Novel Sensitive Probes for Detection in Living cells and Complex Media

NU 2019-117B

INVENTORS

Chad Mirkin*

Sasha Ebrahimi

Devleena Samanta

Ho Fung Cheng

A new class of signaling aptamers which contain a single modification, yet can be used to detect a broad range of targets

ABSTRACT

Aptamers are oligonucleotide sequences that can be evolved to bind to various analytes of interest. Northwestern researchers have developed a general design strategy that transduces an aptamer-target binding event into a fluorescence readout via the use of a viscosity-sensitive dye. Target binding to the aptamer leads to forced intercalation (FIT) of the dye between oligonucleotide base pairs, increasing its fluorescence by up to 20-fold.  The application of this technology is particularly useful in challenging scenarios where studying analytes in living cells and complex media with high sensitivity and specificity remains difficult. Fluorescence-based methods offer advantages like simplicity, low-cost, high-throughput, and ability to multiplex. They demonstrate that FIT-aptamers can report target presence through intramolecular conformational changes, sandwich assays, as well as target-templated reassociation of split-aptamers, showing that the most common aptamer-target binding modes can be coupled to a FIT-based readout. Further, this strategy also can be used to detect the formation of a metallo-base pair within a duplexed strand and is therefore attractive for screening for metal-mediated base pairing events. The strategy can also be used to deliver different molecules, via functionalization of nanoparticles, for diagnostic and theranostic purposes in vitro.  Taken together, FIT-aptamers are a new class of signaling aptamers which contain a single modification, yet detect a broad range of targets.

APPLICATIONS

• Report target presence through intramolecular conformational changes, sandwich assays, and target-templated reassociation of split-aptamers in cells and complex media
  • Detects and images analytes
  • Quantifies analyte levels
  • Regulates and detects analytes for thernostic purposes
• Screening for metal-mediated base pairing events by detection of the formation of a metallo-base pair within a duplexed strand

ADVANTAGES

• Reduces false-positive signals typically associated with fluorophore-quencher based systems (No requirement of quencher)
• Quantitatively outperforms FRET-based probes by providing up to 15-fold higher signal to background ratios and superior readout kinetics
• Allows rapid and highly sensitive target detection (nanomolar range) in complex media such as human serum
• Enables enhanced delivery of detection elements in live cells without transfection agents
• Requires only a single modification in its target of interest to fluoresce
• Allows spatial monitoring and organelle-specific analyte mapping in live cells

PUBLICATIONS

Ebrahimi S, Smanta D, Cheng H, Nathan L and Mirkin C (2019) Forced Intercalation (FIT) Aptamers. Journal of American Chemical Society. 141: 13744-48.

IP STATUS

U.S. and Europe nationalization has been filed.