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Sequence-Specific DNA Binding Agents
By Michael Waring
The Royal Society of Chemistry
Copyright © 2006 The Royal Society of ChemistryAll rights reserved.
ISBN: 978-0-85404-370-5
CHAPTER 1
DNA Recognition by Triple Helix Formation
DAVID A. RUSLING, TOM BROWN AND KEITH R. FOX
1.1 Introduction
Oligonucleotides can bind in the major groove of double-stranded DNA by forming hydrogen bonds with exposed groups on the base pairs, generating a triple-helical structure (Figure 1A). This was first demonstrated nearly 50 years ago by Rich and co-workers by mixing the synthetic polyribonucleotides polyU and polyA in a 2:1 ratio. Further studies showed that polyC and polyG can generate a similar structure under conditions of low pH and a variety of DNA and RNA triple-stranded structures have since been identified.
Since these complexes form in a sequence-specific fashion they can be used to target unique sequences. By knowing the rules that govern triplex formation, it should be possible to design oligonucleotides to interact with any desired DNA sequence. The formation of intermolecular triple-helical DNA therefore has a number of applications including inhibition of gene transcription, site-directed mutagenesis, and various biotechnological applications.
1.1.1 Triplets and Triplex Motifs
Triplex-forming oligonucleotides bind in the DNA major groove and make specific contacts with the purine strand of the duplex. The binding can be either parallel or antiparallel to the target strand, depending on the base composition of the oligonucleotide.
Pyrimidine-rich oligonucleotides bind under low pH conditions in a parallel orientation to the purine strand of the target duplex, with T and protonated C forming Hoogsteen hydrogen bonds with AT and GC base pairs, respectively. This generates the base triplets T.AT and C+.GC as shown in Figure 1B. (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). These triplets are isomorphic, that is if the C-1' atoms of their Watson-Crick base pairs are superimposed, the positions of the C-1' atoms of the third strand are almost identical. This minimizes backbone distortion of both the third strand and duplex between adjacent triplets. It is also possible to form a G.GC triplet within this motif, though this is not isomorphic with T.AT and C+.GC (Figure 1B (iii)). Most of this chapter will concentrate on the parallel motif.
Purine-rich oligonucleotides bind in an antiparallel orientation to the purine strand of the target duplex, with A and G forming reverse-Hoogsteen hydrogen bonds with AT and GC base pairs respectively, generating A.AT and G.GC triplets (see Figure 1C). In contrast to the parallel triplets, A.AT and G.GC triplets are not isomorphic, leading to structural distortions at junctions between each triplet. As a consequence of this and other factors, the antiparallel motif is often less stable than the parallel motif. T.AT triplets can also adopt an antiparallel orientation.
Since G.GC and T.AT triplets can be formed in both the parallel and antiparallel motifs GT-containing oligonucleotides can be designed to bind in either orientation, parallel or antiparallel. The backbone distortion imposed by the non-isomorphic nature of these two triplets means that the most stable orientation is dependent on the number of GpT and TpG steps.
Triplex formation in the parallel motif suffers from the requirement for low pH, which is necessary for protonation of cytosine at N3. Without protonation the C.GC triplet only contains one hydrogen bond between the exocyclic N4 of cytosine and 6-keto group of guanine. The pKa of cytosine is around 4.5, though this is increased on triplex formation, and is higher in the centre than the termini of a triplex. Runs of contiguous cytosine residues are destabilizing as they decrease the pKa. A large number of cytosine analogues have been prepared to overcome this problem and are described in a later section.
Several reports have suggested that C+.GC is more stable than T.AT. This is attributed to electrostatic interactions between the positive charge of cytosine and the negatively charged phosphodiester backbone and/or favourable stacking interactions between the charged base and the π-stack.
1.2 Strategies to Increase Triplex Stability
1.2.1 Sugar Modifications
The affinity of a third strand for its target is affected by its ability to adopt N- or S-type conformations, since the former require less distortion of the duplex purine strand upon triplex formation. This explains why RNA TFOs have a higher affinity for duplex DNA than those composed of DNA. Oligonucleotide modifications that favour N-type sugars are therefore expected to produce more stable triplexes and some examples are shown in Figure 2. The addition of an electronegative group at the 2'-position of the sugar, as in RNA, strongly favours the N-type sugar pucker due to the gauche effect and the 2'-O-methyl modification (Figure 2a) enhances triplex stability. NMR studies have confirmed that the 2'-methoxy group increases triplex stability by reducing the distortion of the duplex purine strand and enhancing the rigidity of the triplex.
Other modifications can also be used to restrict the sugar pucker and reduce the rotational freedom of the sugar phosphate backbone. The best characterized of these modifications is locked nucleic acid (LNA), which is also known as bridged nucleic acid (BNA), in which a 2'-O,4'-C methylene bridge is used to constrain the sugar to N-type (Figure 2b). This modification was developed independently by the Wengel and Imanishi groups for use in antisense or antigene applications, respectively. TFOs that contain LNA residues every 2-3 nucleotides are markedly more stable than their unmodified counterparts. Two further derivatives have also been developed from this. ENA contains an additional carbon in the bridge and unlike LNA can be fully substituted into TFOs (Figure 2c). In addition, 3'-amino-2',4'-LNA combines the LNA sugar with the N3'-P5' modification (considered below), though triplexes with this analogue are no more stable than those formed with LNA.
The bicyclo and tricyclo furanose modifications developed by Leumann and co-workers represent further attempts to restrict the sugar conformation (Figures 2d and 2e). Bicyclo-DNA contains a 3'-O,5'-C ethylene bridge that locks the sugar in an S-type conformation. The tricyclo derivative contains an additional cyclopropane unit locking the sugar in a N-type pucker and studies with TFOs composed of tricyclothymidine showed a 2°C increase in T per modification at pH 7.
1.2.2 Addition of Positive Charges
Triplex stability is limited by charge repulsion between the three negatively charged backbones. The simplest strategy to alleviate this problem is to incorporate positively charged moieties into the TFO by either modifying the backbone, the sugar or the base, and examples of each of these modifications are shown in Figure 3.
1.2.1.1 Addition of Positive Charge to the Backbone
Bruice and co-workers have replaced the phosphodiester backbone with positively charged guanidinium linkages. A pentameric thymidyl oligomer of deoxyriboguanidine (DNG, Figure 3A (i)) with four positive charges exhibited an unprecedented high affinity for poly(dA), forming a 2:1 thymine adenine complex. The synthesis of the ribose derivative has recently been reported but has yet to be studied for its triplex-forming properties. This derivative may combine the stabilizing effect of the additional positive charge with a favourable sugar conformation. Two further modifications that replace the phosphate residues with either cationic dimethylaminopropyl phosphoramidate linkages (PNHDMAP) or N,N-diethyl-ethylenediamine linkages (DEED) (Figure 3A (ii and iii)) have also been reported. TFOs with the PNHD-MAP modification generated a triplex which was more stable than the underlying duplex at pH 7. Surprisingly the α-anomer produced a more stable triplex than the β-anomer though, as for unmodified TFOs, α-anomers bind in the opposite orientation to the normal β-anomers.
1.2.2.2 Addition of Positive Charge to the Sugar
An important strategy has centred on the addition of positively charged groups to various positions of the sugar unit. Modification has been attempted at either the 2' (Figure 3B (i)) or 4'-positions (Figure 3B (ii)). In both cases, the most stable triplexes were formed by the addition of an aminoethoxy or aminoethyl side chain with a Tm increase of 3.5°C and 1°C per modification at pH 7 for the 2' and 4' derivatives, respectively. The greater stabilization by the 2'-derivative has been attributed to the formation of a salt bridge between the positive charge and a pro-R oxygen of a negatively charged phosphate of the purine strand and a favourable N-type sugar pucker. In experiments with psoralen-linked oligonucleotides it has been suggested that the 2-aminoethoxy modification is more effective when the positively charged derivatives are clustered together. Another modification involves substitution of the furanose oxygen with nitrogen, generating pyrrolidine oligonucleotides (Figure 3B (iii)). This positions a positive charge next to the pro-R non-bridging phosphate oxygen in the purine strand. In this instance the effect of the modification depends on the base that is attached to the modified sugar; pseudoisocytosine is stabilizing giving a Tm increase of 2°C per modification while uracil is destabilizing. The addition of guanidines instead of amino functions is another strategy, which offers two further advantages. Firstly, it can be applied post-oligonucleotide synthesis and secondly, the guanidine group is protonated over a greater pH-range than the amine. This modification gives typically the same increase in stability as an amine at neutral pH, but in principle should afford greater triplex stabilization at higher pH.
1.2.1.3 Addition of Positive Charge to the Bases
As mentioned above, the C+.GC triplet is more stable than T.AT due, in part, to the effect of the positive charge. With this in mind 5-propargylamino dU (UP, Figure 3C (i)) was prepared in order to enhance the affinity of T for AT base pairs. This analogue bears a positive charge attached to the 5-position of U rather than in the stacked ring system (as in protonated C). TFOs containing several substitutions of this analogue are markedly more stable than unmodified TFOs, though the complexes are pH-dependent as a result of the requirement for protonation of this amino group. Unlike protonated C, adjacent UP substitutions are not destabilizing. This demonstrates that removing the charge from the π-stacked bases and placing it in the major groove is a useful approach for stabilizing triplexes. The alkylyl moiety of UP also contributes to triplex stability by enhancing stacking interactions.
The covalent attachment of polyamine groups to different bases has also been attempted. The attachment of spermine at the 5-position of uracil and to the N4 position of methylcytosine (Figure 3C (iii and iv)) both increased triplex stability at physiological pH, though the complexes exhibited decreased selectivity.
Combining favourable base modifications with suitable sugar modifications has also been extremely successful. Bis-amino dU (BAU) combines the propargylamino modification at the 5-position of U with addition of an amino-ethoxy group at the 2'-position of the sugar (Figure 3C (ii)). This analogue contains two positive charges at physiological pH and dramatically increases triplex stability with an increase in Tm of 8°C per modification at pH 6, and the stabilization is much greater than when either modification is used alone. This is shown in the melting curves presented in Figure 4, which compare triplex stability at pH 6.0 using oligonucleotides in which the central triplet is either T.AT, UP.AT, or BAU.AT. These two positive charges act in different ways to enhance triplex stability: the 2'-aminoethoxy group interacts with a phosphate on the duplex purine strand, while the 5-propargylamino group interacts with a third-strand phosphate. Interestingly this analogue has greater sequence selectivity than thymidine, with enhanced discrimination against pyrimidine inversions. The high stability of the BAU.AT triplet permits triplex formation at physiological pH, even for sequences that contain several GC base pairs. An example of this effect is shown in Figure 5a for which an 11-mer oligonucleotide containing 5 BAU residues, together with three cytosines and three thymine bases is able to produce a triplex with an apparent dissociation constant of 5.5 nM at pH 7.0.
1.2.3 Backbone Modifications
An alternative method for decreasing the charge repulsion between the three-polyanionic DNA strands is to use TFOs that contain neutral backbones. Examples of these are shown in Figure 6. Several such modifications have been developed. Replacement of the phosphate linkage with a methylphosphonate group (Figure 6 (iii)) was successfully used for triplex formation using short oligonucleotides containing alternating methylphosphonate and phosphodiester linkers. However, subsequent studies with longer fully substituted TFOs showed that this modification was destabilizing. This may have been caused by the diastereoisomeric mixture of the methylphosphonate residues. Other studies showed that α-methylphosphonate TFOs produce stable triplexes but these are much more difficult to synthesize.
The N3'-P5' amidate modification, where O3' of the internucleoside phosphate is replaced by NH (Figure 6 (ii)) increases the binding constant at neutral pH by nearly two orders of magnitude. Triplex binding is probably improved as this modification favours the N-type sugar conformation as discussed above. This modification has also been combined with the addition of a cationic copolymer, which cooperatively stabilizes triplex formation and increases association rates by four orders of magnitude.
Morpholino oligonucleotides are an interesting class of analogues, in which the ribose sugar is replaced with a six-membered morpholino ring and the phosphodiester linkage is replaced by a phosphorodiamidate (Figure 6 (iv)). TFOs containing this modification are less stable than those containing the N3'-P5' amidate modification at high concentration of cations but are more stabilizing at low ionic strength. This modification has also been used with α-oligonucleotides.
The most extensively employed uncharged backbone modification is peptide nucleic acid (PNA). PNA is composed of repeating (2-aminoethyl)glycine units to which nucleobases are linked by methylene bridges (Figure 6 (v)). PNA usually interacts with duplex DNA via a mechanism of strand displacement and P-loop formation, requiring two molecules of PNA, generating a 2:1 PNA: DNA triplex. Two pyrimidine-containing PNA molecules form a local triplex with the purine-containing DNA strand. This leaves the pyrimidine DNA strand looped out as a single strand. The resulting triplex is more stable than the equivalent DNA triplex since there is much lower charge repulsion between the three strands. In a few instances, PNA can form a 1:2 PNA:DNA triplex by simple binding of a PNA third strand to a DNA duplex, though this is usually restricted to cytosine-containing PNAs.
1.2.4 Base Stacking
Base stacking is an important factor that affects the stability and structure of both duplex and triplex DNA and increasing the aromatic surface area of a base might be expected to enhance triplex formation. Most such modifications have been based on thymine, by adding further aromatic rings across the 4–5 or 5–6 positions, which should not affect the hydrogen-bonding groups. Surprisingly triplexes containing these modifications do not show enhanced stability, though the non-natural pyrido[2,3-d] pyrimidine nucleoside (F) recognizes AT base pairs with a similar affinity to T. The addition of a hydrophobic substituent at the 5-position of the pyrimidine base increases hydrophobic stacking interactions within the major groove. The simplest of these is the addition of a methyl group and probably explains why T.AT is more stable than U.AT and MeC+.GC is more stable than C+.GC. The addition of a propyne group to the 5-position of pyrimidines further extends the hydrophobic surface and each propynyl-dU substitution increases the Tm by about 2.5°C relative to thymine and this modification requires lower concentrations of magnesium to achieve a stabilizing effect. Propynyl-dC reduces triplex stability relative to C as it decreases the pKa of the N(3) atom of the heterocycle. A recent study on the properties of four different C5-amino modified deoxyuridines 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.
1.2.5 Triplex-Binding Ligands
Several small molecules have been developed that preferentially bind to triplex over duplex DNA and stabilize triple-stranded structures. These compounds are usually composed of aromatic rings containing heteroatoms for stacking (intercalation) between the base triplets and commonly incorporate a positive charge to partially alleviate the triplex charge repulsion. The first to be described was a benzopyridoindole (BePI), though a wide range of such ligands has now been described. These have recently been reviewed.
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Excerpted from Sequence-Specific DNA Binding Agents by Michael Waring. Copyright © 2006 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Table of Contents
Chapter 1: DNA recognition by triple helix formation; Chapter 2: Interfacial inhibitors of human topoisomerase I; Chapter 3: Diversity of topoisomerase I inhibitors for cancer chemotherapy; Chapter 4: Slow DNA binding; Chapter 5: DNA Gene Targeting using Peptide Nucleic Acid (PNA); Chapter 6: Actinomycin D - Sixty years of progress in characterising a sequence-selective DNA binding agent; Chapter 7: Thermal denaturation of drug-DNA complexes - Tools and tricks; Chapter 8: Computer simulations of drug-DNA interactions - a personal journey; Chapter 9: The discovery of G-quadruplex telomere targeting drugs; Chapter 10: The Mechanism of Action of Telomestatin, a G-Quadruplex-Interactive Compound; Chapter 11: Riboswitches and RibozymesWhat People are Saying About This
An excellent overview of the work being done in the exciting areas of discovering agents that exhibit selectivity and sequence specificity for DNA. Michael J Waring, who has been a pioneer in and made seminal contributions to this particular field. A comprehensive reference on this rapidly expanding field.....is a must read.....an excellent text book for a graduate course on this subject and a useful guide at the post-graduate level for biochemists as well as biophysicists.