Origin of the 260/280 ratio?

Question

This is not a duplicate of all the other 260/280 ratio questions, I already know that DNA is supposed to be 1.8 and RNA is supposed to be 2.0. However, this might be more appropriate for chemistry, and will move it if I have to.

My question has to do with the Poly A tails on mRNA. I've been doing in vitro transcription and tailing for a while now and have noticed that the untailed RNA has a 260/280 of about 2.0, like you'd expect, but successful tailing results in ratios from 2.15 to 2.45, with higher ratios correlating to better band shifts on agarose gels, implying a longer tail. I suspect the reason for this is because the adenine has a much higher 260/280 than the other bases, so if you add a lot of adenines to the 3' end of the RNA you add much more 260 absorbance than 280 absorbance.

After seeing this I tried to come up with a mathematical method to estimate tail length based on 260/280, since gels are annoying, while I can get the ratio directly from the nanodrop.

I started by using the 260/280 for each nucleotide, A: 4.50, G: 1.15, C: 1.51, U: 4.00, T: 1.47 source here: 1. I then took the sequence for the Luciferase gene I'm using for mRNA and counted the nucleotides, 449 A, 566 C, 470 G, 374 U. I tried to get the weighted average of the 260/280 for my RNA.

A: 449 x 4.50 = 2020.50
C: 566 x 1.51 =  854.60
G: 470 x 1.15 =  540.50
U: 374 x 4.00 = 1496.00
Sum:            4911.60

Nucleotides: 449 + 566 + 470 + 374 = 1859
Sum / Nucleotides : 4911.60 / 1859 = 2.64

2.64 is clearly not 2.0. Even accounting for the potential effect of pH and ionic strength on RNA 260/280 ( acidic solutions have lower ratios ) I can't explain why the calculated ratio is so high. Even if we assumed an RNA with 25% of each base, the weighted average is still (1.15 + 4.50 + 1.51 + 4.00)/4 = 2.79, even higher than for my real sequence.

Doing the same calculation for DNA, (1.15 + 4.50 + 1.51 + 1.47)/4 = 2.16.

So why don't my calculated ratios for RNA and DNA 260/280 match the "ideal" ratios of 2.0 and 1.8?

Answer 1

Here's one explanation: It's not the base composition that determines UV absorbance (260 nm), but whether the nucleic acid is in a single- or double stranded state.

Promega cites Griffiths biochemical methods that claims that double-stranded DNA absorbs UV less strongly than denatured DNA due to the stacking interactions between the bases.

I think your calculations must be wrong, since you can not expect a polymer to have the same spectrometric behaviour as the sum of all monomers. The absorbance of a molecule depends on its surroundings (like solvent, or nearby chemical groups). Bases within a DNA strand have much less contact to the solvent (H2O) when compared to a single nucleotide. In general, bases in ssRNA (single stranded RNA) and dsDNA interact with each other via aromatic pi-stacking. This affects the energies of the electrons that are responsible for light absorbance.

Most importantly, each base inside a dsDNA strand is engaged with a hydrogen bond from the other strand, which (I presume) significantly shifts electron energies and hence also its absorbance behavior. This might also be the reason why low pH decreases UV absorption of RNA (260 nm), since hydrogen acceptors are protonated just like in the double stranded state.