Nucleotide Analog Interference Mapping (NAIM) is a chemogenetic
approach that makes it possible to simultaneously, yet individually, probe
the contribution of a particular functional group at almost every RNA
nucleotide position in a single experiment1. The method utilizes
a series of 5’-O-(1-thio)nucleoside analog triphosphates in a modification
interference procedure that is as simple as RNA sequencing. In a NAIM
experiment the smallest mutable unit is not the base pair, but rather
the individual functional groups that comprise the nucleotides. Because
the modification or deletion of a particular functional group within an
RNA can severely affect its activity, this approach makes it possible
to efficiently determine the chemical basis of RNA structure and function.
Instead of synthesizing a series of RNAs with chemical
substitutions at specific sites, NAIM utilizes a combinatorial approach.
Each nucleotide analog is prepared as a triphosphate for incorporation
into the RNA during DNA templated in vitro transcription. The nucleotide
analogs used in NAIM include a specific chemical alternation to the base
or sugar, and an α-phosphorothioate substitution which serves as
a chemical tag. The nucleotide analog triphosphate is randomly incorporated
into an RNA transcript, where the phosphorothioate linkage can be selectively
cleaved by the addition of I2 to produce a series of RNA cleavage
products whose lengths correspond to the sites of analog incorporation2.
By radioactively or fluorescently tagging one end of the RNA transcript,
cleaving the RNA with I2 , and resolving the cleavage products
on a denaturing polyacrylamide gel, the sites of analog incorporation
throughout the RNA can be individually assayed and used for interference
analysis. The phosphorothioate tagged nucleotide analogs make it possible
for all of the positions in the RNA to be assayed individually for functional
group modification in a single experiment.
Because the phosphorothioate chemical tag is independent
of the nucleotide analog whose location it reports, NAIM is generalizable
to any analog that can be incorporated into a transcript by an RNA polymerase.
A typical NAIM experiment is comprised of four steps. (i) The phosphorothioate
tagged nucleotide analog is randomly incorporated throughout the RNA to
create a family of transcripts, each of which contains only a few substitutions.
A different transcription reaction is performed for each analog. (ii)
The functional RNA variants in the population are separated from the inactive
transcripts. The exact nature of the activity assay is specific for the
RNA being studied, but could include affinity chromatography, native gel
electrophoresis, filter binding, selective radiolabeling, etc. (iii) The
phosphorothioate linkages in the active and unselected RNA populations
are cleaved by I2 addition to mark the sites of analog incorporation
within each molecule. (iv) The individual RNA fragments are resolved by
gel electrophoresis and visualized by autoradiography. Sites of analog
substitution that are detrimental to function are scored as gaps in the
sequencing ladder among the active RNA variants. Because every position
in the sequence is a unique and independent band on the sequencing gel,
a single screen can define the effect a particular analog has at every
incorporated position within the RNA. The approach is applicable to any
RNA that can be transcribed in vitro and has an assayable function
that can be used to distinguish active and inactive variants. RNA functions
that are amenable to this approach include catalysis, folding, protein
or ligand binding, and the ability to act as a reaction substrate.
NAIM utilizes α-phosphorothioate tagged nucleotide analogs,
each of which includes an incremental chemical alteration in the base
or ribose sugar. The most completely developed set of analogs are those
of adenosine, for which eight different analogs have been utilized in
NAIM.3 Five analogs modify the nucleotide base and three modify the ribose
sugar. The base analogs include purine riboside (PurαS), N6-methyladenosine
(m6AαS), tubercidin (7dAαS), diaminopurine riboside (DAPαS), and 2-aminopurine
riboside (2APαS). The ribose sugar analogs all modify the 2'-OH group
and include 2'-deoxyadenosine (dAαS), 2'-deoxy-2'-fluoroadenosine (FAαS),
and 2'-O-methyladenosine (OMeAαS). All of the analogs can be randomly
incorporated into an RNA transcript at an ideal 5% level of efficiency
using either the wild-type T7 RNA polymerase or a Y639F RNA polymerase
point mutant4. Each of these analogs provides specific information about
the chemical basis of RNA activity at almost every incorporated position
in the transcript.
α-Thiotriphosphates are sodium salts in TE buffer,
pH7, 10X concentrates. The concentrations shown are optimal for incorporation
during polymerase reactions.
Also See: Nucleoside Triphosphates
(1) S. A. Strobel and K. Shetty, Proc.
Natl. Acad. Sci. U.S.A., 1997, 94, 2903-2908.
(2) G. Gish and F. Eckstein, Science, 1988, 240, 1520-1522.
(3) L. Ortoleva-Donnelly, A. A. Szewczak, R. R. Gutell and S. A. Strobel,
RNA, 1998, 4, 498-519.
(4) R. Sousa and R. Padilla, EMBO J., 1995, 14, 4609-4621.
Products for Nucleotide Analog Interference
Mapping (NAIM) are supplied under license.