Analysis of the Sequence Preference of Saporin by Deep Sequencing

Ribosome-inactivating proteins (RIPs) are RNA:adenosine
glycosidases
that inactivate eukaryotic ribosomes by depurinating the sarcin-ricin
loop (SRL) in 28S rRNA. The GAGA sequence at the top of the SRL or
at the top of a hairpin loop is assumed to be their target motif.
Saporin is a RIP widely used to develop immunotoxins for research
and medical applications, but its sequence specificity has not been
investigated. Here, we combine the conventional aniline cleavage assay
for depurinated nucleic acids with high-throughput sequencing to study
sequence-specific depurination of oligonucleotides caused by saporin.
Our data reveal the sequence preference of saporin for different substrates
and show that the GAGA motif is not efficiently targeted by this protein,
neither in RNA nor in DNA. Instead, a preference of saporin for certain
hairpin DNAs was observed. The observed sequence-specific activity
of saporin may be relevant to antiviral or apoptosis-inducing effects
of RIPs. The developed method could also be useful for studying the
sequence specificity of depurination by other RIPs or enzymes.