AptaBlocks: Accelerating the Design of RNA-Based Drug Delivery Systems;

摘要

Synthetic RNA molecules are increasingly used to alter cellular functions [1-4]. These successful applications indicate that RNA-based therapeutics might be able to target currently undruggable genes [5, 6]. However, to achieve this promise, an effective method for delivering therapeutic RNAs into specific cells is required. Recently, RNA aptamers emerged as promising delivery agents due to their ability of binding specific cell receptors [7, 8]. Crucially, these aptamers can frequently be internalized into the cells expressing these receptors on their surfaces. This property is leveraged in aptamer based drug delivery systems by combining such receptor-specific aptamers with a therapeutic "cargo" such that the aptamer facilitates the internalization of the cargo into the cell [9-11]. The advancement of this technology however is contingent on an efficient method to produce stable molecular complexes that include specific aptamers and cargoes. A recently proposed experimental procedure for obtaining such complexes relies on conjugating the aptamer and the cargo with complementary RNA strands so that when such modified molecules are incubated together, the complementary RNA strands hybridize to form a double-stranded "sticky bridge" connecting the aptamer with its cargo [12, 13]. However, designing appropriate sticky bridge sequences guaranteeing the formation and stability of the complex while simultaneously not interfering with the aptamer or the cargo as well as not causing spurious aggregation of the molecules during incubation has proven highly challenging. To fill this gap, we developed AptaBlocks, a computational method to design sticky bridges to connect RNA-based molecules (blocks). Accounting for the three-step procedure [12, 13], we formulate the sticky bridge sequence design as an optimization problem utilizing an objective function which reflects the biophysical characteristics of the assembly process. Specifically, we designed the objective function considering the equilibrium probabilities of the target structures over all possible structures of the aptamer-stick and cargo-stick, the probability of the interaction between the aptamer-stick and cargo-stick at equilibrium, the hybridization energy between the sticky bridge sequences, and additional sequence constraints including but not limited to the GC content. We further provide a simulated annealing algorithm that enables efficient estimation of the corresponding combinatorial optimization problem. The effectiveness of the algorithm has been verified computationally and experimentally. AptaBlocks can be used in a variety of experimental settings and its preliminary version has already been leveraged to design an aptamer based delivery system for a cytotoxic drug targeting Pancreatic ductal adenocarcinoma cells [14]. It is thus expected that AptaBlocks will play a substantial role in accelerating RNA-based drug delivery design.