Photoactivatable Circular Caged Oligonucleotides for Transcriptome In Vivo Analysis (TIVA)

Abstract

Light activation is an effective way to impart spatiotemporal control over oligonucleotide probes that are widely applied for gene expression regulation and target function investigation. Among the major oligonucleotide caging strategies, cyclization with a photocleavable linker is an elegant design, which affords both atom efficiency and stability in many biological environments. Here, we introduce an improved protocol for circular oligonucleotide synthesis requiring only one round of HPLC purification. With a series of poly‐U oligonucleotide strands of different sizes and backbone modifications, the pre‐photolysis caging stability and post‐photolysis target binding affinity were studied through a denaturing gel assay and melting temperature measurements. A 14 U 2'‐OMe RNA probe was selected, with strong potential application in transcriptome in vivo analysis (TIVA) for mRNA isolation.

Structure-Guided Design of a Potent and Specific Inhibitor against the Genomic Mutator APOBEC3A

“…After 2 h, the azide-modified product was purified with a NAP-5 column (GE Healthcare). The stem loop ligation follows from prior studies , and was adapted from a previously published protocol . A premixed solution of CuSO 4 (10 equiv, 10 mM in H 2 O) and THPTA (100 equiv, 100 mM in H 2 O) was diluted to 100 μM with 10 mM Tris–Cl (pH 7.5) and H 2 O.…”

Circular Antisense Oligonucleotides for Specific RNase-H-Mediated microRNA Inhibition with Reduced Off-Target Effects and Nonspecific Immunostimulation

“…However, we found that it was not successful to enzymatically cyclize such phosphorothioated ODNs even with one or two normal phosphates at two terminals (data not shown). Previously, Dmochowski’s , and Frank Seela’s have improved the great potential of copper­(I)-catalyzed azide-alkyne cycloaddition (CuAAC) in terminal ligation of oligonucleotides. Therefore, we developed another efficient chemical conjugation strategy to prepare small c-ODNs (chemically modified or unmodified) based on CuAAC through intramolecular cross-linking.…”

“…Among more than 48,860 different mature microRNAs (miRBase), miR 21 is one of the most well-known and most studied onco-miRNAs, which is upregulated in many cancers. − In addition, miR 21 also plays an important role in cardiovascular disease, immune response, and organ fibrosis. , miR 222 is another common onco-miRNA, which significantly increases cell proliferation. ,− Previous studies have applied antisense microRNA oligonucleotides against miR 21 and miR 222 to reduce tumors in vivo . − However, few effective studies were investigated to reduce their possible off-target effects. Due to the specific topology of circular oligonucleotides, they have been reported to regulate and/or evaluate oligonucleotide confirmation and gene functions. − Herein, we reported a new strategy to develop small circular AMOs and specifically mediate microRNA inhibition. The circular topology of circular AMOs is expected to greatly reduce general off-target effects of ODNs as well as decrease their nonspecific immunological responses and interspecies crossing.…”

Optical control of CRISPR-Cas editing with cyclically caged guide RNAs

“…[52][53][54] It was reported that the cyclization of oligonucleotides often was accompanied by dimerization or cyclodimerization as the ring size varied. [55][56][57] Hence, these macrocyclizations usually had to be performed under high dilution conditions. Interestingly, it was found that the covalent condensation proceeded smoothly at 75 μM with almost same rates despite the different ring sizes of these two crRNAs (22 nt and 16 nt, respectively) (Fig.…”