I was talking with my graduate mentor and we remembered that when we were first transcribing our molecules a somewhat shady incident occurred.  When we were precipitating the ethanol washes from our newly synthesized mutant RNA molecules some ethanol spilled onto the caps and smeared some of the labels that had been written on the test tubes.  We thought that we had correctly re-labeled the molecules according to the remains of the smeared sharpie labels, however our weird experimental results made us rethink whether or not we actually did relabel the molecules correctly.

Although my attempts at making a self-programmable RNA cubic structure did not work out as planned despite running several polyaccrylimide gel electrophoresis experiments, I have moved on to working on a project that stems from the same motifs I was using when constructing my cube.  My new RNA molecules that I recently finished synthesizing are all mutants of the corner piece of the cube I was initially working on.  There are three different point mutations that cause the corner piece to fold so that each strand is perpendicular to the other two.  I have synthesized six mutants from the corner piece accounting for all of the possible combinations of one or two point mutations in order to see the effects the three mutations have on the folding of the corner piece.  This should help me to grasp a better understanding of what role each of the mutations plays in the tertiary structure of the corner piece.

Last week I ran my first experiments using polyaccrylimide gel electrophoresis to analyze the assembly of my mutant molecules.  We radioactively label or RNA molecules with phosphorus 32 in and expose the dried gels to a phosphor screen overnight before visualizing them on a scanner the next day.  Hence there is always a tinge of suspense as to how the experiment turned out since I can never tell how the experiment went immediately after.  The most anticipation is generally felt the first time I run an experiment with new molecules because you never know how they will behave.  Sometimes I predict what the results will look like and sometimes it goes my way and other times it goes the completely opposite way; but this time I hadn’t really known what to expect which made the process very exciting when I was able to visualize my first experiment and see that my results could be explained in a reasonable fashion, something that does not always happen.

For the majority of the summer I spent my time trying to assemble a fully programmable RNA nano-particle that was shaped like a cube and  could possibly be used as a scaffold for drug delivery.  It would serve a medical purpose because one would be able to attach a quantified dosage to the clean geometrical shape of a cube that could have good potential to easily pass through the body to its target and then it would biodegrade naturally within the body as your own RNA does usually.  In addition exploring the different folding patterns and interactions of the RNA motifs used to make the cube has led us to discover more information on the folding morphologies and language of RNA.  Through running various assembly experiments of my cubic nano-structure I discovered that some interactions were not as favorable as they seemed on the computer model I initially made.  I found that the RNA pieces were not combining into the clean cut looking cube I had designed using Swiss Protein Data Base Viewer on the computer.  After running a plethora of polyacrylimide gel electrophoresis experiments and finding that a no distinct cube band was forming, only the half cubic square structures were able to be seen as a band, I decided to run another experiment to see if there were any unwanted interactions taking place.  In my “dimer” experiment I tested how each of the eight cubic pieces interacted with each other piece and found that some motifs were interacting to form dimers when they should not have been connecting at all.  At this point I realized that while a computer model can look nice, it can also not predict everything and it takes multiple different approaches to figure out how to get the final product that you want.  I am now working on forming a semi-programmable cube that combines two of the same half square species in a step-wise fashion so as to try and eliminate any mismatched pairings from occurring.