Once we make your RNA, we then have to check if it folds how you predicted. To do this, we use one of many RNA structure determination methods (see here for more info). This is based on the principle that base-paired nucleotides are more rigid or protected than free-floating unpaired nucleotides. When we modify our RNAs, we label the unpaired nucleotides.
Having marked these unpaired nucleotides, we need to be able to find which ones were labeled. By borrowing a trick from retroviruses like HIV and Hepatatis B virus (don’t worry, our trick is safe), we can use an enzyme called reverse transcriptase to read the RNA and make a cDNA strand (essentially the reverse of transcription above). However, rather than reading the RNA to the end, the reverse transcription process will stop at the position of the label.
To allow this process to work, not all unpaired nucleotides in a single strand of RNA will be labeled. If they were, the reverse transcriptase would always stop at the first unpaired nucleotide and it wouldn’t be able to make anything beyond it. Instead, we use small amounts of label relative to the amount of RNA present so that each individual strand of RNA contains only a few labeled unpaired nucleotides at random. With all the strands of RNA combined, we can make cDNA of all sizes between the first unpaired nucleotide and the very end of the strand.
So how do we tell these cDNAs apart? Using capillary electrophoresis, which is essentially a super powerful gel electrophoresis, we can separate our cDNAs from each other and, at a single nucleotide resolution, find out the size of cDNA made from the reverse transcriptase. Since all the cDNA was made starting from the same position, the sizes of the cDNA clearly indicate the positions of labeled nucleotides.
