Effect of loop orientation on quadruplex-TMPyP4 interaction

G-quadruplexes are believed to be potential targets for therapeutic intervention and this has resulted in designing of various quadruplex interacting ligands. Moreover, reports about existence of quadruplex forming sequences across the genome have propelled greater interest in understanding their interaction with small molecules. An intramolecular quadruplex sequence can adopt different conformations, owing to different orientation of loops in the structure. The differences in the loop orientation can affect their molecular recognition. Herein, we have studied the interaction of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H, 23H-porphine (TMPyP4), a well-known G quadruplex binding ligand with three DNA quadruplexes differing in loop orientations. Results obtained from UV, ITC, and SPR studies have coherently revealed that the TMPyP4 molecule shows preferential binding to parallel G-quadruplex ( c-myc and c-kit) over its antiparallel counterpart (human telomeric). The binding affinity for parallel quadruplex was (10(7)) 1 order of magnitude higher than that for antiparallel DNA quadruplex (10 ). The study shows two binding modes, stronger binding (10(7)) of TMPyP4 involving end stacking and a weaker external binding (10 ), while TMPyP4 shows only one binding mode with duplex with a binding affinity of the order of 10(6). Overall, the study emphasizes that differences in the loop orientation give rise to different conformations of quadruplex, which in turn govern its binding to small molecules, and thereby play a pivotal role in molecular recognition.     

Adaptive recognition of small molecules by nucleic acid aptamers through a label-free approach

We report a novel label-free method for the investigation of the adaptive recognition of small molecules by nucleic acid aptamers using capillary electrophoresis analysis. Cocaine and argininamide were chosen as model molecules, and the two corresponding DNA aptamers were used. These single-strand DNAs folded into their specific secondary structures, which were mainly responsible for the binding of the target molecules with high affinity and specificity. For molecular recognition, the nucleic acid structures then underwent additional conformational changes, while keeping the target molecules stabilized by intermolecular hydrogen bonds. The intrinsic chemical and physical properties of the target molecules enabled them to act as indicators for adaptive binding. Thus any labeling or modification of the aptamers or target molecules were made obsolete. This label-free method for aptamer-based molecular recognition was also successfully applied to biological fluids and therefore indicates that this approach is a promising tool for bioanalysis.     

Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP

Background: Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP)' with micromolar affinity exhibit distinct binding site sequences and secondary structures. We report below on the tertiary structure of the AMP-DNA aptamer complex in solution and compare it with the previously reported tertiary structure of the AMP-RNA aptamer complex in solution.Results: The solution structure of the AMP-DNA aptamer complex shows, surprisingly, that two AMP molecules are intercalated at adjacent sites within a rectangular widened minor groove. Complex formation involves adaptive binding where the asymmetric internal bubble of the free DNA aptamer zippers up through formation of a continuous six-base mismatch segment which includes a pair of adjacent three-base platforms. The AMP molecules pair through their Watson-Crick edges with the minor groove edges of guanine residues. These recognition G·A mismatches are flanked by sheared G·A and reversed Hoogsteen G·G mismatch pairs.Conclusions: The AMP-DNA aptamer and AMP-RNA aptamer complexes have distinct tertiary structures and binding stoichiometries. Nevertheless, both complexes have similar structural features and recognition alignments in their binding pockets. Specifically, AMP targets both DNA and RNA aptamers by intercalating between purine bases and through identical G·A mismatch formation. The recognition G·A mismatch stacks with a reversed Hoogsteen G·G mismatch in one direction and with an adenine base in the other direction in both complexes. It is striking that DNA and RNA aptamers selected independently from libraries of 10¹⁴ molecules in each case utilize identical mismatch alignments for molecular recognition with micromolar affinity within binding-site pockets containing common structural elements.     

Single-Round DNA Aptamer Selection by Combined Use of Capillary Electrophoresis and Next Generation Sequencing: An Aptaomics Approach for Identifying Unique Functional Protein-Binding DNA Aptamers

In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time. Selection rules based on dissociation equilibria and kinetics were devised to obtain families of analogous sequences. Selected sequences of the same family were shown to bind thrombin with high affinity. Furthermore, data acquired from SR-CE selection was mined by creating sub-libraries that were categorized by the functionality of the aptamers (e. g., pre-organized aptamers versus structure-induced aptamers). Using this approach, a novel fluorescent molecular recognition sensor for thrombin with nanomolar detection limits was discovered. Thus, in this proof-of-concept report, we have demonstrated the potential of a “DNA Aptaomics” approach to systematically design functional aptamers as well as to obtain high affinity aptamers.     

Design of peptide that recognizes double-stranded DNA.

A novel molecular tool for double-stranded (ds) DNA detection using synthetic peptide is described. The peptide was designed based on the DNA binding domain of the lambda phage CRO repressor (CRO). The designed peptides contain helix-turn-helix (HTH), which is DNA binding motif. A cyclic peptide and a mutant peptide based on CRO were also designed, and the resulting affinity for dsDNA was increased. Furthermore, native amino acids of the peptide were replaced with arginine to increase the affinity for dsDNA. The affinity of these peptides for DNA binding was assessed by surface plasmon resonance (SPR) technique.     

Effects of DNA mismatches on binding affinity and kinetics of polymerase-DNA complexes as revealed by surface plasmon resonance biosensor

In this study, surface plasmon resonance (SPR) biosensor techniques were used to obtain quantitative information on the kinetics of the DNA and polymerase I (Klenow fragment) interaction. DNA duplexes containing different base compositions at the binding site were immobilized on the SPR sensor surface via biotin-streptavidin chemistry and the subsequent binding of the polymerase was measured in real time. Various kinetic models were tested and a translocation model was shown to provide the best fit for the binding and dissociation profiles. The results revealed that the enzyme binds to DNA at both the polymerase and the exonuclease domains with different association and dissociation rates as well as affinity constants, depending on the presence of mismatches near the primer 3'-end. Introduction of unpaired bases increases the DNA binding affinity towards the exonuclease domain and promotes the translocation of DNA from the polymerase site to the exonuclease site. The results also demonstrated that SPR biosensors may be used as a sensitive technique for studying molecular recognition events such as single-base discrimination involved in protein-DNA interaction.     

Arrest of Rolling Circle Amplification by Protein‐Binding DNA Aptamers

Certain DNA polymerases, such as ?29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super‐long single‐stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand‐displacement ability of these polymerases. In this work, the ability of ?29DNAP to carry out RCA over circular templates containing a protein‐binding DNA aptamer sequence was investigated. It was found that protein–aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein‐binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.     

The Investigation of Interaction Competition Between ATP and DIPP-Ala,Boc-Ala,or Ala by ESI-MS/MS and Theoretical Calculation

The interactions between ATP and N-(O,O-diisopropyl) phosphoryl-L-alanine (DIPP-Ala), N-(tert-butoxycarbonyl)-L-alanine (Boc-Ala), or L-alanine (Ala) were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The non-covalent complexes between ATP and Boc-Ala or DIPP-Ala were observed, while the complex between ATP and Ala was not found in the mass spectra. The affinity of DIPP-Ala for ATP was confirmed to be stronger than that of Boc-Ala by competition experiment. Through molecular modeling calculations, it was found that the non-covalent complexes were stabilized by intermolecular hydrogen bonds, and the affinity sequence for ATP was DIPP-Ala > Boc-Ala > Ala by comparing their binding energy, ?35.407 kcal/mol, ?15.634 kcal/mol, ?6.555 kcal/mol, respectively. The results implied that a phosphoryl group was a very important functional group to provide an interaction site between amino acids and ATP, and that N-phosphoryl amino acids can be used as a good model of protein in the studies of molecular recognition of ATP.     

High Affinity Recognition of a Selected Amino Acid Epitope within a Protein by Cucurbit[8]uril Complexation

Supramolecular interactions between the host cucurbit[8]uril (CB[8]) and amino acids have been widely interrogated, but recognition of specific motifs within a protein domain have never been reported. A phage display approach was herein used to select motifs with the highest binding affinity for the heteroternary complex with methyl viologen and CB[8] (MV?CB[8]) within a vast pool of cyclic peptide sequences. From the selected motifs, an epitope consisting of three amino acid was extrapolated and incorporated into a solvent‐exposed loop of a protein domain; the protein exhibited micromolar binding affinity for the MV?CB[8] complex, matching that of the cyclic peptide. By achieving selective CB[8]‐mediated conjugation of a small molecule to a recombinant protein scaffold we pave the way to biomedical applications of this simple ternary system.     

光谱法研究金属离子与肿瘤抑制蛋白p53的DNA结合结构域的相互作用

利用荧光滴定法研究了9种金属离子与序列特异性的DNA结合结构域(p53DBD)的结合反应,其结合能力依次为Fe3+＞Zn2+＞Cu2+＞Ca2+＞Mg2+＞Ba2+＞Mn2+＞Ni2+＞Co2+.圆二色谱研究结果表明,Ba2+, Ca2+, Co2+, Mn2+及Ni2+并未引起蛋白二级结构变化; Zn2+, Mg2+及Fe3+诱导蛋白结构细微调整;而Cu2+结合导致蛋白螺旋结构大量丢失.ANS结合研究结果表明, Mg2+与Zn2+相似,诱导p53DBD蛋白表面疏水性增强,而Fe3+引起p53DBD蛋白表面疏水性降低.因此,Mg2+和Fe3+可能是影响或调节p53活性的潜在因子之一.     

Aromatic Rings as Molecular Determinants for the Molecular Recognition of Protein Kinase Inhibitors

Protein kinases are key enzymes in many signal transduction pathways, and play a crucial role in cellular proliferation, differentiation, and various cell regulatory processes. However, aberrant function of kinases has been associated with cancers and many other diseases. Consequently, competitive inhibition of the ATP binding site of protein kinases has emerged as an effective means of curing these diseases. Over the past three decades, thousands of protein kinase inhibitors (PKIs) with varying molecular frames have been developed. Large-scale data mining of the Protein Data Bank resulted in a database of 2139 non-redundant high-resolution X-ray crystal structures of PKIs bound to protein kinases. This provided us with a unique opportunity to study molecular determinants for the molecular recognition of PKIs. A chemoinformatic analysis of 2139 PKIs resulted in findings that PKIs are “flat” molecules with high aromatic ring counts and low fractions of sp³ carbon. All but one PKI possessed one or more aromatic rings. More importantly, it was found that the average weighted hydrogen bond count is inversely proportional to the number of aromatic rings. Based on this linear relationship, we put forward the exchange rule of hydrogen bonding interactions and non-bonded π-interactions. Specifically, a loss of binding affinity caused by a decrease in hydrogen bonding interactions is compensated by a gain in binding affinity acquired by an increase in aromatic ring-originated non-bonded interactions (i.e., π–π stacking interactions, CH–π interactions, cation–π interactions, etc.), and vice versa. The very existence of this inverse relationship strongly suggests that both hydrogen bonding and aromatic ring-originated non-bonded interactions are responsible for the molecular recognition of PKIs. As an illustration, two representative PKI–kinase complexes were employed to examine the relative importance of different modes of non-bonded interactions for the molecular recognition of PKIs. For this purpose, two FDA-approved PKI drugs, ibrutinib and lenvatinib, were chosen. The binding pockets of both PKIs were thoroughly examined to identify all non-bonded intermolecular interactions. Subsequently, the strengths of interaction energies between ibrutinib and its interacting residues in tyrosine kinase BTK were quantified by means of the double hybrid DFT method B2PLYP. The resulting energetics for the binding of ibrutinib in tyrosine kinase BTK showed that CH–π interactions and π–π stacking interactions between aromatic rings of the drug and hydrophobic residues in its binding pocket dominate the binding interactions. Thus, this work establishes that, in addition to hydrogen bonding, aromatic rings function as important molecular determinants for the molecular recognition of PKIs. In conclusion, our findings support the following pharmacophore model for ATP-competitive kinase inhibitors: a small molecule features a scaffold of one or more aromatic rings which is linked with one or more hydrophilic functional groups. The former has the structural role of acting as a scaffold and the functional role of participating in aromatic ring-originated non-bonded interactions with multiple hydrophobic regions in the ATP binding pocket of kinases. The latter ensure water solubility and form hydrogen bonds with the hinge region and other hydrophilic residues of the ATP binding pocket.     

Exploring Weak Ligand–Protein Interactions by Long‐Lived NMR States: Improved Contrast in Fragment‐Based Drug Screening

Ligands that have an affinity for protein targets can be screened very effectively by exploiting favorable properties of long‐lived states (LLS) in NMR spectroscopy. In this work, we describe the use of LLS for competitive binding experiments to measure accurate dissociation constants of fragments that bind weakly to the ATP binding site of the N‐terminal ATPase domain of heat shock protein 90 (Hsp90), a therapeutic target for cancer treatment. The LLS approach allows one to characterize ligands with an exceptionally wide range of affinities, since it can be used for ligand concentrations [L] that are several orders of magnitude smaller than the dissociation constants K_D. This property makes the LLS method particularly attractive for the initial steps of fragment‐based drug screening, where small molecular fragments that bind weakly to a target protein must be identified, which is a difficult task for many other biophysical methods.     

Scaffold-independent analysis of RNA-protein interactions: the Nova-1 KH3-RNA complex

We describe a method to analyze the sequence specificity of an RNA-binding domain. The method, which we have named scaffold-independent analysis, reports on the specificity for each nucleotide position within an RNA target, uncoupled from the surrounding structural and sequence context. We expect this information to improve our understanding of protein-RNA interfaces in ssRNA binding domains (e.g., KH or RRM domains) and to be useful to the design of novel protein-RNA recognition surfaces. Our NMR binding assays using the third KH domain of the Nova-1 protein provide a proof-of-principle for the method and novel information on the specificity of this domain for its RNA targets.     

Structure-switching allosteric deoxyribozymes

DNA aptamers and DNA enzymes (DNAzymes or deoxyribozymes) are single-stranded DNA molecules with ligand-binding and catalytic capabilities, respectively. Allosteric DNA enzymes (aptazymes) are deoxyribozymes whose activity can be regulated by the binding state of an appended aptamer domain and have many potential uses in the fields of drug discovery and diagnostics. In this report, we describe a simple, yet potentially general, DNA aptazyme rational design strategy that requires no structural characterization of the constituent deoxyribozymes and aptamers. It is based on the concept originally developed in our laboratory for the design of structure-switching signaling aptamers that change structural states from a DNA-DNA duplex to a DNA-target complex upon target binding. In our new strategy, an antisense oligonucleotide is used to regulate the enzymatic activity of a linked aptamer-deoxyribozyme by annealing with a stretch of nucleotides on each side of the aptamer-DNAzyme junction. Structural reorganization of the aptamer domain upon target binding relieves the suppressive effect of this regulatory oligonucleotide on the attached DNA enzyme. Consequently, the target-binding event triggers the catalytic action of the aptazyme. We have demonstrated this concept using two RNA-cleaving deoxyribozymes, each adjoined to a DNA aptamer that binds ATP. These allosteric DNA enzymes exhibit the same ligand-binding specificity as the parental DNA aptamer and show up to 30-fold rate enhancement in the presence of ATP. The described methodology provides a convenient approach for rationally designing catalytic DNA-based biosensors.     

Coupling a natural receptor protein with an artificial receptor to afford a semisynthetic fluorescent biosensor

An artificial receptor and a signal transducer have been engineered on a lectin (saccharide-binding protein) surface by a post-photoaffinity labeling modification method. Saccharide binding can be directly and selectively read out by the fluorescence changes of the fluorophore via photoinduced electron transfer (PET) mode. Fluorescence titration with various saccharides reveals that molecular recognition by the artificial receptor is successfully coupled to the native binding site of the lectin, producing a novel fluorescent saccharide biosensor showing modulated specificity and enhanced affinity. Designed cooperativity between artificial and native molecular recognition modules was quantitatively demonstrated by the comparison of the binding affinities, and it represents a new strategy in molecular recognition. By using appropriate artificial receptors and various native lectins, this approach may provide many new semisynthetic biosensors for saccharide derivatives such as glycolipids and glycopeptides/proteins. An extended library of lectin-based biosensors is envisioned to be useful for glycome research, a newly emerging field of the post-genomic era.     

Towards a rational spacer design for bivalent inhibition of estrogen receptor

Estrogen receptors are known drug targets that have been linked to several kinds of cancer. The structure of the estrogen receptor ligand binding domain is available and reveals a homodimeric layout. In order to improve the binding affinity of known estrogen receptor inhibitors, bivalent compounds have been developed that consist of two individual ligands linked by flexible tethers serving as spacers. So far, binding affinities of the bivalent compounds do not surpass their monovalent counterparts. In this article, we focus our attention on the molecular spacers that are used to connect the individual ligands to form bivalent compounds, and describe their thermodynamic contribution during the ligand binding process. We use computational methods to predict structural and entropic parameters of different spacer structures. We find that flexible spacers introduce a number of effects that may interfere with ligand binding and possibly can be connected to the low binding affinities that have been reported in binding assays. Based on these findings, we try to provide guidelines for the design of novel molecular spacers.     

Cell-specific internalization study of an aptamer from whole cell selection

Nucleic acid aptamers have been shown many unique applications as excellent probes in molecular recognition. However, few examples are reported which show that aptamers can be internalized inside living cells for aptamer functional studies and for targeted intracellular delivery. This is mainly due to the limited number of aptamers available for cell-specific recognition, and the lack of research on their extra- and intracellular functions. One of the major difficulties in aptamers' in vivo application is that most of aptamers, unlike small molecules, cannot be directly taken up by cells without external assistance. In this work, we have studied a newly developed and cell-specific DNA aptamer, sgc8. This aptamer has been selected through a novel cell selection process (cell-SELEX), in which whole intact cells are used as targets while another related cell line is used as a negative control. The cell-SELEX enables generation of multiple aptamers for molecular recognition of the target cells and has significant advantages in discovering cell surface binding molecules for the selected aptamers. We have studied the cellular internalization of one of the selected aptamers. Our results show that sgc8 is internalized efficiently and specifically to the lymphoblastic leukemia cells. The internalized sgc8 aptamers are located inside the endosome. Comparison studies are done with the antibody for the binding protein of sgc8, PTK7 (Human protein tyrosine kinase-7) on cell surface. We also studied the internalization kinetics of both the aptamer and the antibody for the same protein on the living cell surface. We have further evaluated the effects of sgc8 on cell viability, and no cytotoxicity is observed. This study indicates that sgc8 is a promising agent for cell-type specific intracellular delivery.     

Conformational fluctuations of a protein-DNA complex and the structure and ordering of water around it

Protein-DNA binding is an important process responsible for the regulation of genetic activities in living organisms. The most crucial issue in this problem is how the protein recognizes the DNA and identifies its target base sequences. Water molecules present around the protein and DNA are also expected to play an important role in mediating the recognition process and controlling the structure of the complex. We have performed atomistic molecular dynamics simulations of an aqueous solution of the protein-DNA complex formed between the DNA binding domain of human TRF1 protein and a telomeric DNA. The conformational fluctuations of the protein and DNA and the microscopic structure and ordering of water around them in the complex have been explored. In agreement with experimental studies, the calculations reveal conformational immobilization of the terminal segments of the protein on complexation. Importantly, it is discovered that both structural adaptations of the protein and DNA, and the subsequent correlation between them to bind, contribute to the net entropy loss associated with the complex formation. Further, it is found that water molecules around the DNA are more structured with significantly higher density and ordering than that around the protein in the complex.