Specific Targeting and Imaging of RNA G-Quadruplex (rG4) Structure Using Non-G4-Containing l-RNA Aptamer and Fluorogenic l-Aptamer.

RNA G-quadruplex structures (rG4s) play important roles in the regulation of biological processes. So far, all the l-RNA aptamers developed to target rG4 of interest contain G4 motif itself, raising the question of whether non-G4-containing l-RNA aptamer can be developed to target rG4. Furthermore, it is unclear whether an l-Aptamer-based tool can be generated for G4 detection in vitro and imaging in cells. Herein, a new strategy is designed using a low GC content template library to develop a novel non-G4-containing l-RNA aptamer with strong binding affinity and improved binding specificity to rG4 of interest. The first non-G4-containing l-Aptamer, l-Apt.1-1, is identified with nanomolar binding affinity to amyloid precursor protein (APP) D-rG4. l-Apt.1-1 is applied to control APP gene expression in cells via targeting APP D-rG4 structure. Moreover, the first l-RNA-based fluorogenic bi-functional aptamer (FLAP) system is developed, and l-Apt.1-1_Pepper is engineered for in vitro detection and cellular imaging of APP D-rG4. This work provides an original approach for developing non-G4-containing l-RNA aptamer for rG4 targeting, and the novel l-Apt.1-1 developed for APP gene regulation, as well as the l-Apt.1-1_Pepper generated for imaging of APP rG4 structure can be further used in other applications in vitro and in cells.

Genomics-driven derivatization of the bioactive fungal sesterterpenoid variecolin: Creation of an unnatural analogue with improved anticancer properties.

A biosynthetic gene cluster for the bioactive fungal sesterterpenoids variecolin (1) and variecolactone (2) was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues (5-7). Analysis of the compounds' anticancer activity in vitro and in vivo revealed that although 5 and 1 had comparable activities, 5 was associated with significantly reduced toxic side effects in cancer-bearing mice, indicating its potentially broader therapeutic window. Our study describes the first tests of variecolin and its analogues in animals and demonstrates the utility of synthetic biology for creating molecules with improved biological activities.

Capture-SELEX: Selection Strategy, Aptamer Identification, and Biosensing Application.

Small-molecule contaminants, such as antibiotics, pesticides, and plasticizers, have emerged as one of the substances most detrimental to human health and the environment. Therefore, it is crucial to develop low-cost, user-friendly, and portable biosensors capable of rapidly detecting these contaminants. Antibodies have traditionally been used as biorecognition elements. However, aptamers have recently been applied as biorecognition elements in aptamer-based biosensors, also known as aptasensors. The systematic evolution of ligands by exponential enrichment (SELEX) is an in vitro technique used to generate aptamers that bind their targets with high affinity and specificity. Over the past decade, a modified SELEX method known as Capture-SELEX has been widely used to generate DNA or RNA aptamers that bind small molecules. In this review, we summarize the recent strategies used for Capture-SELEX, describe the methods commonly used for detecting and characterizing small-molecule-aptamer interactions, and discuss the development of aptamer-based biosensors for various applications. We also discuss the challenges of the Capture-SELEX platform and biosensor development and the possibilities for their future application.