Nucleic acid triplex structures

Triple-helical nucleic acids were first described in 1957 (52). Triplex structures form between three nucleic acid strands. They occur in purine-rich DNA strands that form Hoogsteen hydrogen bonds. Two different triplex motifs have been described: 1. the purine motif and 2.

the pyrimidine motif (10). Both require a purine rich Watson-Crick duplex binding the third strand in the major groove. In the purine (R) motif the third strand has an antiparallel orientation to the duplex purine strand and forms A(T)AT and GGC triplexes in reverse Hoogsteen configuration (see Figure 1.3 A). The pyrimidine (Y) motif contains TAT and CGC triplets in Hoogsteen configuration, thus having the third strand in parallel orientation (see Figure 1.3. B). The cytosine containing Y motif is stabilized under acidic conditions (cytosine in third strand is protonated C⁺) (see Figure 1.3. B).

Figure 1.3: Purine and Pyrimidine type triplexes.

A Purine motif base triplets. Purine bases are colored red, pyrimidine bases are colored blue and reverse Hoogsteen bases are shown in green. B Pyrimidine motif base triplets. Purine bases are colored red, pyrimidine bases are colored blue and Hoogsteen pairing bases are shown in green. C Strand orientations for parallel and antiparallel triplex motifs. Color code as in A/B, Y: pyrimidine rich strand; R: purine rich strand; H: strand bound by Hoogsteen base pairing; RH: strand bound by reverse Hoogsteen base pairing. A and B modified from (10).

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Steric properties make the triplex of the pyrimidine motif more stable compared to the purine motif triplex, especially for intermolecular formations (53). Triplex structures can be formed intra- and intermolecularly. Intermolecular structures are formed out of two or three distinct DNA strands – most often between a DNA duplex and a single stranded triplex-forming oligonucleotide (TFO) (54) (see Figure 1.4. A). In intramolecular triplexes the third strand is physically tethered to the DNA duplex. Most studies investigating intramolecular triplexes focus on H-DNA. For the formation of a H-DNA (see Figure 1.4 B) the homopurine-homopyrimidine sequence must be a mirror repeat (see Chapter 1.3). That way, half of the pyrimidine tract swivels its backbone towards the purine strand of the duplex or the purine strand binds to the purine part of the underlying duplex, forming a parallel or antiparallel H-DNA structure, respectively (55). Different H-H-DNA isoforms can occur, depending on whether the 3’ half or the 5’ half of the third strand is involved in triplex structure formation (see Figure 1.4 B).

Figure 1.4: Schematics of inter- and intramolecular triplex structures.

A Schematics of an intermolecular purine motif triplex with antiparallel strand orientation formed by 3 distinct DNA strands. B Examples of intramolecular H-DNA structures that can form from a mirror repeat sequence within a DNA double strand. Pyrimidine motif H-DNA with parallel strand orientation is shown on the left side. Purine motif H-DNA with antiparallel orientation is shown on the right side. Pyrimidine-rich strands are shown in blue, purine-rich strands are depicted in red and the reverse Hoogsteen strand is colored green. Figure modified from (56).

Different groups have reported the existence of imperfect triplexes with mismatches between the strands (57-60) which lead to destabilizing effects. Such effects increase with the number of contiguous mismatches (61) and further depend on their position in the triplex:

Mismatches in the center of a triplex are more disruptive than those at terminal sites (62).

Furthermore, triplex stability is influenced by the presence of multivalent cations. They compensate the negative electrostatic repulsion of the three nucleic acid backbones and therefore stabilize triplex structures (63-65). G-rich triplex sequences can compete with

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quadruplex formation, which is favored in the presence of potassium (66). Other factors that can influence triplex stability are pH, temperature, solvent and the presence of charge-neutralizing polyamines (67) or polypeptides (68). Apart from DNA, triplex structures also form in combination with RNA strands. RNA triplexes are found in different structured RNAs:

They occur in pseudoknots (minor groove triplexes) (69,70), riboswitches (71) and other structured RNAs (72). Pyrimidine motif triplexes have been the main focus of most investigations, but other types may form as well (73,74).

1.1.2.1 Intrastrand triplexes

A different type of intramolecular triplex structure arises from the folding of polypurine/polypyrimidine units along one single strand of DNA or RNA. Although those intrastrand triplexes have been investigated in vitro (75-84), studies investigating their in vivo occurrence (10) and function (85) are sparse. Intrastrand triplex motifs have been assigned to four different conformational classes, depending on the strand orientation of their base triplets (10) (see Figure 1.5). Class I and II refer to purine motif triplexes, with class I having the reverse Hoogsteen domain at the 5’ terminus, followed by the purine- and the pyrimidine-rich domain. Class II triplexes have the pyrimidine-pyrimidine-rich domain at the 5’ end, followed by the purine rich domain and the reverse Hoogsteen domain at the 3’ end of the sequence.

Figure 1.5: Intrastrand triplex classes.

A R motif triplexes: class I and class II. B Y motif triplexes: class III and class IV. Pyrimidine rich strands are shown in blue, purine rich strands are depicted in red and (reverse) Hoogsteen strands are colored green.

Dashed lines represent arbitrary spacer sequences. Figure modified from (10).

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Class III and IV correspond to the Y motif triplex structures: Class III triplexes progress from the 5’ purine-rich domain through the pyrimidine-rich domain to the 3’ Hoogsteen domain, and class IV triplexes start with the Hoogsteen domain at the 5’ terminus, followed by the pyrimidine-rich domain and ending with the purine rich domain at the 3’ terminus. It has been shown that both purine and pyrimidine type triplex DNA structures can form under physiological conditions. Pyrimidine-type intrastrand triplex structures even occur on the RNA level (11).