Fluorescent 2‐Aminopyridine Nucleobases for Triplex‐Forming Peptide Nucleic Acids

Development of new fluorescent peptide nucleic acids (PNAs) is important for fundamental research and practical applications. The goal of this study was the design of fluorogenic nucleobases for incorporation in triplex‐forming PNAs. The underlying design principle was the use of a protonation event that accompanied binding of a 2‐aminopyridine (M) nucleobase to a G‐C base pair as an on switch for a fluorescence signal. Two fluorogenic nucleobases, 3‐(1‐phenylethynyl)‐M and phenylpyrrolo‐M, were designed, synthesized and studied. The new M derivatives provided modest enhancement of fluorescence upon protonation but showed reduced RNA binding affinity and quenching of fluorescence signal upon triple‐helix formation with cognate double‐stranded RNA. Our study illustrates the principal challenges of design and provides guidelines for future improvement of fluorogenic PNA nucleobases. The 3‐(1‐phenylethynyl)‐M may be used as a fluorescent nucleobase to study PNA–RNA triple‐helix formation.     

Bivalent Display of Dicysteine on Peptide Nucleic Acids for Homogenous DNA/RNA Detection through in Situ Fluorescence Labelling

Fluorogenic probes that signal the presence of specific DNA or RNA sequences are key enabling tools for molecular disease diagnosis and imaging studies. Usually, at least one fluorophore is attached through covalent bonding to an oligonucleotide probe. However, the additional conjugation step increases costs. Here we introduce a method that avoids the requirement for the preparation of fluorescence‐labelled oligonucleotides and provides the opportunity to alter the fluorogenic reporter dye without resynthesis. The method is based on adjacent hybridization of two dicysteine‐containing peptide nucleic acid (PNA) probes to form a bipartite tetracysteine motif that binds profluorescent bisarsenical dyes such as FIAsH, ReAsH or CrAsH. Binding is accompanied by strong increases in fluorescence emission (with response factors of up to 80‐fold and high brightness up to 50 mL mol^?1 cm^?1). The detection system provides sub‐nanomolar limits of detection and allows discrimination of single nucleotide variations through more than 20‐fold changes in fluorescence intensity. To demonstrate its usefulness, the FIAsH‐based readout of the bivalent CysCys‐PNA display was interfaced with a rolling‐circle amplification (RCA) assay used to detect disease‐associated microRNA let‐7a.     

Cationic DOPC–Detergent Conjugates for Safe and Efficient in Vitro and in Vivo Nucleic Acid Delivery

The ability of a nonviral nucleic acid carrier to deliver its cargo to cells with low associated toxicity is a critical issue for clinical applications of gene therapy. We describe biodegradable cationic DOPC–C₁₂E₄ conjugates in which transfection efficiency is based on a Trojan horse strategy. In situ production of the detergent compound C₁₂E₄ through conjugate hydrolysis within the acidic endosome compartment was expected to promote endosome membrane destabilization and subsequent release of the lipoplexes into cytosol. The transfection efficiency of the conjugates has been assessed in vitro, and associated cytotoxicity was determined. Cellular uptake and intracellular distribution of the lipoplexes have been investigated. The results show that direct conjugation of DOPC with C₁₂E₄ produces a versatile carrier that can deliver both DNA and siRNA to cells in vitro with high efficiency and low cytotoxicity. SAR studies suggest that this compound might represent a reasonable compromise between the membrane activity of the released detergent and susceptibility of the conjugate to degradation enzymes in vitro. Although biodegradability of the conjugates had low impact on carrier efficiency in vitro, it proved critical in vivo. Significant improvement of transgene expression was obtained in the mouse lung tuning biodegradability of the carrier. Importantly, this also allowed reduction of the inflammatory response that invariably characterizes cationic‐lipid‐mediated gene transfer in animals.     

Peptide Nucleic Acid Conjugated with Ruthenium‐Complex Stabilizing Double‐Duplex Invasion Complex Even under Physiological Conditions

Peptide nucleic acid (PNA) can form a stable duplex with DNA, and, accordingly, directly recognize double‐stranded DNA through the formation of a double‐duplex invasion complex, wherein a pair of complementary PNA strands form two PNA/DNA duplexes. Because invasion does not require prior denaturation of DNA, PNA holds great potential for in cellulo or in vivo applications. To broaden the applicability of PNA invasion, we developed a new conjugate of PNA with a ruthenium complex. This Ru–PNA conjugate exhibits higher DNA‐binding affinity, which results in enhanced invasion efficiency, even under physiological conditions.     

Gene Delivery by Liposomes

In gene therapy, genetic materials, such as plasmid DNA, antisense oligonucleotides (asODNs), and small interfering RNA (siRNA), can be used to treat or prevent disease. This includes replacing a mutated gene, inactivating a mutated gene, or introducing a new gene. Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections), successful gene therapy is hampered by the lack of effective delivery systems (viral and nonviral). There are several nonviral gene carriers, such as lipids, polymers, and peptides, that can be used for this purpose. Liposomal delivery of nucleic acids, such as plasmid DNA, asODNs, and siRNAs, represents a very promising nanocarrier system that is relatively safe and effective in delivering genes to targeted locations in the body. Lipoid-based delivery systems have also been shown to be stable in serum and plasma, have improved biodistribution, prolonged circulation half-life, and enhanced target tissue selectivity. The most common lipids used in liposomes are cationic lipids, which facilitate binding between their positively charged head group(s) to negatively charged nucleic acids. This review is focused on the most common methods of lipoid-based nanocarriers of nucleic acids for gene therapy in vivo.     

混合表面活性剂介质中微量核酸的光谱测定

健那绿（Janus Green B,JGB）与核酸形成离子性缔合物增溶于混合非离子性表面活性剂中,导致吸光度降低,降低程度与加入的核酸浓度成线性关系,在最佳反应条件下,小牛胸腺DNA（ctDNA）、鱼精子DNA（fsDNA）及酵母RNA（yRNA）的线性范围分别为0．0～4．2μg／mL,0．0～2．4μg／mL及0．0～1．9μg／mL,检测限分别为ctDNA：57ng／mL,fsDNA：96ng／mL及yRNA：42ng／mL,用双倒数直线方程求出各种核酸与JGB的结合常数,均为10^4数量级,本法已用于实际样品分析。     

Design and Application of Antimicrobial Peptide Conjugates

Antimicrobial peptides (AMPs) are an interesting class of antibiotics characterized by their unique antibiotic activity and lower propensity for developing resistance compared to common antibiotics. They belong to the class of membrane-active peptides and usually act selectively against bacteria, fungi and protozoans. AMPs, but also peptide conjugates containing AMPs, have come more and more into the focus of research during the last few years. Within this article, recent work on AMP conjugates is reviewed. Different aspects will be highlighted as a combination of AMPs with antibiotics or organometallic compounds aiming to increase antibacterial activity or target selectivity, conjugation with photosensitizers for improving photodynamic therapy (PDT) or the attachment to particles, to name only a few. Owing to the enormous resonance of antimicrobial conjugates in the literature so far, this research topic seems to be very attractive to different scientific fields, like medicine, biology, biochemistry or chemistry.     

On the in vitro and in vivo properties of four locked nucleic acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide

Locked nucleic acid (beta-D-LNA) monomers are conformationally restricted nucleotides bearing a methylene 2'-O, 4'-C linkage that have an unprecedented high affinity for matching DNA or RNA. In this study, we compared the in vitro and in vivo properties of four different LNAs, beta-D-amino LNA (amino-LNA), beta-D-thio LNA (thio-LNA), beta-D-LNA (LNA), and its stereoisomer alpha-L-LNA in an antisense oligonucleotide (ODN). A well-known antisense ODN design against H-Ras was modified at the 5'- and 3'-ends with the different LNA analogues (LNA-DNA-LNA gapmer design). The resulting gapmers were tested in cancer-cell cultures and in a nude-mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H-Ras ODNs were very efficient in H-Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti-H-Ras ODN containing alpha-L-LNA had clearly the highest efficacy in H-Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino-LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both alpha-L-LNA and LNA gapmer ODNs had a high efficacy of tumor-growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor-growth inhibition could be observed at dosages as low as 0.5 mg kg(-1) per day. These results indicate that alpha-L-LNA is a very promising member of the family of LNA analogues in antisense applications.     

Native Chemical Ligation Directed by Photocleavable Peptide Nucleic Acid (PNA) Templates

A novel peptide–peptide ligation strategy is introduced that has the potential to provide peptide libraries of linearly or branched coupled fragments and will be suited to introduce simultaneous protein modifications at different ligation sites. Ligation is assisted by templating peptide nucleic acid (PNA) strands, and therefore, ligation specificity is solely encoded by the PNA sequence. PNA templating, in general, allows for various kinds of covalent ligation reactions. As a proof of principle, a native chemical ligation strategy was elaborated. This PNA‐templated ligation includes easy on‐resin procedures to couple linkers and PNA to the respective peptides, and a traceless photocleavage of the linker/PNA oligomer after the ligation step. A 4,5‐dimethoxy‐2‐nitrobenzaldehyde‐based linker that allowed the photocleavable linkage of two bio‐oligomers was developed.     

Antibody–Bactericidal Macrocyclic Peptide Conjugates To Target Gram‐Negative Bacteria

To combat antimicrobial infections, new active molecules are needed. Antimicrobial peptides, ever abundant in nature, are a fertile starting point to develop new antimicrobial agents but suffer from low stability, low specificity, and off‐target toxicity. These drawbacks have limited their development. To overcome some of these limitations, we developed antibody–bactericidal macrocyclic peptide conjugates (ABCs), in which the antibody directs the bioactive macrocyclic peptide to the targeted Gram‐negative bacteria. We used cysteine S_NAr chemistry to synthesize and systematically study a library of large (>30‐mer) macrocyclic antimicrobial peptides (mAMPs) to discover variants with extended proteolytic stability in human serum and low hemolytic activity while maintaining bioactivity. We then conjugated, by using sortase A, these bioactive variants onto an Escherichia coli targeted monoclonal antibody. We found that these ABCs had minimized hemolytic activity and were able to kill E. coli at nanomolar concentrations. Our findings suggest macrocyclic peptides if fused to antibodies may facilitate the discovery of new agents to treat bacterial infections.     

New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods

For decades the detection of nucleic acids and their interactions at low abundances has been a challenging task that has thus far been solved by enzymatic target amplification. In this work we aimed at developing efficient tools for amplification‐free nucleic acid detection, which resulted in the synthesis of new fluorescent nanoparticles. Here, the fluorescent nanoparticles were made by simple and inexpensive radical emulsion polymerization of butyl acrylate in the presence of fluorescent dyes and additional functionalization reagents. This provided ultra‐bright macrofluorophores of 9–84 nm mean diameter, modified with additional alkyne and amino groups for bioconjugation. By using click and NHS chemistries, the new nanoparticles were attached to target‐specific DNA probes that were used in fluorimetry and fluorescence microscopy. Overall, these fluorescent nanoparticles and their oligonucleotide derivatives have higher photostability, brighter fluorescence and hence dramatically lower limits of target detection than the individual organic dyes. These properties make them useful in approaches directed towards ultrasensitive detection of nucleic acids, in particular for imaging and in vitro diagnostics of DNA.     

Effects of Thymoquinone–Fatty Acid Conjugates on Cancer Cells

More than the sum of its parts : The seeds of Nigella sativa have been a traditional herbal remedy for ailments such as asthma, diarrhea, and cancer. Their essential oil is rich in polyunsaturated fatty acids and thymoquinone. By linking them covalently, a tremendous boost in anticancer activity can be achieved in resistant tumor cells.

     

Advances in Quantitative FRET‐Based Methods for Studying Nucleic Acids

Förster resonance energy transfer (FRET) is a powerful tool for monitoring molecular distances and interactions at the nanoscale level. The strong dependence of transfer efficiency on probe separation makes FRET perfectly suited for “on/off” experiments. To use FRET to obtain quantitative distances and three‐dimensional structures, however, is more challenging. This review summarises recent studies and technological advances that have improved FRET as a quantitative molecular ruler in nucleic acid systems, both at the ensemble and at the single‐molecule levels.