Oligonucleotides containing abasic threoninol-terpyridine residues as potential artificial ribonucleases.

Artificial ribonucleases, also known as synthetic ribozymes, were synthesized with an internal, stereochemically-pure, abasic threoninol backbone-residue to which the RNA transesterification catalyst copper (II) terpyridine was covalently linked. These oligonucleotide conjugates were constructed to determine if the stereochemistry of the abasic threoninol backbone residue influences the transesterification rate of complementary RNA oligonucleotides. Following synthesis, these compounds were reacted with complementary 28-mer and 159-mer RNA substrates and their relative transesterification efficiencies were determined. The transesterification kinetics were also compared with previously synthesized oligonucleotides that incorporated copper (II) terpyridine via a serinol-residue. It was determined that oligonucleotides that contained copper (II) terpyridine linked via a (2S,3S)-threoninol backbone were more efficient at RNA transesterification than their (2R,3R)-stereoisomer counterpart.

Size matters: DNA binding site kinetics as a function of polyamide size.

Hairpin polyamides (PAs) are remarkable minor groove-binding DNA ligands that demonstrate high affinity and sequence selectivity. Following extensive studies of 6-8 ring hairpin PAs have been more recent descriptions of larger PAs (14 rings or more) and their distinguishing properties and biological activities. However, there are no comparative kinetic studies of PA DNA binding behaviors over a range of PA sizes, making it difficult to understand important structure-activity relationships related to PA size. Described herein is the first comparative kinetic study as a function of hairpin PA size that examines the complexities of PA-DNA binding behaviors with unprecedented detail. DNA binding kinetics of PAs with 6 (PA6) and 20 (PA20) rings are extensively characterized by fluorescence spectroscopy, and the properties compared with those of 8 and 14 ring hairpin PAs. PA6 has a 1:1 binding site stoichiometry but exhibits biphasic association kinetics, consistent with populating more than one binding mode. One decay constant is at the diffusion controlled limit, and the other is 400-fold slower. In contrast, PA20 has high binding site stoichiometry (2.5:1) but displays a much simpler association kinetic trace with a decay constant of 1e6 M-1s-1. Due to the variability and complexity of association kinetics, it is difficult to identify trends in this behavior as a function of PA size. However, even though hairpin PAs of 8 or more rings bind DNA with similar affinities, residence times increase as PA size increases, ranging from 20 s to over 2500 s. Particularly compelling is that the antiviral PA20 shows little to no dissociation from DNA when challenged with competitor DNA, suggesting that high residence times are important for this biological activity.

DNA binding site kinetics of a large antiviral polyamide.

Polyamides (PAs) are powerful DNA ligands that can bind the minor groove of DNA with high affinity and specificity. While the characterization of PA-DNA behavior has focused principally on hairpin PAs 6-8 rings in size, there is increasing evidence that their behavior does not necessarily reflect the complexities that are emerging from studies of larger hairpin PAs, particularly concerning sequence mismatch tolerance and observed but unaddressed high PA-target site binding stoichiometries. To explore these complexities in more detail, kinetics studies of binding a large anti-HPV hairpin polyamide to an isolated DNA recognition site are described. Using a fluorescence assay, two distinct binding phases are observed for the first time in hairpin PA literature. PA14 concentration dependence analysis indicates that the faster binding event is diffusion-controlled; the apparent, second event is significantly slower (350-1500 fold). Both association phases are sampled in 1:1 complexes, consistent with cooperative binding of two PA molecules even under this condition. Fitting of the slow phase to a biexponential model yields two λon,app that differ by 4-5-fold, which is consistent with the high mismatch tolerance and binding site stoichiometry previously observed. A/T patterns in the recognition sequence do not affect these decay constants significantly. Dissociation decay constants are among the slowest reported for hairpin PAs (10-3 s-1), independent of A/T pattern, and may point to the efficacy of PA14 as an antiviral.

DNA binding thermodynamics and site stoichiometry as a function of polyamide size.

The interactions of 6-8 ring hairpin polyamides (PAs) with the minor groove of DNA have been investigated extensively. More recent studies of large antiviral PAs (14-20 rings) active against small DNA tumor viruses lead to questions regarding the extent to which the DNA binding behaviors of the well studied, smaller PAs can be reliably extrapolated to the larger ones. Described here is the first reported study of hairpin PA-DNA binding thermodynamics as a function of PA size (6-20 rings). All PAs exhibit binding affinity in the low nM to upper pM range, which indicates that affinity is not a discriminator of antiviral activity. Unlike the smaller PAs, a 20-ring PA does not appreciably dissociate from DNA in competition experiments, which indicates very long residence time that is consistent with antiviral activity. While the DNA binding thermodynamics for the smaller antivirally inactive 6- and 8-ring PAs is clearly enthalpically driven, the larger antiviral PAs (14- and 20-rings) exhibit strongly entropically-driven DNA binding. These distinct energetic signatures indicate that different types of interactions drive these associations. In DNA binding site stoichiometry experiments conducted at both nM and µM concentrations, all PAs except the 6-ring PA bind an isolated site with site stoichiometry of at least two PAs per recognition sequence. Electrostatic contributions to DNA binding affinity are small for all PAs and not correlated with PA size but weakly correlated with the number of imidazole residues. Altogether, these results indicate that DNA binding behaviors of smaller hairpin PAs do not necessarily reflect those of larger PAs. These are vital considerations in the development of hairpin PAs for biological use.

Modulating DNA by polyamides to regulate transcription factor PU.1-DNA binding interactions.

Hairpin polyamides are synthetic small molecules that bind DNA minor groove sequence-selectively and, in many sequences, induce widening of the minor groove and compression of the major groove. The structural distortion of DNA caused by polyamides has enhanced our understanding of the regulation of DNA-binding proteins via polyamides. Polyamides have DNA binding affinities that are comparable to those proteins, therefore, can potentially be used as therapeutic agents to treat diseases caused by aberrant gene expression. In fact, many diseases are characterized by over- or under-expressed genes. PU.1 is a transcription factor that regulates many immune system genes. Aberrant expression of PU.1 has been associated with the development of acute myeloid leukemia (AML). We have, therefore, designed and synthesized ten hairpin polyamides to investigate their capacity in controlling the PU.1-DNA interaction. Our results showed that nine of the polyamides disrupt PU.1-DNA binding and the inhibition capacity strongly correlates with binding affinity. One molecule, FH1024, was observed forming a FH1024-PU.1-DNA ternary complex instead of inhibiting PU.1-DNA binding. This is the first report of a small molecule that is potentially a weak agonist that recruits PU.1 to DNA. This finding sheds light on the design of polyamides that exhibit novel regulatory mechanisms on protein-DNA binding.

Thermodynamics and site stoichiometry of DNA binding by a large antiviral hairpin polyamide.

PA1 (dIm-PyPyßPyPyPy-γ-PyPyßPyPyPyPyß-Ta) is a large (14-ring) hairpin polyamide that was designed to recognize the DNA sequence 5'-W2GW7-3', where W is either A or T. As is common among the smaller 6-8-ring hairpin polyamides (PAs), it binds its target recognition sequence with low nM affinity. However, in addition to its large size, it is distinct from these more extensively characterized PAs in its high tolerance for mismatches and antiviral properties. In ongoing attempts to understand the basis for these distinctions, we conducted thermodynamics studies of PA1-DNA interactions. The temperature dependence of binding affinity was measured using TAMRA-labeled hairpin DNAs containing a single target sequence. PA1 binding to either an ATAT/TATA or an AAAA/TTTT pattern is consistently entropically driven. This is in contrast to the A/T pattern-dependent driving forces for DNA binding by netropsin, distamycin, and smaller hairpin polyamides. Analysis of the salt dependence of PA1-DNA binding reveals that within experimental error, there is no dependence on ionic strength, indicating that the polyelectrolyte effect does not contribute to PA1-DNA binding energetics. This is similar to that observed for smaller PAs. PA1-DNA recognition sequence binding stoichiometries were determined at both nM (fluorescence) and µM (circular dichroism) concentrations. With all sequences and under both conditions, multiple PA1 molecules bind the small DNA hairpin that contains only a single recognition sequence. Implications for these observations are discussed.

A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid.

Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus.IMPORTANCE Negative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs.

ß-Alanine and N-terminal cationic substituents affect polyamide-DNA binding.

Minor-groove binding hairpin polyamides (PAs) bind specific DNA sequences. Synthetic modifications can improve PA-DNA binding affinity and include flexible modules, such as ß-alanine (ß) motifs to replace pyrroles (Py), and increasing compound charge using N-terminal cationic substituents. To better understand the variations in kinetics and affinities caused by these modifications on PA-DNA interactions, a comprehensive set of PAs with different numbers and positions of ß and different types of N-cationic groups was systematically designed and synthesized to bind their cognate sequence, the λB motif. The λB motif is also a strong binding promoter site of the major groove targeting transcription factor PU.1. The PA binding affinities and kinetics were evaluated using a spectrum of powerful biophysical methods: thermal melting, biosensor surface plasmon resonance and circular dichroism. The results show that ß inserts affect PA-DNA interactions in a number and position dependent manner. Specifically, a ß replacement between two imidazole heterocycles (ImßIm) generally strengthens binding. In addition, N-terminal cationic groups can accelerate the association between PA and DNA, but the bulky size of TMG can cause steric hindrance and unfavourable repulsive electrostatic interactions in some PAs. The future design of stronger binding PA requires careful combination of ßs and cationic substituents.

Interactions of two large antiviral polyamides with the long control region of HPV16.

PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes (DNA) in cell culture, and PA25 has broad spectrum activity against three cancer-causing forms of HPV (Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok, K., Helmus, M., Bashkin, J. K., Fisher, C., Antiviral Res. 91 (2011) 177-186). Described here are the interactions of these PAs with sequences in the long control region (LCR) of HPV16 (7348-122). Using an FeEDTA conjugate of PA1 (designed to recognize 5'-W2GW7-3'; W = A or T), 34 affinity cleavage (AC) patterns were detected for this fragment. These sites can be rationalized with sequences featuring perfect, single, double, triple and quadruple mismatches. Quantitative DNase I footprinting analysis indicates that perfect sites bind PA1 with Kds between 0.7 and 2.2 nM. Kds for single, double, triple and quadruple mismatch sites range from 1-3 nM-20 nM. Using AC and EDTA conjugates, we report that unlike smaller 8-ring hairpin PAs, introduction of a chiral turn in this large polyamide has no effect on binding orientation (forward vs. reverse). Despite its design to recognize 5'-W2GW5GW4-3' via two Im residues, a motif not represented in this HPV sequence, a PA25-EDTA conjugate yielded 31 affinity cleavage sites on the region. Low nM Kds for PA25 without EDTA indicates a high tolerance for triple and quadruple mismatches. While there is extensive coverage of the sequence examined, AC cleavage patterns for the two PAs show discrete binding events and do not overlap significantly. This indicates that within the context of A/T rich sequences, these PAs do not recognize a simple shared sequence-related feature of the DNA. These insights continue to inform the complex nature of large hairpin PA-DNA interactions and antiviral behavior.

Heterogeneous dynamics in DNA site discrimination by the structurally homologous DNA-binding domains of ETS-family transcription factors.

The ETS family of transcription factors exemplifies current uncertainty in how eukaryotic genetic regulators with overlapping DNA sequence preferences achieve target site specificity. PU.1 and Ets-1 represent archetypes for studying site discrimination by ETS proteins because their DNA-binding domains are the most divergent in sequence, yet they share remarkably superimposable DNA-bound structures. To gain insight into the contrasting thermodynamics and kinetics of DNA recognition by these two proteins, we investigated the structure and dynamics of site discrimination by their DNA-binding domains. Electrophoretic mobilities of complexes formed by the two homologs with circularly permuted binding sites showed significant dynamic differences only for DNA complexes of PU.1. Free solution measurements by dynamic light scattering showed PU.1 to be more dynamic than Ets-1; moreover, dynamic changes are strongly coupled to site discrimination by PU.1, but not Ets-1. Interrogation of the protein/DNA interface by DNA footprinting showed similar accessibility to dimethyl sulfate for PU.1/DNA and Ets-1/DNA complexes, indicating that the dynamics of PU.1/DNA complexes reside primarily outside that interface. An information-based analysis of the two homologs' binding motifs suggests a role for dynamic coupling in PU.1's ability to enforce a more stringent sequence preference than Ets-1 and its proximal sequence homologs.

Modulation of DNA-polyamide interaction by ß-alanine substitutions: a study of positional effects on binding affinity, kinetics and thermodynamics.

Hairpin polyamides (PAs) are an important class of sequence-specific DNA minor groove binders, and frequently employ a flexible motif, ß-alanine (ß), to reduce the molecular rigidity to maintain the DNA recognition register. To better understand the diverse effects that ß can have on DNA-PA binding affinity, selectivity, and especially kinetics, which have rarely been reported, we have initiated a detailed study for an eight-heterocyclic hairpin PA and its ß derivatives with their cognate and mutant sequences. With these derivatives, all internal pyrroles of the parent PA are systematically substituted with single or double ßs. A set of complementary experiments have been conducted to evaluate the molecular interactions in detail: UV-melting, biosensor-surface plasmon resonance, circular dichroism and isothermal titration calorimetry. The ß substitutions generally weaken the binding affinities of these PAs with cognate DNA, and have large and diverse influences on PA binding kinetics in a position- and number-dependent manner. The DNA base mutations have also shown positional effects on the binding of a single PA. Besides the ß substitutions, the monocationic Dp group [3-(dimethylamino)propylamine] in parent PA has been modified into a dicationic Ta group (3,3'-diamino-N-methyldipropylamine) to minimize the frequently observed PA aggregation with ITC experiments. The results clearly show that the Ta modification not only maintains the DNA binding mode and affinity of PA, but also significantly reduces PA aggregation and allows the complete thermodynamic signature of eight-ring hairpin PA to be determined for the first time. This combined set of results significantly extends our understanding of the energetic basis of specific DNA recognition by PAs.

DNA Binding Polyamides and the Importance of DNA Recognition in their use as Gene-Specific and Antiviral Agents.

There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyamides (PAs) that are higher homologs of natural products such as distamycin A and netropsin. This work has been pursued by many groups, with the Dervan and Sugiyama groups responsible for many breakthroughs. We have studied PAs since about 1999, partly in industry and partly in academia. Early in this program, we reported methods to control cellular uptake of polyamides in cancer cell lines and other cells likely to have multidrug resistance efflux pumps induced. We went on to discover antiviral polyamides active against HPV31, where SAR showed that a minimum binding size of about 10 bp of DNA was necessary for activity. Subsequently we discovered polyamides active against two additional high-risk HPVs, HPV16 and 18, a subset of which showed broad spectrum activity against HPV16, 18 and 31. Aspects of our results presented here are incompatible with reported DNA recognition rules. For example, molecules with the same cognate DNA recognition properties varied from active to inactive against HPVs. We have since pursued the mechanism of action of antiviral polyamides, and polyamides in general, with collaborators at NanoVir, the University of Missouri-St. Louis, and Georgia State University. We describe dramatic consequences of ß-alanine positioning even in relatively small, 8-ring polyamides; these results contrast sharply with prior reports. This paper was originally presented by JKB as a Keynote Lecture in the 2nd International Conference on Medicinal Chemistry and Computer Aided Drug Design Conference in Las Vegas, NV, October 2013.

Binding studies of a large antiviral polyamide to a natural HPV sequence.

PA1 is a large hairpin polyamide (dImPyPy-ß-PyPyPy-γ-PyPy-ß-PyPyPyPy-ß-Ta; Py = pyrrole, Im = imidazole, ß = beta alanine) that targets the sequence 5'-WWGWWWWWWW-3' (W = A or T) and is effective in eliminating HPV16 in cell culture (Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok, K., Helmus, M., Bashkin, J. K., Fisher, C., Antiviral Res. 91 (2011) 177-186). Described here are its DNA binding properties toward a natural DNA, a 523 bp portion of HPV16 (2150-2672) containing three predicted perfect match sites. Strategies for obtaining binding data on large fragments using capillary electrophoresis are also described. Using an Fe EDTA conjugate of PA1, 19 affinity cleavage (AC) patterns were detected for this fragment. In many cases, there are multiple possible binding sequences (perfect, single and double mismatch sites) consistent with the AC data. Quantitative DNase I footprinting analysis indicates that perfect and most single mismatch sites bind PA1 with Kds between 0.7 and 4 nM, indicating excellent tolerance for the latter. Double mismatch sites exhibit Kds between 12 and 62 nM. A large fraction of the accessible sequence is susceptible to PA1 binding, much larger than predicted based on the literature of polyamide-DNA recognition rules.

DNA damage repair genes controlling human papillomavirus (HPV) episome levels under conditions of stability and extreme instability.

DNA damage response (DDR) genes and pathways controlling the stability of HPV episomal DNA are reported here. We set out to understand the mechanism by which a DNA-binding, N-methylpyrrole-imidazole hairpin polyamide (PA25) acts to cause the dramatic loss of HPV DNA from cells. Southern blots revealed that PA25 alters HPV episomes within 5 hours of treatment. Gene expression arrays identified numerous DDR genes that were specifically altered in HPV16 episome-containing cells (W12E) by PA25, but not in HPV-negative (C33A) cells or in cells with integrated HPV16 (SiHa). A siRNA screen of 240 DDR genes was then conducted to identify enhancers and repressors of PA25 activity. Serendipitously, the screen also identified many novel genes, such as TDP1 and TDP2, regulating normal HPV episome stability. MRN and 9-1-1 complexes emerged as important for PA25-mediated episome destruction and were selected for follow-up studies. Mre11, along with other homologous recombination and dsDNA break repair genes, was among the highly significant PA25 repressors. The Mre11 inhibitor Mirin was found to sensitize HPV episomes to PA25 resulting in a ∼5-fold reduction of the PA25 IC50. A novel assay that couples end-labeling of DNA to Q-PCR showed that PA25 causes strand breaks within HPV DNA, and that Mirin greatly enhances this activity. The 9-1-1 complex member Rad9, a representative PA25 enhancer, was transiently phosphorylated in response to PA25 treatment suggesting that it has a role in detecting and signaling episome damage by PA25 to the cell. These results establish that DNA-targeted compounds enter cells and specifically target the HPV episome. This action leads to the activation of numerous DDR pathways and the massive elimination of episomal DNA from cells. Our findings demonstrate that viral episomes can be targeted for elimination from cells by minor groove binding agents, and implicate DDR pathways as important mediators of this process.

Different thermodynamic signatures for DNA minor groove binding with changes in salt concentration and temperature.

The effects of salt concentration and temperature on the thermodynamics of DNA minor groove binding have quite different signatures: binding enthalpy is salt concentration independent but temperature dependent. Conversely, binding free energy is salt dependent but essentially temperature independent through enthalpy-entropy compensation.

Mapping small DNA ligand hydroxyl radical footprinting and affinity cleavage products for capillary electrophoresis.

The mapping of DNA footprints and affinity cleavage sites for small DNA ligands is affected by the choice of sequencing chemistry and end label, and the potential for indexing errors can be significant when mapping small ligand-DNA interactions. Described here is a mechanism for avoiding such errors based on a summary of standard labeling, cleavage, and indexing chemistries and a comparison among them for analysis of these interactions by capillary electrophoresis. The length dependence of the difference between Sanger and Maxam-Gilbert indexing is examined for a number of duplexes of mixed sequence.

Human papillomavirus episome stability is reduced by aphidicolin and controlled by DNA damage response pathways.

A highly reproducible quantitative PCR (Q-PCR) assay was used to study the stability of human papillomavirus (HPV) in undifferentiated keratinocytes that maintain viral episomes. The term "stability" refers to the ability of episomes to persist with little copy number variation in cells. In investigating the mechanism of action of PA25, a previously published compound that destabilizes HPV episomes, aphidicolin was also found to markedly decrease episome levels, but via a different pathway from that of PA25. Since aphidicolin is known to activate DNA damage response (DDR) pathways, effects of inhibitors and small interfering RNAs (siRNAs) acting within DDR pathways were investigated. Inhibitors of Chk1 and siRNA directed against ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia Rad3-related (ATR) pathways significantly reduced viral episomes, suggesting that these pathways play a role in maintaining HPV episome stability. Inhibitors of Chk2 and DNA-PK had no effect on episome levels. Pharmacological inhibition of ATM proteins had no effect on episome levels, but ATM knockdown by siRNA significantly reduced episome levels, suggesting that ATM proteins are playing an important role in HPV episome stability that does not require kinase activity. These results outline two pathways that trigger episome loss from cells and suggest the existence of a little-understood mechanism that mediates viral DNA elimination. Together, our results also indicate that HPV episomes have a stability profile that is remarkably similar to that of fragile sites; these similarities are outlined and discussed. This close correspondence may influence the preference of HPV for integration into fragile sites.

Promoter scanning of the human COX-2 gene with 8-ring polyamides: unexpected weakening of polyamide-DNA binding and selectivity by replacing an internal N-Me-pyrrole with ß-alanine.

Rules for polyamide-DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal ß-alanines as polyamide building blocks in place of N-methylpyrrole; ß-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327-339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with ß-alanine in 8-ring polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with ß-alanine to generate a ß/Im pairing in two 8-ring polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that ß-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide-DNA recognition.

Correlation of local effects of DNA sequence and position of ß-alanine inserts with polyamide-DNA complex binding affinities and kinetics.

To improve our understanding of the effects of ß-alanine (ß) substitution and the number of heterocycles on DNA binding affinity and selectivity, we investigated the interactions of an eight-ring hairpin polyamide (PA) and two ß derivatives as well as a six-heterocycle analogue with their cognate DNA sequence, 5'-TGGCTT-3'. Binding selectivity and the effects of ß have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV melting, biosensor surface plasmon resonance, isothermal titration calorimetry, circular dichroism, and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔC(p) and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that ß substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in the binding of PA to the mutant DNAs further confirm the position-dependent effects on the PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T·A versus A·T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of ß substitution on binding tell a clear and very important story about sequence-dependent binding of PAs to DNA.