Targeting of single‐stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides

Abstract

The aim of this work was to construct an anti‐messenger targeted to the pim‐1 oncogene transcript, based on circular or clamp oligodeoxyribonucleotides. The formation of bimolecular triplexes by clamp or circular oligonucleotides was investigated using single‐stranded targets of both DNA (5′‐CCCTCCTTTGAAGAA‐3′) and RNA type (5′‐CCCUCCUUUGAAGAA‐3′). The third, ‘Hoogsteen’ strand of the triplex was represented by G,T‐rich sequences. The secondary structures of the complexes were determined by thermal denaturation, circular dichroism and gel mobility shift experiments and shown to depend on the nature of the target strand. With DNA as target, the sequence of a clamp (or circular) oligonucleotide that formed the triple helix was 3′‐GGGAGGAAACTTCTTTT‐TTGTTGTTT‐TT‐GGTGGG‐5′, where the first TT dinucleotide (in italics) is a linker and the second TT (bold) represents the bridge through which the ‘Hoogsteen’ strand switches from one strand of the Watson–Crick duplex to the other, once the duplex is formed by the corresponding portion of the anti‐messenger (underlined). The portion of the ‘Hoogsteen’ sequence of the triplex between the two TT dinucleotides binds to the 3′ extremity of the target strand and runs parallel to it. The portion situated at the 5′ end of the oligonucleotide switches to the purine tract of the complementary strand of the duplex and is antiparallel to it. In contrast, with RNA as target, for a branched clamp oligonucleotide that formed a triple helix over its entire length (5′‐TTCTTCAAAGGAGGG‐3′^3′‐GGGTGGTTT‐T‐GTTGTT‐5′) the portion of the ‘Hoogsteen’ sequence that bound to the 3′ extremity of the target strand had to be antiparallel to it.

Circular RNAs: Regulators of Cancer-Related Signaling Pathways and Potential Diagnostic Biomarkers for Human Cancers

“…CircRNAs are novel non-coding RNAs which appear to function as oncogenes or tumor suppressor genes in human cancers. One of the molecular mechanisms through which some circRNAs regulate cancer progression is by targeting specific miRNAs 116 , 141 . The role of circRNAs in cellular transcriptional control needs to be further explored with respect to cancer development.…”

Synthesis and Triplex-Forming Properties of Cyclic Oligonucleotides with (G,A)-Antiparallel Strands

“…However, most of this work has been undertaken using T-/C-rich cyclic oligonucleotides designed to bind the polypurine Watson-Crick strand [8,9]. In contrast, there is little information available on purine-rich cyclic oligonucleotides designed to bind the polypyrimidine Watson-Crick strand, and available data has been obtained with cyclic oligonucleotides carrying the (G,T)-antiparallel strand [10][11][12].…”

Targeted Gene Knockout by 2′-O-Aminoethyl Modified Triplex Forming Oligonucleotides

“…The specific interactions can be described as a third strand binding code (9), and are the basis for the high stringency of TFO specificity (10,11). Triplexes can be quite stable with half-lives of many hours or days (12)(13)(14)(15). Although current oligonucleotide chemistry limits triplex formation to polypurine:polypyrimidine stretches, these elements are overrepresented in the mammalian genome (16), and most genes contain appropriate target sequences.…”