RNA, despite being really powerful, is really delicate and has to be treated with extreme TLC. And, yes, kept really cold unless it has some serious protection. RNA is similar in a lot of ways to its molecular “cousin” DNA, but it’s different in ways that make it more “fragile” – good for making temporary copies of protein instructions in your cells (called messenger RNAs (mRNAs) but not so good for long-term use. Here’s why…
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). A & G have 2 rings in them and are called “purines” and C, T, & U have a single ring and are called “pyrimidines” – you can remember this with the mnemonic “pure As Gold” (and then think of “pure” as having it all – 2 rings instead of just one). http://bit.ly/nucleicacidstructure
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 grab one (get protonated). Once protonated, they don’t need sugar to satisfy their needs, so they break off 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 a letter should be there, but it has to guess what that missing letter is, and 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. BUT when you’re working in a test tube you have to make sure that pH is “safe” too! In science-y terms, the nucleobases have a pKa that’s substantially higher than the cellular pH.
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/phacidbase
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.
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, (electrons are the negatively-charged counterparts to protons – atoms share pairs of them to form covalent bonds). 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 (really low pH) because the phosphate groups are happy being negative since 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. https://bit.ly/rnaseadepc
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
If you want to learn more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 ⠀