Base Pair Recognition Research Articles

Structural characteristics requisite for the ligand-based selective detection of i-motif DNA.

Here, we have explored the light-up property of coumarin 343 (C343) selectively towards various i-motif DNAs based on the recognition of hemi-protonated cytosine-cytosine base pairing, unlike other DNA structures. We have also demonstrated the versatile ability of this i-motif ligand, i.e., the affinity of C343 towards both intermolecular and intramolecular i-motif DNA differing in the chain lengths, molecularity and sizes, which is observed in various oncogene promoters. A systematic study between i-motif DNA and various coumarin derivatives helps in understanding the structural characteristics required for an ideal ligand, which can be useful for future design of any i-motif DNA ligands.

Selective Small Molecule Recognition of RNA Base Pairs.

Many types of RNAs exist in the human transcriptome, yet only the bacterial ribosome has been exploited as a small molecule drug target. Aside from rRNA, other cellular RNAs such as noncoding RNAs have primarily secondary structure and limited tertiary structure. Within these secondary structures of noncanonically paired and unpaired regions, more than 50% are base paired, with most efforts to target these structures focused on looped regions. A void exists in the availability of small molecules capable of targeting RNA base pairs. Using chemoinformatics, an RNA-focused library enriched for nitrogen-containing heterocycles was developed and tested for binding RNA base pairs, leading to the identification of six selective and previously unknown binders. While all binders were derivatives of benzimidazoles, those with expanded aromatic polycycles bound selectively to AU pairs, while those with flexible urea side chains bound selectively to GC pairs.Two of the three selective GC pair binders candistinguish between two different orientations,5'GG/3'CC and 5'GC/3'CG pairs. Furthermore, all six molecules showed >50-fold selectivity for RNA over DNA. These studies provide foundational knowledge to better exploit RNA as targets for small molecule chemical probes or lead therapeutics by using modules that target RNA base pairs.

Internal transcribed spacer-based CAPS marker development for Lilium hansonii identification from wild Lilium native to Korea

Abstract We have developed a cleaved amplified polymorphic sequence (CAPS) marker with the aim of identifying the best species among the different wild Lilium species native to Korea for breeding purpose. The CAPS marker is based on single nucleotide polymorphisms (SNPs) in the nucleotide sequences of the internal transcribed spacer genes, which are widely used in the breeding of angiosperms. We cloned and sequenced the ITS regions of nuclear ribosomal genes from 11 Lilium species and deduced the phylogenetic relationships among them. Phylogenetic analysis based on the maximum likelihood method showed that Lilium species formed a monophyletic clade and were divided into two groups that linked to the two sections, Martagon and Sinomartagon, out of seven sections of Lilium. The lengths of the ITS1 and ITS2 regions in the wild native Lilium species were distinct, and nucleotide polymorphisms were distinguished in these regions. A conserved region in the sequence was found from position 330–340 in all the species. We analyzed the SNP sites and found a suitable restriction endonuclease, Eco52I, having six base pair recognition site, to be suitable for the development of the CAPS marker. Among the 11 Lilium species, only the amplicon from the ITS region of L. hansonii contained the restriction site and produced two distinct bands of around 623 and 268 bp upon digestion with Eco52I. The genotyping of the 11 wild Lilium species by the developed marker can be recommended for breeding programs as it provides an indirect selection of plants and can effectively differentiate wild native lilies. The method using CAPS marker is simple, quick, and highly reliable for identifying the best species for efficient breeding of Lilium.

Egg-shell derived carbon dots for base pair selective DNA binding and recognition.

The development of base pair selective fluorescent binding probes and their interaction mode with nucleic acids have created great interest for sensing and biomedical applications. Herein, we have used chicken egg shell membrane (ESM) as a cost effective easily available protein source for the synthesis of highly fluorescent carbon dots. The detailed characterizations have confirmed the in situ formation of heteroatom doped graphitic carbon nanodots (CDs) from ESM. The intrinsic fluorescence property of the material has been utilized for the label free binding of duplex deoxyribonucleic acid (DNA). The interaction of different natural and synthetic DNAs with carbon dots resulted in the enhancement of fluorescence characteristics of the latter. Analysis of the binding data obtained from steady state fluorescence studies revealed a selective and stronger affinity of CDs to the adenine-thymine (AT) base pair rich double stranded DNA (ds DNA) than that of the guanine-cytosine (GC) pair rich ds DNA. Base pair specific binding was further validated from isothermal titration calorimetry (ITC) and melting temperature data. The thermodynamic profile revealed endothermic binding that was driven by the hydrophobic interaction at the nano-bio interfaces. The results reveal the potential of carbon dots as a new and promising fluorescent probe for base pair selective and sequence specific DNA recognition.

Faulty RNA splicing: consequences and therapeutic opportunities in brain and muscle disorders.

Alternative splicing is a powerful mechanism that largely expands the coding potential of eukaryotic genomes. Indeed, its complex and flexible regulation is exploited by cells to adapt to various environmental conditions, through production of protein variants displaying different functions. Such flexibility, however, is accompanied by high risk of errors, and dysregulation of splicing is now recognized as an important factor in human diseases. Notably, the RNA-based nature of splicing, which involves high specificity through base pair recognition, offers a remarkable therapeutic opportunity by allowing design of tools with elevated target selectivity. Herein, we illustrate examples of how defective splicing, obtained by mutations affecting multiple layers of regulation, can result in pathology. In particular, we focus on splicing-related defects occurring in brain and muscle diseases and describe therapeutic approaches currently available for these pathologies.

ITRAQ-MS/MS Proteomic Analysis Reveals Differentially Expressed Proteins During Post-harvest Maturation of the White Button Mushroom Agaricus bisporus

Agaricus bisporus (J. E. Lange) Imbach, well known as the common cultivated mushroom or white button mushroom, is widely cultivated all over the world. We used iTRAQ-MS/MS (i.e., isobaric tags for relative and absolute quantification-coupled two-dimensional liquid chromatography-tandem mass spectrometry) to identify protein expression changes that occur during the post-harvest maturation of Agaricus bisporus fruitbodies at 0, 6, 12, and 48h after harvest. A total of 5878 unique peptides, representing 1063 proteins, were identified. Quantitative data were obtained from 1012 out of the 1063 proteins. A total of 102, 106, and 160 differentially expressed proteins (>twofolds differences) were identified from samples collected at 6, 12, and 48h compared to sample from 0h post harvest, accounting for 10.1, 10.5, and 15.8% of the total proteins identified, respectively. All identified proteins including the differentially expressed proteins among different post-harvest stages were subjected to bioinformatic analysis. Furthermore, seven representative proteins either up or down-regulated at different post-harvest stages were analyzed by real-time PCR. Three out of the seven proteins, the mismatched base pair and cruciform DNA recognition protein, hydrophobin-B and protein transporter, exhibited similar expression patterns as their corresponding proteins. The 260 differentially expressed proteins identified from our study laid a foundation for future studies aiming to understand the post-harvest maturation process of A. bisporus and eventually, might help in the development of breeding program to identify strains with extended shelf life.

Synthesis of Geranyl-2-Thiouridine-Modified RNA.

This unit describes the chemical synthesis of the S-geranyl-2-thiouridine (ges2 U) phosphoramidite and its incorporation into RNA oligonucleotides through solid-phase synthesis. Starting from the 2-thiouracil nucleobase and the protected ribose, the 2-thiouridine is synthesized and the geranyl functionality is introduced into the 2-thio position by using geranyl bromide as the geranylating reagent before the conversion of this modified nucleoside into a phosphoramidite building block. The modified phosphoramidite is used to make the geranyl-RNA oligonucleotides with a solid-phase DNA synthesizer. These RNA strands are then purified by ion-exchange HPLC before further structural and functional studies, such as base pairing and enzyme recognition, can be done. © 2017 by John Wiley & Sons, Inc.

The developments of gene expression regulating PI polyamide by the based on DNA base pair recognition

The developments of gene expression regulating PI polyamide by the based on DNA base pair recognition

Molecular self-assembly using peptide nucleic acids.

Peptide nucleic acids (PNAs) are extensively studied for the control of genetic expression since their design in the 1990s. However, the application of PNAs in nanotechnology is much more recent. PNAs share the specific base-pair recognition characteristic of DNA together with material-like properties of polyamides, both proteins and synthetic polymers, such as Kevlar and Nylon. The first application of PNA was in the form of PNA-amphiphiles, resulting in the formation of either lipid integrated structures, hydrogels or fibrillary assemblies. Heteroduplex DNA-PNA assemblies allow the formation of hybrid structures with higher stability as compared with pure DNA. A systematic screen for minimal PNA building blocks resulted in the identification of guanine-containing di-PNA assemblies and protected guanine-PNA monomer spheres showing unique optical properties. Finally, the co-assembly of PNA with thymine-like three-faced cyanuric acid allowed the assembly of poly-adenine PNA into fibers. In summary, we believe that PNAs represent a new and important family of building blocks which converges the advantages of both DNA- and peptide-nanotechnologies.

A stretched conformation of DNA with a biological role?

We have discovered a well-defined extended conformation of double-stranded DNA, which we call Σ-DNA, using laser-tweezers force-spectroscopy experiments. At a transition force corresponding to free energy change ΔG = 1·57 ± 0·12 kcal (mol base pair)-1 60 or 122 base-pair long synthetic GC-rich sequences, when pulled by the 3'-3' strands, undergo a sharp transition to the 1·52 ± 0·04 times longer Σ-DNA. Intriguingly, the same degree of extension is also found in DNA complexes with recombinase proteins, such as bacterial RecA and eukaryotic Rad51. Despite vital importance to all biological organisms for survival, genome maintenance and evolution, the recombination reaction is not yet understood at atomic level. We here propose that the structural distortion represented by Σ-DNA, which is thus physically inherent to the nucleic acid, is related to how recombination proteins mediate recognition of sequence homology and execute strand exchange. Our hypothesis is that a homogeneously stretched DNA undergoes a 'disproportionation' into an inhomogeneous Σ-form consisting of triplets of locally B-like perpendicularly stacked bases. This structure may ensure improved fidelity of base-pair recognition and promote rejection in case of mismatch during homologous recombination reaction. Because a triplet is the length of a gene codon, we speculate that the structural physics of nucleic acids may have biased the evolution of recombinase proteins to exploit triplet base stacks and also the genetic code.

Aminopyridinyl-Pseudodeoxycytidine Derivatives Selectively Stabilize Antiparallel Triplex DNA with Multiple CG Inversion Sites.

The sequence-specific formation of triplex DNA offers a potential basis for genome-targeting technologies. In an antiparallel triplex DNA, the sequence-specificity is established by the formation of specific base triplets (G-GC, A-AT, and T-AT) between a triplex-forming oligonucleotide (TFO) and a duplex DNA. However, there are no natural nucleosides that can selectively recognize the inverted CG and TA base pairs. Therefore, the recognition of the CG and TA inversion sites to form a stable triplex DNA has been a long-standing goal for the triplex-forming technology. We now describe the design and synthesis of pseudo-deoxycytidine (ΨdC) derivatives for selective recognition of the CG base pair to expand the triplex-forming sequence. The aminopyridine-bearing ΨdC derivatives showed high selectivity and affinity toward the CG base pair in all neighboring base contexts. Remarkably, 3-methyl-2-aminopyridinyl-ΨdC ((Me) AP-ΨdC) formed a stable triplex with the promoter sequence of the hTERT gene containing four CG inversion sites, and effectively inhibited its transcription in human cancer cells. Thus, (Me) AP-ΨdC is expected to serve as a new starting point of triplex-forming oligonucleotides for a wide variety of genome-targeting applications.

Aminopyridinyl–Pseudodeoxycytidine Derivatives Selectively Stabilize Antiparallel Triplex DNA with Multiple CG Inversion Sites

AbstractThe sequence‐specific formation of triplex DNA offers a potential basis for genome‐targeting technologies. In an antiparallel triplex DNA, the sequence‐specificity is established by the formation of specific base triplets (G−GC, A−AT, and T−AT) between a triplex‐forming oligonucleotide (TFO) and a duplex DNA. However, there are no natural nucleosides that can selectively recognize the inverted CG and TA base pairs. Therefore, the recognition of the CG and TA inversion sites to form a stable triplex DNA has been a long‐standing goal for the triplex‐forming technology. We now describe the design and synthesis of pseudo‐deoxycytidine (ΨdC) derivatives for selective recognition of the CG base pair to expand the triplex‐forming sequence. The aminopyridine‐bearing ΨdC derivatives showed high selectivity and affinity toward the CG base pair in all neighboring base contexts. Remarkably, 3‐methyl‐2‐aminopyridinyl−ΨdC (MeAP−ΨdC) formed a stable triplex with the promoter sequence of the hTERT gene containing four CG inversion sites, and effectively inhibited its transcription in human cancer cells. Thus, MeAP−ΨdC is expected to serve as a new starting point of triplex‐forming oligonucleotides for a wide variety of genome‐targeting applications.

Imino proton NMR guides the reprogramming of A•T specific minor groove binders for mixed base pair recognition.

Sequence-specific binding to DNA is crucial for targeting transcription factor-DNA complexes to modulate gene expression. The heterocyclic diamidine, DB2277, specifically recognizes a single G•C base pair in the minor groove of mixed base pair sequences of the type AAAGTTT. NMR spectroscopy reveals the presence of major and minor species of the bound compound. To understand the principles that determine the binding affinity and orientation in mixed sequences of DNA, over thirty DNA hairpin substrates were examined by NMR and thermal melting. The NMR exchange dynamics between major and minor species shows that the exchange is much faster than compound dissociation determined from biosensor–surface plasmon resonance. Extensive modifications of DNA sequences resulted in a unique DNA sequence with binding site AAGATA that binds DB2277 in a single orientation. A molecular docking result agrees with the model representing rapid flipping of DB2277 between major and minor species. Imino spectral analysis of a 15N-labeled central G clearly shows the crucial role of the exocyclic amino group of G in sequence-specific recognition. Our results suggest that this approach can be expanded to additional modules for recognition of more sequence-specific DNA complexes. This approach provides substantial information about the sequence-specific, highly efficient, dynamic nature of minor groove binding agents.

Functional interplay between NTP leaving group and base pair recognition during RNA polymerase II nucleotide incorporation revealed by methylene substitution.

RNA polymerase II (pol II) utilizes a complex interaction network to select and incorporate correct nucleoside triphosphate (NTP) substrates with high efficiency and fidelity. Our previous ‘synthetic nucleic acid substitution’ strategy has been successfully applied in dissecting the function of nucleic acid moieties in pol II transcription. However, how the triphosphate moiety of substrate influences the rate of P-O bond cleavage and formation during nucleotide incorporation is still unclear. Here, by employing β,γ-bridging atom-‘substituted’ NTPs, we elucidate how the methylene substitution in the pyrophosphate leaving group affects cognate and non-cognate nucleotide incorporation. Intriguingly, the effect of the β,γ-methylene substitution on the non-cognate UTP/dT scaffold (∼3-fold decrease in kpol) is significantly different from that of the cognate ATP/dT scaffold (∼130-fold decrease in kpol). Removal of the wobble hydrogen bonds in U:dT recovers a strong response to methylene substitution of UTP. Our kinetic and modeling studies are consistent with a unique altered transition state for bond formation and cleavage for UTP/dT incorporation compared with ATP/dT incorporation. Collectively, our data reveals the functional interplay between NTP triphosphate moiety and base pair hydrogen bonding recognition during nucleotide incorporation.

Virus-like supramolecular assemblies formed by cooperation of base pairing interaction and peptidic association

A peptide nucleic acid (PNA)-peptide conjugated molecule, T′3(AKAE)2, was designed to have both a PNA segment for oligonucleotide binding and an ionic self-complementary peptide sequence for self-association. T′3(AKAE)2 could co-assemble with oligoadenines (d(A) x ) to form virus-like supramolecular structures whose morphology showed dependence on the chain length and rigidity of the d(A) x molecules. Smaller nanospheres with diameters of 13.0±2.0 nm were produced in the case of d(A)6. Wormlike aggregates with lengths of 20–50 nm and diameters of 15.0±2.5 nm were found in the cases of d(A)12, d(A)18, d(A)24 and d(A)30. And larger spherical aggregates with diameters of 18±5 nm came into presence in the cases of d(A)36 and d(A)42. These nanostructures were suggested to be formed under a cooperative effect of base pair recognition and peptidic association. The study provides insights into the programmed assembly of a multi-components system as well as control of the size and shape of the co-assembled structures, which is of great significance in developing gene/drug delivery systems.

Minor-Groove Binding Agents: Rational Design of Carboxamide Bond Isosteres.

Distamycin and netropsin analogues have been designed for targeting specific sequences in DNA. Numerous reviews have been centered on the replacement of N-methylpyrrole with heteroaromatic rings in order to induce better fitting to improve the binding efficiency and the introduction of additional interactions for recognition of GC base pairs at the minor groove of DNA. Most of these designed analogs retained the use of carboxamide-bond for interconnecting the heteroaromatic rings. Computer simulations of netropsin and distamycin has pinpointed the advantages of designing isosteres of the carboxamide-bond for amplifying or attenuating particular interactions to DNA, but have been less studied. The key challenges that must be overcome to realize this goal are the development of feasible synthetic methodologies. This review examined in detail for the first time the electronic, structural, and conformational attributes of the various carboxamide isosteres: (i) neutral isostere-alkenyl and alkyl, (ii) hydrogen donating isostere-urea and carbonylurea, and (iii) hydrogen acceptor isosteres-diazene and diketone. In particular, the ability of these isosteres to participate in non-covalent interactions by tuning the shape and hydrogen bonding to the floor of the minor groove is compared with that of the carboxamide bond. We hope this review will encourage the development of a library of modified isosteres of the carboxamide bond which target DNA with excellent sequence specificity, stronger binding affinity and exhibit improved biological properties. Another goal is to develop synthetic methodolgies for the ready synthesis of poly-isosteric bond used in mimicking of the poly-carboxamide bond for DNA minor groove binding agents, the area in which progress has been slow.

Structural insights into specific crRNA G-rich sequence binding by Meiothermus ruber Cse2.

CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)-mediated defense against invading nucleic acids is a process recently discovered in prokaryotes, which includes recognition and incorporation of invading genetic elements, transcription and processing of CRISPR-RNA (crRNA) and targeting the invaders through base pair recognition. In the type I-E CRISPR-Cas system, Cse2 is proposed to provide a platform to facilitate the targeting of the invading dsDNA by crRNA. Here we report the crystal structure of Meiothermus ruber Cse2 at 2.8Å. M. ruber Cse2 adopts an α-helical bundle scaffold, harbors a positive surface for nucleic acid binding and a conserved dimer interface with strikingly low buried surface area. M. ruber Cse2 selectively binds to G-rich crRNA sequence, which is stripped off from the Cse2-crRNA and Cascade-crRNA complexes by ssDNA or dsDNA with complementary sequence. Stable M. ruber Cascade is readily formed by co-expression of M. ruber Cascade proteins together with G-rich crRNA in vitro. Docking of M. ruber Cse2 structures into the Escherichia coli Cascade Cryo-EM envelope reveals a curved elongated shallow groove for ssRNA binding, which adopts a similar dimer interface discovered by high-resolution crystal structure of Cse2 within E. Coli Cascade. Taken together, our data provides the structural insights into crRNA G-rich sequence recognition by M. ruber Cse2 and reveals the potential structural mechanism for M. ruber Cascade assembly and function.

Light-emitting self-assembled peptide nucleic acids exhibit both stacking interactions and Watson-Crick base pairing.

The two main branches of bionanotechnology involve the self-assembly of either peptides or DNA. Peptide scaffolds offer chemical versatility, architectural flexibility and structural complexity, but they lack the precise base pairing and molecular recognition available with nucleic acid assemblies. Here, inspired by the ability of aromatic dipeptides to form ordered nanostructures with unique physical properties, we explore the assembly of peptide nucleic acids (PNAs), which are short DNA mimics that have an amide backbone. All 16 combinations of the very short di-PNA building blocks were synthesized and assayed for their ability to self-associate. Only three guanine-containing di-PNAs-CG, GC and GG-could form ordered assemblies, as observed by electron microscopy, and these di-PNAs efficiently assembled into discrete architectures within a few minutes. The X-ray crystal structure of the GC di-PNA showed the occurrence of both stacking interactions and Watson-Crick base pairing. The assemblies were also found to exhibit optical properties including voltage-dependent electroluminescence and wide-range excitation-dependent fluorescence in the visible region.

Base Pair Recognition Ability of 2-(Methylamino)pyrimidin-4-yl Nucleobase in Parallel Triplex DNA

Base Pair Recognition Ability of 2-(Methylamino)pyrimidin-4-yl Nucleobase in Parallel Triplex DNA

Protein-DNA Binding in the Absence of Consensus Binding Motif

Until now, it has been reasonably assumed that specific base-pair recognition is the only mechanism controlling the specificity of transcription factor (TF)-DNA binding. In our study we show that nonspecific DNA sequences possessing certain repeat symmetries, when present outside of specific TF binding sites (TFBSs), statistically control TF-DNA binding preferences. We used high-throughput protein-DNA binding assays to measure the binding levels for several human TFs to tens of thousands of short DNA sequences with varying repeat symmetries. Based on statistical mechanics modeling, we identify a new protein-DNA binding mechanism induced by DNA sequence symmetry in the absence of consensus binding motif, and experimentally demonstrate that this mechanism indeed highly affects protein-DNA binding preferences.