Abstract:
:Fluorogenic RNAs that are based on the complex formed by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) derivatives and the RNA aptamer named Spinach were used to engineer a new generation of in vitro and in vivo sensors for bioanalytics. With the resolved crystal structure of the RNA/small molecule complex, the engineering map becomes available, but comprehensive information regarding the thermodynamic profile of the molecule is missing. Here, we reconstructed the full thermodynamic binding and stability landscapes between DFHBI and a truncated sequence of first-generation Spinach. For this purpose, we established a systematic screening procedure for single- and double-point mutations on a microfluidic large-scale integrated chip platform for 87-nt long RNAs. The thermodynamic profile with single base resolution was used to engineer an improved fluorogenic spinach generation via a directed rather than evolutional approach.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Ketterer S,Fuchs D,Weber W,Meier Mdoi
10.1093/nar/gkv944subject
Has Abstractpub_date
2015-10-30 00:00:00pages
9564-72issue
19eissn
0305-1048issn
1362-4962pii
gkv944journal_volume
43pub_type
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