Nucleic Acid Structure Determination - Nucleotide Analog Interference Mapping

Nucleotide Analog Interference Mapping

Nucleotide analog interference mapping (NAIM) is the process of using nucleotide analogs, molecules that are similar in some ways to nucleotides but lack function, to determine the importance of a functional group at each location of an RNA molecule. The process of NAIM is to insert a single nucleotide analog into a unique site. This can be done by transcribing a short RNA using T7 RNA polymerase, then synthesizing a short oligonucleotide containing the analog in a specific position, then ligating them together on the DNA template using a ligase. The nucleotide analogs are tagged with a phosphorothioate, the active members of the RNA population are then distinguished from the inactive members, the inactive members then have the phosphorothioate tag removed and the analog sites are identified using gel electrophoresis and autoradiography. This indicates a functionally important nucleotides, as cleavage of the phosphorothioate by iodine results in an RNA that is cleaved at the site of the nucleotide analog insert. By running these truncated RNA molecules on a gel, the nucleotide of interest can be identified against a sequencing experiment Site directed incorporation results indicate positions of importance where when running on a gel, functional RNAs that have the analog incorporated at that position will have a band present, but if the analog results in non-functionality, when the functional RNA molecules are run on a gel there will be no band corresponding to that position on the gel. This process can be used to evaluate an entire area, where analogs are placed in site specific locations, differing by a single nucleotide, then when functional RNAs are isolated and run on a gel, all areas where bands are produced indicate non-essential nucleotides, but areas where bands are absent from the functional RNA indicate that inserting a nucleotide analog in that position caused the RNA molecule to become non-functional