Recognition of RNA duplexes by chemically modified triplex-forming oligonucleotides

Abstract

Triplex is emerging as an important RNA tertiary structure motif, in which consecutive non-canonical base pairs form between a duplex and a third strand. RNA duplex region is also often functionally important site for protein binding. Thus, triplex-forming oligonucleotides (TFOs) may be developed to regulate various biological functions involving RNA, such as viral ribosomal frameshifting and reverse transcription. How chemical modification in TFOs affects RNA triplex stability, however, is not well understood. Here, we incorporated locked nucleic acid, 2-thio U- and 2′-O methyl-modified residues in a series of all pyrimidine RNA TFOs, and we studied the binding to two RNA hairpin structures. The 12-base-triple major-groove pyrimidine–purine–pyrimidine triplex structures form between the duplex regions of RNA/DNA hairpins and the complementary RNA TFOs. Ultraviolet-absorbance-detected thermal melting studies reveal that the locked nucleic acid and 2-thio U modifications in TFOs strongly enhance triplex formation with both parental RNA and DNA duplex regions. In addition, we found that incorporation of 2′-O methyl-modified residues in a TFO destabilizes and stabilizes triplex formation with RNA and DNA duplex regions, respectively. The (de)stabilization of RNA triplex formation may be facilitated through modulation of van der Waals contact, base stacking, hydrogen bonding, backbone pre-organization, geometric compatibility and/or dehydration energy. Better understanding of the molecular determinants of RNA triplex structure stability lays the foundation for designing and discovering novel sequence-specific duplex-binding ligands as diagnostic and therapeutic agents targeting RNA.

Incorporation of thio-pseudoisocytosine into triplex-forming peptide nucleic acids for enhanced recognition of RNA duplexes

“…Previous research shows that the p K a decrease toward neutral for N 3 in 2-thio U (p K a = 8.8) relative to U (p K a = 9.3) monomer (Figure 1D) may enhance, respectively, the Watson–Crick (14,54) and Hoogsteen hydrogen bonding (38). Consistently, our quantum mechanics calculations have revealed that the gas phase local p K a values for N 3 atoms decrease upon thiolation (L versus J, 2-thio T versus T and 2-thio C versus C; Supplementary Tables S2 and S3).…”

Mimicking Ribosomal Unfolding of RNA Pseudoknot in a Protein Channel

“…13,33,82–88 To investigate the effect of the loop-stem tertiary contacts on the unfolding kinetics of the T2 pseudoknot, we substituted L2 with a poly(U) loop. Elastic property studies have shown that a single-stranded RNA poly(U) chain tends to be more disordered with weaker loopstem tertiary interactions.…”

Incorporating a guanidine-modified cytosine base into triplex-forming PNAs for the recognition of a C-G pyrimidine–purine inversion site of an RNA duplex

“…For example, even though the major groove is relatively deep and narrow, an RNA double helix can accommodate a third strand in the major groove to form a modestly stable triplex (11–13). In a major-groove triplex, the pyrimidine bases in the third strand recognize the sequence of the purine strand of the double helical RNA by Hoogsteen hydrogen bonding, forming U·A-U or T·A-U and C + ·G-C base triples (Figure 1A and B) (8–10).…”

“…For example, we have shown previously (15) that PNAs incorporating a modified neutral base thio-pseudoisocytosine (L) (Figure 1C) can recognize a Watson–Crick G-C pair in an RNA duplex by forming a Hoogsteen L·G pair with improved stability and minimized pH dependence compared to a Hoogsteen C + ·G pair (Figure 1B). The enhanced Hoogsteen L·G pair formation is presumably due to improved van der Waals interaction between the thio group in L and H8 in G, as well as base stacking and hydrogen bonding interactions (9,13,15). PNAs incorporating L-modified residues show selective binding to RNA duplexes over single-stranded RNAs, because a Watson–Crick-like G-L pair (Figure 1E) is destabilized compared to a Watson–Crick G-C pair due to the steric clash between the relatively bulky thio group in L and the amino group in G. Furthermore, PNAs show enhanced binding to RNA duplexes over DNA duplexes (9,15–20).…”