DEVELOPMENT OF E-SRS (ENVIRONMENT-SENSING RESPONSE SYSTEM) AS A NOVEL METHOD TO DISTINGUISH GENETIC ENVIRONMENTS AND RESOLVE CLOSELY RELATED NUCLEIC ACID SEQUENCES

Abstract

Existing limitations of conventional Nucleic Acid (NA) detection prompted us to conduct a Proof-of-Concept of a novel NA sensing platform called environment-Sensing Response System (e-SRS), which could deliver a physical response upon sensing its NA Target Sequence (NTS). e-SRS is a NA sensing and response system with two components: 1) An RNA based sensor that changes conformation and activates upon binding specific NTS; 2) A Response System that is triggered by the activated sensor to initiate some physical response, such as emitting a fluorescent signal to indicate presence of the NTS, or other biomolecular actions like induction of gene expression. Its modular nature, whereby the sensor is separate from the Response System, allows e-SRS flexibility in adapting to different formats and applications. The ability to activate Response System after sensing enables the e-SRS sensor to serve as a signal transducer, which passes a signal of one form from the environment (e.g. presence of specific NA), to that of another form as produced by the Response System (e.g. activation of inducible expression system). A biomolecular signal transducer could function in more diverse ways than a biomolecular probe, and could be a powerful tool in research, diagnostics and therapy. After initial tests, an early design known as e-SIGE was unworkable, likely because the sensor¿s RNA folding was designed without computational secondary structure prediction. The RNA folding likely did not occur as intended. We redesigned the physical implementation to create the current e-SRS, adapting an existing allosteric ribozyme, the Maxizyme, as e-SRS sensor, employing computational secondary structure prediction. We were able to successfully test e-SRS in the test tube environment via Ribozyme Trans-cleavage Assays (RTA). Unsatisfied with RNA cleavage assays via Denaturing Polyacrylamide Gel Electrophoresis, we developed the Reporter Substrate (RS), which provided real time fluorescence reporting of e-SRS sensor activity, and served as a gene detection Response System. Our attempts to activate an inducible gene system as the Response System within cell lines were unsuccessful, likely due to interfering RNA secondary structure in the cellular environment. e-SRS sensor with RS for fluorescence based real-time test tube detection and resolution of closely related RNA sequences was tested on 7 NTS from 2 categories: 1) 3 strains of Malaria parasites (Plasmodium falciparum), denoted as Mfs, Mfr1, and Mfr2; 2) 4 common serotypes of Dengue viruses, denoted as D1, D2, D3, and D4. We developed a computational algorithm in Perl that greatly automated the design and assessment of e-SRS Mz-based sensors. Our seven e-SRS sensors were optimised to specifically detect their sNTS (19 to 24 nt synthesised RNA). Addition of a 24 nt ¿competitor nucleotide¿ (sNTS-Mfr2) allowed Mfr1 e-SRS sensor to distinguish a single nucleotide difference out of 24 nt between Mfs and Mfr1. For Malaria, we created long NTS (120 nt) from genomic sequences using NASBA (isothermal RNA amplification). Addition of antisense oligonucleotides allowed the detection of otherwise undetectable long NTS. Mfs and Mfr2 long NTS were specifically detected, while the same for Mfr1 required further work to establish.