This week I’m “at” the RNA Society’s Annual Meeting where I “gave” my first ever official oral presentation. Conferences like these are usually big, crowded events where scientists from around the world jam into packed rooms to hear talks and squeeze through tangles of people to get a look at the hundreds of research posters tacked up on corkboards spaced out around exhibit halls. Not exactly social distancing… So this year, in order to protect all of our coworkers, and everyone else we could possibly accidentally infect, the conference was held online, with posters and talks hosted on a web platform. This layout may be unusual, but RNA researchers are used to protecting our coworkers – our molecular coworkers that is – because RNA, despite being really powerful, is really delicate and has to be treated with extreme TLC. A bit about why and then a few notes about the conference.
First off, what is RNA? It’s a nucleic acid – like the nucleic acid you’re probably more familiar with, DNA, it’s a chain of NUCLEOTIDES which have a generic “sugar-phosphate” part that allows them to link together, connected to a unique nitrogenous base or “base” part (C, G, A, and T (in DNA) or U (in RNA) which allow for specific base-pairing between strands (C to G and A to T or U). These bases of your RNA and DNA really are bases (as in things that can accept a proton (H⁺) – thankfully only weak ones! If a solution is acidic, meaning there are lots of protons around (low pH), A or G bases can get protonated. Now, it doesn’t need sugar to satisfy its needs, so it breaks that relationship off which can lead them to leave the sugar-phosphate backbone they’re attached to -> DEPURINATION
This leaves you with a sort of “hangman” like situation where you’re left with something like S_METHING. Your cell knows that something should be there, but it has to guess what that missing letter is, but it might get it wrong. Is that supposed to be “sOmething” or “sAmething” – which are NOT the “same thing”!
Thankfully, your cells maintain a pH of about 7.4, which is too high for them to protonate much. In science-y terms, the nucleobases have a pKa that’s substantially higher than the cellular pH. The pKa tells you the pH at which 1/2 of the groups will be deprotonated. So a higher pKa means that you have to swamp them with more protons (make conditions more acidic) to get them to take one. BUT when you’re working in a test tube you have to make sure that pH is “safe” too!
pH is a measure of how many protons (H⁺) there are in a solution. The more protons, the lower the pH because it’s an inverse log scale and the more acidic. And the fewer the number of protons, the higher the pH & the more basic. Unlike most covalent bonds, which are strong, many bonds to hydrogen are somewhat “looser” – the H⁺ can come and go depending on the pH. More here: http://bit.ly/2CcLQX7
We usually think of nucleic acids as having a negatively charged backbone because, under physiological (typically bodily) conditions, the phosphate groups are negatively charged because they have “extra” electrons, the negatively-charged counterparts to protons, which atoms share pairs of in covalent bonds. But the phosphates are only negatively charged because they’re in their deprotonated form. If they grab onto a proton (protonate) they’ll become neutral because the positive charge of the proton will cancel out their negative charge. But they’ll only do this if there are TONS of free protons around because the phosphate groups are happy being negative because they have something called resonance stabilization – they kinda play “hot potato” with the “extra” electrons, evenly distributing that charge. So you have to get things super acidic before you have to worry about this.
So normally you have a negatively charged backbone & NEUTRAL nucleobases. BUT the nucleobases can also give and take protons. When they do, it disrupts the base pairing – this denatures double stranded DNA or RNA – it removes it’s “natural” shape and separates the strands. But the strands remain strand-y and unbroken and, unlike with the depurination, the nucleobase stays attached to the sugar.
So, even though we don’t have to worry about the backbone protonating until we reach crazy-low pHs, milder acidic conditions can still cause problems – both with disrupted base-pairing and increased depurination -> low pH is no good.
But you don’t want to go too far the other way either (too few protons (too high a pH)) or you’ll pull off the protons that need to be there for the bases to bind each other. And in RNA you have an additional, very important one to worry about
The main difference between RNA & DNA is the thing that makes them “R” or “D” -> RNA has a RIBOSE sugar and DNA has a DEOXYribose sugar -> RNA has an “extra” oxygen. Usually this oxygen is bonded to a hydrogen to give you a hydroxyl (OH) group, but if there aren’t better sources around, an OH⁻ can pull that H⁺ off…
That leads you with a O⁻ on the hunt for some + charge. And it doesn’t have to look far. That phosphate group might be negative overall, but the Phosphorus (P) at its center doesn’t get to participate in that electron hot potato fun, so that P is actually slightly positive. So the O⁻ attacks it. But then that phosphate would have too many bonds, so it kicks off one of it’s old oxygens, the one connected to the base below it -> the chain breaks
Initially you get a 2’,3’-cyclic monophosphate derivative (the sugar’s “legs” kinda playing with each other criss cross applesauce style), but this can then react with water to form a mix of 2’ and 3’ monophosphate derivatives -> basically the sugar takes back a proton. but it can do this on either “leg” so you get 2 products. Note that neither of these are where that phosphate was before. In “normal” nucleotides that phosphate is at the 5’ position (like the “left arm”) but now it’s at one of the legs – the left one or the right one. It’s never on the “right arm” because that’s where the nitrogenous base goes.
So, when working with RNA or DNA, pH is one thing that researchers have to be careful to monitor. But with RNA, there’s another really important thing to worry about – RNA can get “chewed up” by enzymes called RiboNucleases (RNases)) which are basically everywhere because they offer a sort of “generic” protection from RNA viruses that try to get near us
🔹 we secrete them in our tears, saliva, mucus, & sweat
🔹 bacteria & fungi also secrete RNAses to protect themselves
But RNAses DON’T protect us from experimental failure! So we have to be super careful when working w/RNA. We use precautions including special cleaners or just good ole ethanol to keep our work area RNase-free. Our lab even has a designated RNAse-free room (that doubles as the hot room (where we do radioactive (“hot”) work) and an RNAse-free microcentrifuge in our main lab.
So -> At low pH you have to worry about depurination or RNA & DNA, as well as disrupted base pairing. At high pH you have to worry about disrupted base pairing of DNA & RNA as well as hydrolysis of the backbone of RNA
So, now that I’ve told you about some of the steps RNA scientists take all the time to protect our RNA friends, some more notes about the conference.
Firstly, I want to give a huge shoutout of gratitude to the International Union of Biochemistry and Molecular Biology (IUBMB) and the RNA Society for providing me funding to attend. Even though I didn’t end up getting to travel to Vancouver and meet a bunch of awesome people in person, it’s still been a tremendous opportunity and it was an honor (albeit a bit weird and still super nerve-wracking) to be able to give a talk on my work studying a protein-production regulation method called RNAi).
But the real highlights of the conference for me were the two IUBMB-sponsored keynote lectures (whereas most talks are short and selected from submitted abstracts, keynotes are long and the speakers are invited based on their record of awesomeness, without them having to “apply”). Dr. Jack Szostak, a professor at Harvard, gave a molecular-mind-boggling talk about how RNA could have evolved from non-life things and given life to things. Speaking of Dr. Szostak – one of my favorite grad school interviews was with him – when he asked if I had any questions, he was probably referring to questions about the school, but I asked him about whether he thought life could evolve on other planets without water…
The second keynote lecture was from Dr. Melissa Moore from Moderna (yeah, *that* Moderna). She talked about how mRNAs (the messenger RNAs that serve as copies of gene recipes that get read by the protein-making complexes called ribosomes to make protein) can be used as therapeutics. I was afraid it was going to be all coorporate-y, but Dr. Moore was totally “down to earth” and hard core science-y. She actually had a highly successful academic career before joining Moderna, and it showed. She also had an inspiring message about the importance of basic research (research aimed at “just” understanding things rather than putting them to immediate use) and how Moderna couldn’t be doing what it’s doing if it weren’t for all the basic scientists who have done and continue to do what we’re doing.
Now, more than ever, as we face an international (and biochemistry-related) crisis, I am incredibly grateful to be able to serve as Student Ambassador for the International Union of Biochemistry and Molecular Biology (@theIUBMB) that has helped me recruit translators and share the translated versions around the world. This post was just one in my series of weekly “Bri*fings” – this week a “Bri*fing from RNA2020!”
If you want to learn more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0