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RNA triplexes: from structural principles to biological and biotech applications

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The diverse biological functions of RNA are determined by the complex structures of RNA stabilized by both secondary and tertiary interactions. An RNA triplex is an important tertiary structure motif that is found in many pseudoknots and other structured RNAs. A triplex structure usually forms through tertiary interactions in the major or minor groove of a Watson–Crick base‐paired stem. A major‐groove RNA triplex structure is stable in isolation by forming consecutive major‐groove base triples such as U·A‐U and C+·G‐C. Minor‐groove RNA triplexes, e.g., A‐minor motif triplexes, are found in almost all large structured RNAs. As double‐stranded RNA stem regions are often involved in biologically important tertiary triplex structure formation and protein binding, the ability to sequence specifically target any desired RNA duplexes by triplex formation would have great potential for biomedical applications. Programmable chemically modified triplex‐forming oligonucleotides (TFOs) and triplex‐forming peptide nucleic acids (PNAs) have been developed to form TFO·RNA2 and PNA·RNA2 triplexes, respectively, with enhanced binding affinity and sequence specificity at physiological conditions. Here, we (1) provide an overview of naturally occurring RNA triplexes, (2) summarize the experimental methods for studying triplexes, and (3) review the development of TFOs and triplex‐forming PNAs for targeting an HIV‐1 ribosomal frameshift‐inducing RNA, a bacterial ribosomal A‐site RNA, and a human microRNA hairpin precursor, and for inhibiting the RNA–protein interactions involving human RNA‐dependent protein kinase and HIV‐1 viral protein Rev. WIREs RNA 2015, 6:111–128. doi: 10.1002/wrna.1261 This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Interactions with Proteins and Other Molecules > Small Molecule–RNA Interactions Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
Standard major‐groove base triples of U·A‐U (a), s2U·A‐U (b), C+·G‐C (c), and U·G‐C (d). The hydrogen, carbon, nitrogen, oxygen, and sulfur atoms are shown in white, cyan, blue, red, and yellow, respectively. The Watson–Crick and Hoogsteen base pairs are indicated by green arrows in the U·A‐U base triple (a). According to Leontis/Westhof base pairing classification, in a Watson–Crick pair, the Watson–Crick edges of two bases are involved in hydrogen bonding. In a Hoogsteen pair, the Hoogsteen edges of A or G (containing atoms 5–8) are exposed in the major groove and involved in hydrogen bonding with the Watson–Crick edges of U or C, respectively. The letter R represents the ribose‐phosphate backbone, which separates the Hoogsteen edge and the sugar edge. The sugar edge also contains the 2′‐OH group in the riboses. Most of the hydrogen bonds are indicated by gray dashed lines. In the base triple C+·G‐C (c), the hydrogen bond formed between protonated N3 in C+ and N7 in G is indicated by a red dashed line. The U·A‐U, C+·G‐C, and U·G‐C base triple structures are taken from the RNA Base Triple Database (http://rna.bgsu.edu/triples). In the base triple s2U·A‐U (b; modeled from the U·A‐U base triple structure), the van der Waals interaction between the sulfur atom of s2U (2‐thio U) and H8 atom of adenine is indicated by a green dashed line.
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Examples of RNA duplexes that have been targeted by triplex‐forming oligonucleotides (TFOs) and triplex‐forming peptide nucleic acids (PNAs). TFOs and PNAs are shown in blue. Watson–Crick and non‐Watson–Crick pairs are indicated by solid lines and dots, respectively. (a) Formation of a major‐groove triplex between an unmodified 20‐nucleotide RNA TFO (shown in blue) and a model RNA duplex, at near‐physiological condition, inhibits RNA‐dependent protein kinase (PKR) binding and activity. (b) A conjugate of unmodified DNA (shown in orange) and RNA (shown in blue) strands forms an 8‐base‐pair DNA–RNA duplex and a 12‐base‐triple TFO·RNA2 triplex with a variant of viral Rev response element (RRE) at physiological buffer condition. The simultaneous duplex and triplex formation can inhibit viral Rev protein binding. The sequence and secondary structure of wild‐type stem IIB are shown in the box. (c) A short PNA binds to an HIV‐1 frameshift stimulatory RNA by forming a 6‐base‐triple PNA·RNA2 triplex at near‐physiological buffer condition. (d and e) An 8‐mer or 9‐mer triplex‐forming PNA binds to the duplex region of the small subunit rRNA helix 44, which contains the bacterial ribosomal A‐site with a 1 × 2 internal loop (shown in red). (f) A 10‐mer PNA incorporating M and E residues (Figure (d) and (i)) binds to a duplex region of a model hairpin derived from pre‐miRNA‐215 at physiological buffer condition.
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Major‐groove base triples formed between peptide nucleic acid (PNA) bases (shown in blue) and RNA Watson–Crick pairs (A‐U, G‐C, C‐G, and U‐A, shown in black). The letter R represents the sugar‐phosphate backbone of RNA. Most of the hydrogen bonds between the bases are indicated by gray dashed lines. The hydrogen bonds formed between protonated N3 in C+ and N7 in G (b), and protonated N1 in M+ and N7 in G (d) are indicated by red dashed lines. Hydrogen bonds involving the backbones may also be present and need to be confirmed by future structural studies. (a and b) Unmodified bases thymidine (T) and cytosine (C) can be utilized to form T·A‐U and C+·G‐C PNA·RNA2 base triples, respectively. J (c) is for pseudoisocytosine. M+ (d) is for the protonated form of 2‐aminopyridine. L (e) is for thio‐pseudoisocytosine. In the base triple L·G‐C, the van der Waals interaction between the sulfur atom of L and H8 atom of G is indicated by a green dashed line. iC (f) is for 5‐methylisocytosine. P and Pex (g and h) are for 2‐pyrimidinone base with carbonyl methylene and carbonyl ethylene linkages to the PNA backbone, respectively. E (i) is for 3‐oxo‐2,3‐dihydropyridazine.
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Chemical structures of (a) DNA, (b) RNA, (c) 2′‐OMe RNA, (d) locked nucleic acid (LNA), and (e) peptide nucleic acid (PNA).
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Representative minor‐groove base triples. The carbon atoms are shown in cyan or green. The distance cutoff for the dashed lines representing hydrogen bonds is 4 Å between two heteroatoms. (a and b) Standard A‐minor interactions formed between a truncated expression and nuclear retention element (ENE) core from KSHV PAN long noncoding RNA (lncRNA) and a 9‐nucleotide oligo(A) sequence (see Figure (e) and (f), PDB: 3P22). In a standard A‐minor base triple, adenosine residues in the third strand form hydrogen bonds with a stem utilizing sugar and/or Watson–Crick edges. Type II and type I A‐minor interactions are present in panels a and b, respectively. (c) Packing of two duplexes facilitated by A‐minor motif interactions between two sheared A·A pairs (A58·A86 and A87·A57) in J4/5 loop and a wobble pair [formed between G and 5‐methyl U (m5U)] in P1 stem in the 5′ exon of a group I intron (PDB: 1U6B). A58 and A87 are present in two opposite strands with cross‐strand stacking, and are involved in type II and type I‐like A‐minor interactions, respectively. (d and e) ‘Twisted A‐minor’ base triple formed between stem 1 and loop 2 in human telomerase RNA pseudoknot (see Figure (a) and (b), PDB: 2K96). A171 (d) and A172 (e) in loop 2 rotate along the minor groove of stem 1 and form minor‐groove base triples using Hoogsteen edge (amino group) only, and Hoogsteen and Watson–Crick edges (amino group and N1), respectively. (f–h) ‘Twisted A‐minor’ base triples formed between stem 1 and loop 2 in an S‐adenosylmethionine (SAM)‐II riboswitch in complex with SAM (see Figure (c) and (d), PDB: 2QWY). A33 (f) and A35 (g) in loop 2 form minor‐groove base triples with stem 1 using Watson–Crick edges (amino group and/or N1). In panel h, A35, A36, and A37 in loop 2 are observed to rotate along the minor groove of stem 1 and form minor‐groove base triples using Hoogsteen edge (amino group) only, Watson–Crick (amino group and N1), and Watson–Crick and sugar edges (N1 and N3), respectively. (i) A minor‐groove base triple involving U38 residue in loop 2 and two consecutive base pairs G6·U26 and C7‐G25 in stem 1 present in a SAM‐II riboswitch (see Figure (c) and (d), PDB: 2QWY).
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Representative major‐groove and other base triples formed involving stem 2 in H‐type pseudoknots found in human telomerase RNA (a; see Figure (a) and (b), PDB: 2K96) and a S‐adenosylmethionine (SAM)‐II riboswitch in complex with SAM (b–f; see Figure (c) and (d), PDB: 2QWY). The distance cutoff for the dashed lines representing hydrogen bonds is 4 Å between two heteroatoms. (a) A major‐groove U·G‐C base triple formed between stem 2 and loop 1 in human telomerase RNA pseudoknot. The standard major‐groove U·A‐U base triple structures (see Figure (a)) are not shown. (b) A nonstandard major‐groove base triple G·G‐C. (c) A nonstandard major‐groove base triple A·C‐G. (d) A base triple U·U·A formed involving the adenine moiety of SAM. (e) A standard major‐groove U·A‐U base triple is shown to highlight the backbone–backbone hydrogen bond formed between 2′‐OH in U12 and a nonbridging phosphate oxygen in A46, in addition to the base–base hydrogen bonds shown in Figure . (f) A base triple formed between A13 in loop 1 and a non‐Watson–Crick A48·U18 pair.
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Triplex and pseudoknot structures. Watson–Crick pairs are indicated by solid lines. Non‐Watson–Crick pairs are indicated by dots or dashed lines. The distance cutoff for the hydrogen bonds is 4 Å between two heteroatoms. (a and b) H‐type pseudoknot structure found in human telomerase RNA (PDB: 2K96). The bulge U177 residue (between A176 and G178) was deleted to stabilize the structure for the initial nuclear magnetic resonance (NMR) studies. (c and d) Crystal structure of an H‐type pseudoknot of S‐adenosylmethionine (SAM)‐II riboswitch in complex with SAM (PDB: 2QWY). (e and f) Crystal structure of a trans pseudoknot formed between a truncated expression and nuclear retention element (ENE) core from KSHV PAN long noncoding RNA (lncRNA) and a 9‐nucleotide oligo(A) sequence (PDB: 3P22). The standard major‐groove triplex with five U·A‐U base triples is shown in panel e.
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RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry
Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
RNA Interactions with Proteins and Other Molecules > Small Molecule–RNA Interactions
RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems

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