Easy to use RNA circuits for detection and quantification of isothermal amplification reactions

Life Sciences : Diagnostics

Available for licensing

Inventors

  • Andrew Ellington, Ph.D. , Molecular Biosciences
  • Sanchita Bhadra , Molecular Genetics and Microbiology

Background/unmet need

Isothermal amplification (IA) reactions are rapid, one-pot assays that produce higher DNA yield than polymerase chain reaction (PCR) without the need for expensive thermal cycling equipment. Because of this, IA reactions are emerging as affordable and practical assays that may be especially suitable for point-of-care molecular diagnostics where PCR is not ideal—for example, the rapid diagnosis of infectious diseases. The main challenge for this to become a reality is to detect specific IA products and quantify those products. Certain types of DNA nucleic acids circuits—e.g., catalytic hairpin assembly (CHA)—have been proposed as a tool to address this challenge; however, preparation of such circuits with high enough purity and fidelity is expensive and cumbersome.

Invention Description

The Ellington lab at UT Austin has developed a user friendly RNA CHA circuit for highly specific detection and quantification of amplicons produced by IA reactions. The RNA CHA circuit, like DNA CHA, is based on toehold-mediated strand displacement reactions, which allow the efficient and specific detection of IA amplicons. Moreover, these RNA circuits can be produced cheaply and easily without employing laborious purifications schemes. Toward this end, the researchers have developed a co-transcriptional enzymatic polymerasation method that simultaneously allows the efficient production of the RNA hairpins and the seamless transformation into functional circuitry. Finally, the researchers optimized the signal-to-noise ratio over 100 fold, by incorporating strategically designed mismatched bases in specific hairpins domains. A set of rules for mismatch design have been characterized and are being developed into an algorithm. In total, this package has the potential to make IA reactions for point-of-care diagnostics a reality. 

Benefits/Advantages

  • Can be used for highly specific end-point and real-time detection of isothermal amplification reactions
  • Improved sensitivity by as much as 25- to 10,000-fold over comparable real-time detection methods
  • Can be used in solution and on disposable printed sensors
  • Can be produced large-scale with high-fidelity enzymatic synthesis that is time and cost effective
  • No purification of the circuits needed
  • Can be prepared in real-time or as needed during diagnostic application
  • Long-term circuit storage in the form of double-stranded DNA templates
  • RNA circuits can be used as sequence-specific signal transducers not only in solution but also on solid platforms such as paper-fluidics aimed for point-of-care devices.
  • The mismatch rules can be used with any DNA based CHA circuit platform.