N-terminal Cationic Modification of Linear Pyrrole-Imidazole Polyamide Improves Its Binding to DNA

Pyrrole-imidazole polyamides (PIPs) bind to double-stranded DNA (dsDNA) with varied sequence selectivity. We synthesized linear PIPs that can bind to narrow minor grooves of polypurine/polypyrimidine sequences and target long recognition sequences but have lower molecular weights than commonly used hairpin PIPs. We modified the N-terminus of linear PIPs using several groups, including β-alanine extension and acetyl capping. Melting curve analysis of dsDNA demonstrated that cationic modifications improved the binding affinity of the PIPs to the targeted dsDNA. In addition, circular dichroism assays revealed the characteristic spectra depending on the binding stoichiometry of the N-cationic linear PIP and dsDNA (1 : 1, monomeric; 2 : 1, dimeric). Surface plasmon resonance assays confirmed the high binding affinities of linear PIPs. These findings may aid in the design of effective linear PIPs.     

吡咯-咪唑聚酰胺固相合成载体应用研究进展

吡咯-咪唑聚酰胺是一类人工合成的能够在B-DNA小沟特异性识别碱基序列的有机小分子;并且能通过细胞膜进入细胞,调控基因的表达。固相合成吡咯-咪唑聚酰胺是一种快速而有效的方法,且对较难合成的聚酰胺比较适用。本文主要阐述树脂在聚酰胺固相合成中的应用。     

Label-Free in Situ Monitoring of the DNA Hybridization Chain Reaction by Using Sequence-Selective Minor-Groove-Binding Fluorophores

A new label-free in situ monitoring system for the hybridization chain reaction (HCR) based on DNA minor-groove-binding fluorophores [Hoechst 33258 (Hoe) or quinone cyanine-dithiazole (QCy-DT)] has been developed. Use of two unmodified hairpin oligodeoxyribonucleotides containing incomplete double-stranded AATT sequences enabled target-dependent formation of probe binding sites—that is, AATT double strand—in the HCR product, together with fluorescence enhancement of minor-groove-binding fluorophores in situ. This system allows target DNA to be detected through the fluorescence enhancement of Hoe and QCy-DT in real time and in situ. Further development of a label-free, isothermal detection system might provide a cost-effective and user-friendly method for nucleic acid detection.     

Precisely Programmed and Robust 2D Streptavidin Nanoarrays by Using Periodical Nanometer‐Scale Wells Embedded in DNA Origami Assembly

A new punched DNA origami assembly with periodic nanometer‐scale wells has been successfully designed and constructed. Through the attachment of two biotins at the two edges of each well, just one streptavidin (SA) tetramer (d=5 nm) was size‐selectively captured in each 6.8×12×2.0 nm well; this allowed formation of a 28 nm‐period SA nanoarray of individual molecules. The position of SA capture can be fully controlled by placement of biotins in the nanoarray well. Moreover, construction of a 2D nanoarray of individual SA tetramers through selective positioning of SA tetramers in any desired wells in a complex of such punched origami motifs is also possible. The stability of the SA captured by this fixation strategy (DNA wells and two biotin linkers) was directly compared on the same molecule with the stability of SA captured with other possible strategies that do not employ wells or two linkers. In this way, the robustness of this means of fixation was clearly established.     

Sequence-Independent Logical Regulation of the Assembly and Disassembly of DNA Polyhedra

Controlling the self-assembly of DNA nanostructures using rationally designed logic gates is a major goal of dynamic DNA nanotechnology, which could facilitate the development of biomedicine, molecular computation, et al. In previous works, the regulations mostly relied on either toehold-mediated strand displacement or stimuli-driven conformational switch, requiring elaborately-designed or specific DNA sequences. Herein, we reported a facile, base-sequence-independent strategy for logically controlling DNA self-assembly through external molecules. The INHIBIT and XOR logic controls over the assembly/disassembly of DNA polyhedra were realized through cystamine ( Cyst ) and ethylenediamine ( EN ) respectively, which were further integrated into a half subtractor circuit thanks to the sharing of the same inputs. Our work provides a sequence-independent strategy in logically controlling DNA self-assembly, which may open up new possibilities for dynamic DNA nanotechnology.     

Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation

The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py–Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py–Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py–Im polyamides. The effect of Py–Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py–Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py–Im-polyamide conjugates. The practical use of protein-Py–Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established.     

Study on the Thermal Stabilization Effect of Polyamide on Polyoxymethylene

The thermal stabilization effect of polyamide (PA) on polyoxymethylene (POM) was studied by the isothermal weight loss analysis and thermogravimetric analysis (TGA), which shows that with the increase of PA content, the thermal weight loss and degradation rate decrease, the characteristic thermal degradation temperatures increase, and the thermal stability of POM can be improved remarkably. The nonisothermal degradation kinetics evaluated from TGA by the Coats-Redfern method shows that the PA-modified POM has higher activation energy than the virgin POM. The yellow index (YI), the melt index (MI), and the mechanical property measurements for both the virgin and the modified POM by multiple-processing further demonstrate that the PA applied in this study shows remarkable thermal stabilization effect on POM.     

Structure and Dynamic Mechanical Properties of Melt Intercalated Polyamide 6—Montmorillonite Nanocomposites

Summary: Polymer‐layered silicate nanocomposites (PLSN), based on polyamide 6 (PA6) and montmorillonite (MMT) modified with an octadecylammonium salt, were produced via melt compounding in a co‐rotating twin‐screw extruder. Wide angle X‐ray diffraction (WAXD) and TEM revealed a PLSN containing 3.3% by weight (wt.‐%) of MMT to exhibit a mixed exfoliated/intercalated morphology, consisting mainly of individual silicate lamellae together with some intercalated stacks, resulting in a mean value of 1.8 lamellae per particle. In contrast, a PLSN containing a higher level of 7.2 wt.‐% MMT exhibited a more ordered intercalated structure, consisting mainly of a distribution of lamellae stacks with a mean value of 3.8 lamellae per particle. The dispersion of MMT in the PLSN generated very large polymer–filler interfacial areas, resulting in significant increase in the volume of constrained PA6 chain segments. Consequently, significant changes in the ratio of α/γ crystallites and in the thermal behaviour of the matrix PA6 were observed during WAXD, DSC and dynamic‐mechanical thermal analysis (DMTA) studies of the PLSN. In particular, damping data from DMTA showed relaxations between T_g and T_m resulting from amorphous polymer chain segments constrained at the polymer–filler interface, indicating the formation of a continuous phase of constrained polymer. In contrast, a PA6 microcomposite formed using unmodified MMT generated much lower polymer–filler interfacial area, with most of the MMT residing within large, poorly wetted aggregates. Consequently, changes to the thermal behaviour of the matrix PA6 were much less significant than those induced in the PLSN.

Shear storage modulus (G′) versus temperature data for the matrix PA6, the 5T and 10T PLSN and the 5P microcomposite.     

Ultraviolet Oxidative Stabilization Effect of the Rare Earth Compound on Polyamide

Rare earth compounds, including rare earth stearate (RESt) and rare earth epoxydised fatty acid (REEFA), were applied for the purpose of further improving the ultraviolet-oxidative stability of polyamide (PA6). The results showed that the rare earth compounds can inhibit the increase of the gel concentration and the reduced viscosity, the formation and the increase of the carboxylic group concentration, and the decrease of the terminal amine group concentration on the molecular chain of PA6, and delay the decrease of the mechanical properties of PA6 during the UV aging. The sample of PA6 with the rare earth compounds had higher absorbance and lower transmittance of ultraviolet band compared with pure PA6, indicating that the rare earth compounds can protect PA6 from ultraviolet degradation.     

Detection of HER2+ Breast Cancer Cells using Bioinspired DNA-Based Signal Amplification

Circulating tumor cells (CTC) are promising biomarkers for metastatic cancer detection and monitoring progression. However, detection of CTCs remains challenging due to their low frequency and heterogeneity. Herein, we report a bioinspired approach to detect individual cancer cells, based on a signal amplification cascade using a programmable DNA hybridization chain reaction (HCR) circuit. We applied this approach to detect HER2⁺ cancer cells using the anti-HER2 antibody (trastuzumab) coupled to initiator DNA eliciting a HCR cascade that leads to a fluorescent signal at the cell surface. At 4 °C, this HCR detection scheme resulted in highly efficient, specific and sensitive signal amplification of the DNA hairpins specifically on the membrane of the HER2⁺ cells in a background of HER2⁻ cells and peripheral blood leukocytes, which remained almost non-fluorescent. The results indicate that this system offers a new strategy that may be further developed toward an in vitro diagnostic platform for the sensitive and efficient detection of CTC.     

Modified DNA bearing 5(methoxycarbonylmethyl)-2'-deoxyuridine: preparation by PCR with thermophilic DNA polymerase and postsynthetic derivatization

A thymidine analogue bearing a methyl ester at the C5 position was accepted as a substrate by the thermophilic family B DNA polymerases, KOD Dash, Pwo, and Vent(exo-), to form the corresponding PCR product, but not by the thermophilic family A DNA polymerases, Taq, Tth, and T7 thermosequenase. Modified DNA containing this analogue was prepared by PCR on a large scale with KOD Dash DNA polymerase and 5(methoxycarbonylmethyl)-2'-deoxyuridine 5'-triphosphate as a substrate. The methyl ester of the modified DNA was further allowed to react with tris(2-aminoethyl)amine or histamine by an ester-amide exchange reaction to form the corresponding derivatized DNA bearing a tris(2-aminoethyl)amine or histamine moiety. Hydrolysis of the methyl ester of the modified DNA gave a functionalized DNA bearing an anionic carboxyl group. The derivatized DNA could act as a template for the PCR with KOD Dash DNA polymerase and the natural 2'-deoxythymidine 5'-triphosphate or the modified thymidine analogue as a substrate. The postsynthetic derivatization of the modified DNA may expand the variety of structurally modified DNA produced by PCR.     

Salting-Out of DNA Origami Nanostructures by Ammonium Sulfate

DNA origami technology enables the folding of DNA strands into complex nanoscale shapes whose properties and interactions with molecular species often deviate significantly from that of genomic DNA. Here, we investigate the salting-out of different DNA origami shapes by the kosmotropic salt ammonium sulfate that is routinely employed in protein precipitation. We find that centrifugation in the presence of 3 M ammonium sulfate results in notable precipitation of DNA origami nanostructures but not of double-stranded genomic DNA. The precipitated DNA origami nanostructures can be resuspended in ammonium sulfate-free buffer without apparent formation of aggregates or loss of structural integrity. Even though quasi-1D six-helix bundle DNA origami are slightly less susceptible toward salting-out than more compact DNA origami triangles and 24-helix bundles, precipitation and recovery yields appear to be mostly independent of DNA origami shape and superstructure. Exploiting the specificity of ammonium sulfate salting-out for DNA origami nanostructures, we further apply this method to separate DNA origami triangles from genomic DNA fragments in a complex mixture. Our results thus demonstrate the possibility of concentrating and purifying DNA origami nanostructures by ammonium sulfate-induced salting-out.     

Using a PCR-Based Method To Analyze and Model Large,Heterogeneous Populations of DNA

The study of populations of large size and high diversity is limited by the capability of collecting data. Moreover, for a pool of individuals, each associated with a unique characteristic feature, as the pool size grows, the possible interactions increase exponentially and quickly go beyond the limit of computation and experimental studies. Herein, the design of DNA libraries with various diversity is reported. By using a facile analytical method based on real-time PCR, the diversity of a pool of DNA can be evaluated to allow extraordinarily high heterogenicity (e.g., >1 trillion). It is demonstrated that these DNA libraries can be used to model heterogeneous populations; these libraries exhibit functions such as self-protection, suitability for biased expansion, and the possibility to evolve into amorphous structures. The method has shown the remarkable power of parallel computing with DNA, since it can resemble an analogue computer and be applied in selection-based biotechnology methods, such as DNA-encoded chemical libraries. As a chemical approach to solve problems traditionally for genetic and statistical analysis, the method provides a quick and cost-efficient evaluation of library diversity for intermediate steps through a selection process.     

Molecular recognition of T:G mismatched base pairs in DNA as studied by electrospray ionization mass spectrometry

Postreplicative mismatch repair (MMR) is a cellular system involved in the recognition and correction of DNA polymerase errors that escape detection in proofreading. Of the various mismatched bases, T:G pairing in DNA is one of the more common mutations leading to the formation of tumors in humans. In addition, the absence of the MMR system can generate resistance to several chemotherapeutic agents, particularly DNA-damaging substances. The main purpose of this study was the setup and validation of an electrospray ionization (ESI) mass spectrometry method for the identification of small molecules that are able to recognize T:G mismatches in DNA targets. These findings could be useful for the discovery of new antitumor drugs. The analytical method is based on the ability of electrospray to preserve the noncovalent adducts present in solution and transfer them to the gas phase. Lexitropsin derivatives (polyimidazole compounds) have been previously described as selective for T:G mismatch binding by NMR and ITC studies. We synthesized and tested various polyimidazole derivatives, one of which in particular (NMS-057) showed a higher affinity for an oligonucleotide DNA sequence containing a T:G mismatched base pair. To rationalize these findings, molecular docking studies were performed using available NMR structures. Moreover, ESI-MS experiments, performed on an orbitrap mass spectrometer, highlighted the formation of heterodimeric complexes between DNA sequences, distamycin A, and polyimidazole compounds. Our results confirm that this ESI method could be a valuable tool for the identification of new molecules able to specifically recognize T:G mismatched base pairs.     

DNA Three‐Way Junctions Stabilized by Hydrophobic Interactions for Creation of Functional Nanostructures

The construction of nanomaterials from oligonucleotides by modular assembly invariably requires the use of branched nucleic acid architectures such as three‐ and four‐way junctions (3WJ and 4WJ). We describe the stabilization of DNA 3WJ by using non‐nucleotide lipophilic spacers to create a hydrophobic pocket within the junction space. Stabilization of nucleic acid junctions is of particular importance when constructing nanostructures in the “ultra‐nano” size range (<20 nm) with shorter double‐stranded regions. UV thermal melting studies show that lipophilic spacers strategically placed within the junction space significantly increased thermal stability. For a 3WJ with eight base pair arms, thermal stability was increased from 30.5 °C for the unmodified junction to a maximum stability of 55.0 °C. The stability of the junction can be modulated within this temperature range by using the appropriate combinations of spacers.     

Chemical restriction: strand cleavage by ammonia treatment at 8-oxoguanine yields biologically active DNA

Cleavage of DNA single and double strands at an 8-oxoguanine-containing nucleotide occurs in 90 % yield if the modified oligonucleotide is treated with NH(3) and O(2) at 60 degrees C. The mechanism of this oxidative cleavage reaction was studied, and the reaction was applied to the generation of single-stranded overhangs on PCR-amplified DNA that can be ligated. As an example, the lac Z' gene was amplified by PCR with 8-oxoguanine modified primers, restricted by ammonia treatment, ligated into a plasmid vector, transformed in Escherischia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure with restriction enzymes, and it allows the design of any 3'-overhang independent of the sequence of the cloned DNA.     

Analysis of Modified Nucleotide Aptamer Library Generated by Thermophilic DNA Polymerases

One of the pivotal steps in aptamer selection is the amplification of target-specific oligonucleotides by thermophilic DNA polymerases; it can be a challenging task if nucleic acids possessing modified nucleotides are to be amplified. Hence, the identification of compatible DNA polymerase and modified nucleotide pairs is necessary for effective selection of aptamers with unnatural nucleotides. We present an in-depth study of using 5-indolyl-AA-dUTP (TAdUTP) to generate oligonucleotide libraries for aptamer selection. We found that, among the eight studied DNA polymerases, only Vent(exo-) and KOD XL are capable of adapting TAdUTP, and that replacing dTTP did not have a significant effect on the productivity of KOD XL. We demonstrated that water-in-oil emulsion PCR is suitable for the generation of aptamer libraries of modified nucleotides. Finally, high-throughput sequence analysis showed that neither the error rate nor the PCR bias was significantly affected by using TAdUTP. In summary, we propose that KOD XL and TAdUTP could be effectively used for aptamer selection without distorting the sequence space of random oligonucleotide libraries.