Read an Excerpt
CHAPTER 1
DNA Recognition by Parallel Triplex Formation
KEITH R. FOX, TOM BROWN AND DAVID A. RUSLING
1.1 Why Triplexes?
Triplex-forming oligonucleotides (TFOs) bind in the duplex major groove by forming hydrogen bonds with exposed groups on the Watson–Crick (W–C) base pairs, generating a triple-helical structure (e.g., Figure 1.1). The unique base–base recognition properties of these molecules can be exploited as a means to target duplex sequences present or embedded within natural or synthetic DNA. Unlike most DNA-recognition agents, such as polyamides, TFOs are capable of targeting extended sequences, with a relatively low propensity to bind to non-target sites. In this way TFOs have been exploited as gene-targeting agents for modulating gene expression, as a means to detect and/or isolate plasmid and genomic DNA for molecular biology or diagnostics, and as a tool to introduce functionality into DNA nanostructures engineered for bionanotechnology or synthetic biology. Despite this, the applications of TFOs that contain natural nucleotides are often restricted by their low binding affinity and slow association kinetics at neutral pH, as well as a requirement for oligopurine–oligopyrimidine duplex target sequences. To overcome these limitations a variety of base, sugar and phosphate modifications have been developed to allow triplex formation at mixed-sequence targets with high affinity at neutral pH. This chapter will review the developments and current state-of-the-art of nucleotide modifications used to improve the triplex-forming properties of oligonucleotides.
1.1.1 Triplets and Triplex Motifs
Triplexes were first observed experimentally over 60 years ago by Rich and co-workers after mixing the polyribonucleotides poly-U and poly-A in a 2 : 1 ratio. Additional studies demonstrated that poly-C and poly-G could generate a similar structure under low-pH conditions, and since then a variety of DNA and RNA triplexes have been identified. The binding of an oligonucleotide within the major groove is asymmetric and can occur in either a parallel or antiparallel orientation relative to the oligopurine-containing strand of the target duplex. Pyrimidine-rich oligonucleotides bind in a parallel orientation under slightly acidic conditions (pH<6.0), with T and protonated C forming Hoogsteen hydrogen bonds with AT and GC base pairs, generating the base triplets T–AT and C+–GC, respectively (Figure 1.1b). (In this chapter the notation X–ZY refers to a triplet, in which the third strand base X interacts with the duplex base pair ZY, forming hydrogen bonds to base Z.) In contrast, purine-rich oligonucleotides bind in an antiparallel orientation, with A and G forming reverse-Hoogsteen hydrogen bonds with AT and GC base pairs respectively, generating A–AT and G–GC triplets.
In theory, both triplex motifs could be usefully exploited for the recognition of unique duplex sequences but the greater stability of the parallel motif has meant it has been more widely adopted. Parallel triplexes are intrinsically more stable than their antiparallel counterparts because T–AT and C+–GC triplets are structurally isomorphic; that is, if the C-1' atoms of their W–C base pairs are superimposed, the positions of the C-1' atoms of the third strand are almost identical. This minimises backbone distortions of both the third strand and duplex between adjacent triplets. In contrast, antiparallel triplets are not isomorphic and lead to structural distortions at the junctions between consecutive triplets. The use of the antiparallel motif is also hampered by the tendency of purine-containing oligonucleotides to self-associate into structures such as G-quadruplexes and GA-duplexes, which compete with triplex formation and reduce the effective TFO concentration. It should also be noted that both G–GC and T–AT triplets can be generated in both binding motifs, and GT-containing oligonucleotides can therefore be designed to bind in either orientation. However, the non-isomorphic nature of these two triplets means that the most stable orientation is dependent on the number of GpT and TpG steps. This chapter will therefore focus on triplexes generated through the parallel binding motif using pyrimidine-rich oligonucleotides.
1.1.2 Base, Sugar and/or Phosphate Modifications
We and others have characterised a variety of novel base, sugar and phosphate modifications designed to improve the triplex-forming properties of oligonucleotides and a wealth of data has been generated on the affinity, kinetics and selectivity of triplexes containing these modifications. However, it is often hard to compare the effectiveness of a given modification between studies, since its properties will depend on its positioning within the third strand, the sequence context, length of third strand and/or duplex, as well as the pH and other solution conditions, such as the presence of divalent cations. Most experiments have involved the characterisation of a single substitution within a third strand by examining its interaction with duplexes containing each of the four base pairs at the same position. In this way an X–ZY triplet is generated, where X is the analogue under study and ZY is either an AT, TA, GC or CG base pair in turn (e.g., Figure 1.1c). Often the modification is compared with the most effective natural nucleotide at the same position, i.e., with T, C1, G or T opposite an AT, GC, TA or CG base pair respectively. The formation of G–TA and T–CG triplets for recognising pyrimidine–purine base pairs will be discussed in Section 1.4.2.
The simplest and most commonmeans for characterising triplex stability is by ultraviolet melting, in which the triplex thermal stability is determined from the temperature-dependent change in absorbance at 260 nm, generating a melting curve, from which the melting temperature (Tm) is estimated. However, the analysis of such melting curves is not always straightforward, since the triplex–duplex and duplex–single-strand transitions often overlap. We find that a better approach is to use synthetic oligonucleotides that contain molecular beacons and tomeasure the fluorescence melting curves. This works best when the fluorescence quencher (e.g., dabcyl) is attached to the TFO and the fluorescent group (e.g., fluorescein) is attached to one of the duplex strands (as shown in Figure 1.1c). In this way, the concentration of the TFO can be varied without altering the background fluorescence. We have used this strategy to characterise 15 of the nucleotide analogues described in this chapter using the same model triplex (Figure 1.1c), with experiments undertaken using the same buffer conditions. (Experiments were performed in 50 mM sodium acetate buffer (pH 6.0) containing 200mMsodiumchloride using a temperature gradient of 0.2 1C min-1 and no hysteresis between melting annealing curves was observed.) To allow the reader to make a useful, and unbiased, comparison between these modifications we have included a table of Tm values later in this chapter (Table 1.1).
1.2 Stabilising Triplexes
Triplex stability stems from the formation of two Hoogsteen hydrogen bonds between each base in the third strand and its duplex partner as well as favourable stacking interactions between consecutive bases. Under low-pH conditions the stability of a parallel triplex can be greater than that of its underlying duplex, i.e., the affinity of a third strand for its duplex target is greater than the affinity of a duplex strand for its W–C partner. However, the majority of applications proposed for TFOs require that they bind with high affinity at neutral pH. Although a variety of base analogues have been used to alleviate, at least in part, the pH dependence of the C+–GC triplet (see Section 1.3), the affinity of the third strand can also be improved by increasing the stability of the canonical T–AT and C+–GC triplets.
1.2.1 Enhancing Stacking and Hydrophobic Interactions
Base stacking and hydrophobic interactions are important factors that influence the structure and stability of both duplex and triplex DNA. Consequently, several thymine analogues have been prepared with additional aromatic rings across the 4–5 or 5–6 positions, which should increase the aromatic surface area of the base without affecting the hydrogen bonding groups. However, and somewhat surprisingly, triplexes containing these analogues did not demonstrate any enhanced stability. The best of these, a non-natural pyrido[2,3-d] pyrimidine nucleoside (F; Figure 1.2a), was shown to recognise AT base pairs with an affinity that was similar to, but not greater than, that of unmodified T. However, these studies were undertaken with isolated substitutions and it is likely that only multiple adjacent substitutions will improve stability through stacking interactions. A more successful strategy has been to introduce hydrophobic substituents at the 5-position of the base to increase hydrophobic interactions within the major groove. The simplest addition is a methyl group and probably explains why T–AT triplets are more stable than U–AT and MeC+–GC triplets are more stable than C+–GC. The addition of a propynyl group (PdU; Figure 1.2b) further extends the hydrophobic surface and it has been shown that each PdU substitution increases the Tm of the triplex by ca. 2.5 °C relative to an unmodified third strand. Subsequent NMR studies revealed that, as expected, the extended aromatic electron cloud of the PdU nucleotide stacks well over the 5'-neighboring nucleotides, and is most probably the cause of the increased stabilisation. A further study examined the properties of four different C5-amino modified deoxyuridines and showed that the order of stability produced by 5-substitutions is alkyne >E-alkene > alkane > Z-alkene. This order must result from steric factors as well as stacking interactions. The same strategy cannot be applied for increasing the affinity of C for a GC base pair, since the addition of a propynyl group to the 5-position lowers the pKa of the base and increases the pH dependence of the triplet. Indeed the attachment of a propargylamino chain to the 5-position (APdC), a similar modification that is discussed further below, generated triplexes of equivalent stability to those formed by cytosine at pH 6.0 (Table 1.1).
More recently the introduction of thiocarbonyl groups to the 2-position of thymine (s2T; Figure 1.2c) and 5-methylcytosine (m5s2C; Figure 1.2d), as well as the 8-position of adenine (s8A; Figure 1.2e), has proved to be a useful strategy for increasing triplex stability. Molecular modelling of a parallel triplex containing s2T in the third strand indicated that the 2-thiocarbonyl group of the 5'-upstream base could interact with the nitrogen atom at the 1 position of the 3'-downstream pyrimidine ring and result in strong stacking effects. Indeed triplexes containing multiple substitutions of s2T led to a Tm increase of around 5 °C per modification at pH 7.0. A further enhancement in affinity was seen when combined with m5s2C and particularly evident for TFOs containing multiple, adjacent substitutions. The m5s2C and s8A analogue were developed for the pH-independent recognition of GC base pairs and are discussed further below (see Sections 1.3.1 and 1.3.2, respectively).
1.2.2 Locking the Sugar Pucker
The affinity of a TFO for its duplex target is affected by its ability to adopt N- or S-type sugar pucker conformations and it has been proposed that the former require less distortion of the duplex purine strand upon triplex formation. This is thought to explain why TFOs composed of ribonucleotides exhibit a higher affinity for duplex DNA than those composed of deoxyribonucleotides. In general, oligonucleotide modifications that favour N-type sugars produce more stable triplexes than their S-type counterparts. The addition of an electronegative group at the 2'-position of the sugar, as in RNA, strongly favours the N-type sugar pucker predominantly due to the gauche effect. Consequently various groups have been attached to this position to promote or restrict the sugar to an N-type configuration.
The first chemical moiety to be added to 2'-position that resulted in improved TFO binding was a methoxy group (2'-OMe; Figure 1.3a). Subsequent NMR studies confirmed that this resulted from a reduced distortion of the duplex purine strand, enhancing the rigidity of the triplex. A better modification that locks the sugar pucker in an N-type configuration and reduces the rotational freedom of the sugar phosphate backbone is bridged/ locked nucleic acid (BNA or LNA; Figure 1.3b). This modification exploits a 2'-O,4'-C methylene bridge to constrain the sugar to N-type and was developed independently by the Wengel and Imanishi groups for use in antisense or antigene applications, respectively. TFOs that contain BNA/LNA residues are markedly more stable than their unmodified counterparts but only when substituted every 2–3 nucleotides. Further BNA/LNA derivatives have been developed to overcome this sequence restriction. Substitution of the bridge with an ethylene moiety (ENA; Figure 1.3c), which contains an additional carbon, allows triplex formation with fully modified TFOs. Whilst the introduction of an O–N bond (BNANC; Figure 1.3d) further improved the nuclease resistance of the oligonucleotide. TFOs containing the N-methyl derivative of BNANC were stable in serum for over 90 minutes compared with an unmodified oligonucleotide, which was completely degraded in 5 minutes. Such modifications are likely to be useful for any applications that require the use of TFOs within a physiological setting. Further thermodynamic and kinetic studies revealed that the enhancement in affinity stemmed from a decrease in the dissociation constant of the TFO. An additional advantage of the sugar analogue is that the presence of the nitrogen atom allows functionalisation with other chemical groups, such as fluorophores. Lastly, 3'-amino-2',4'-BNA has been developed in which the BNA/LNA modification is combined with the N3'-P5' modification considered below (Figure 1.3e, see Section 1.2.4). Although triplexes with this analogue were more stable than their duplex equivalents, they were no more stable than those formed with BNA/LNA alone. Further attempts to constrain the sugar pucker to either S-type or N-type include the bicyclo and tricyclo furanose modifications developed by Leumann and co-workers (Figure 1.3f and g). Bicyclo-DNA contains a 3'-O,5'-C ethylene bridge that locks the sugar in an S-type conformation, while the tricyclo derivative contains an additional cyclopropane unit locking the sugar in an N-type pucker. Studies with TFOs composed of tricyclo-modified thymidine showed an increase in Tm of 2 °C per modification at pH 7.0.
1.2.3 Adding Positive Charge(s)
The formation of a triplex brings three polyanionic strands into close proximity, increasing the negative charge density by 50%, and leads to a high degree of charge repulsion. This can be partially screened using high concentrations of monovalent ions (e.g., up to 200 mM of sodium) and lower concentrations of divalent or polycationic ions (e.g., up to 10 mM magnesium or spermine). Consequently, the incorporation of positively charged moieties into the TFO by their addition to the phosphate, sugar or base has helped increase triplex stability by alleviating in part, some of this charge repulsion.
1.2.3.1 Phosphate
One means to incorporate charges into a TFO is by their appendage to the phosphodiester backbone. For example, Bruice and co-workers have shown that the addition of positively charged guanidinium linkages (DNG; Figure 1.4a) causes a dramatic increase in TFO affinity. The synthesis of the ribose derivative has also been reported but to our knowledge this has not yet been studied for its triplex-forming properties. Two further modifications that replace the phosphate residues with either cationic dimethylaminopropyl phosphoramidate linkages (PNHDMAP) or N,N-diethyl-ethylenediamine linkages (DEED; Figure 1.4b) have also been characterised. TFOs with these modifications generated triplexes which were more stable than the underlying duplex at pH 7.0.56 More recently, oligonucleotides containing non-nucleosidic monomers composed of partially protonated amines have been prepared. When incorporated at the TFO termini such modifications lead to a significant enhancement of triplex stability, particularly when positioned at the 5'-end of the TFO.
(Continues…)
Excerpted from "DNA-targeting Molecules as Therapeutic Agents"
by .
Copyright © 2018 The Royal Society of Chemistry.
Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.
