Lipid domain specific recruitment of lipophilic nucleic acids: a key for switchable functionalization of membranes

Lipid domains in mammalian plasma membranes serve as platforms for specific recruitment or separation of proteins involved in various functions. Here, we have applied this natural strategy of lateral separation to functionalize lipid membranes at micrometer scale in a switchable and reversible manner. Membrane-anchored peptide nucleic acid and DNA, differing in their lipophilic moieties, partition into different lipid domains in model and biological membranes. Separation was visualized by hybridization with the respective complementary fluorescently labeled DNA strands. Upon heating, domains vanished, and both lipophilic nucleic acid structures intermixed with each other. Reformation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. By linking appropriate structures/functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces.     

Interaction of nucleic acids with the glycocalyx

Mammalian cells resist the uptake of nucleic acids. The lipid bilayer of the plasma membrane presents one barrier. Here, we report on a second physicochemical barrier for uptake. To create a sensitive probe for nucleic acid-cell interactions, we synthesized fluorescent conjugates in which lipids are linked to DNA oligonucleotides. We found that these conjugates incorporate readily into the plasma membrane but are not retained there. Expulsion of lipid-oligonucleotide conjugates from the plasma membrane increases with oligonucleotide length. Conversely, the incorporation of conjugates increases markedly in cells that lack the major anionic components of the glycocalyx, sialic acid and glycosaminoglycans, and in cells that had incorporated highly cationic lipids into their plasma membrane. We conclude that anionic oligosaccharides provide a formidable barrier to the uptake of nucleic acids by mammalian cells. This conclusion has implications for genomic stability, as well as the delivery of genes and siRNAs into mammalian cells.     

Fluorescent Probes Based on Charge and Proton Transfer for Probing Biomolecular Environment

Fluorescent probes for sensing fundamental properties of biomolecular environment, such as polarity and hydration, help to study assembly of lipids into biomembranes, sensing interactions of biomolecules and imaging physiological state of the cells. Here, we summarize major efforts in the development of probes based on two photophysical mechanisms: (i) an excited-state intramolecular charge transfer (ICT), which is represented by fluorescent solvatochromic dyes that shift their emission band maximum as a function of environment polarity and hydration; (ii) excited-state intramolecular proton transfer (ESIPT), with particular focus on 5-membered cyclic systems, represented by 3-hydroxyflavones, because they exhibit dual emission sensitive to the environment. For both ICT and ESIPT dyes, the design of the probes and their biological applications are summarized. Thus, dyes bearing amphiphilic anchors target lipid membranes and report their lipid organization, while targeting ligands direct them to specific organelles for sensing their local environment. The labels, amino acid and nucleic acid analogues inserted into biomolecules enable monitoring their interactions with membranes, proteins and nucleic acids. While ICT probes are relatively simple and robust environment-sensitive probes, ESIPT probes feature high information content due their dual emission. They constitute a powerful toolbox for addressing multitude of biological questions.     

Structural effects of lipophilic methotrexate conjugates on model phospholipid biomembranes

Lipophilic conjugates of the antitumor drug methotrexate (MTX) with lipoamino acids (LAAs) have been previously described as a tool to enhance MTX passive entrance into cells, overcoming a form of transport resistance which makes tumour cells insensitive to the antimetabolite. A knowledge of the mechanisms of interaction of such lipophilic derivatives with cell membranes could be useful for planning further lipophilic MTX derivatives with an optimal antitumour activity. To this aim, a calorimetric study was undertaken using a biomembrane model made from synthetic 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) multilamellar liposomes. The effects of MTX and conjugates on the phase transition of liposomes were investigated using differential scanning calorimetry.

The interaction of pure MTX with the liposomes was limited to the outer part of the phospholipid bilayers, due to the polar nature of the drug. Conversely, its lipophilic conjugates showed a hydrophobic kind of interaction, perturbing the packing order of DPPC bilayers. In particular, a reduction of the enthalpy of transition from the gel to the liquid crystal phase of DPPC membranes was observed. Such an effect was related to the structure and mole fraction of the conjugates in the liposomes.

The antitumour activity of MTX conjugates was evaluated against cultures of a CCRF–CEM human leukemic T-cell line and a related MTX resistant sub-line. The in vitro cell growth inhibitory activity was higher for bis(tetradecyl) conjugates than for both the other shorter- and longer-chain derivatives. The biological effectiveness of the various MTX derivatives correlated very well with the thermotropic effects observed on the phase transition of DPPC biomembranes.     

Lipophilic oligonucleotides spontaneously insert into lipid membranes, bind complementary DNA strands, and sequester into lipid-disordered domains

For the development of surface functionalized bilayers, we have synthesized lipophilic oligonucleotides to combine the molecular recognition mechanism of nucleic acids and the self-assembly characteristics of lipids in planar membranes. A lipophilic oligonucleotide consisting of 21 thymidine units and two lipophilic nucleotides with an alpha-tocopherol moiety as a lipophilic anchor was synthesized using solid-phase methods with a phosphoramadite strategy. The interaction of the water soluble lipophilic oligonucleotide with vesicular lipid membranes and its capability to bind complementary DNA strands was studied using complementary methods such as NMR, EPR, DSC, fluorescence spectroscopy, and fluorescence microscopy. This oligonucleotide inserted stably into preformed membranes from the aqueous phase. Thereby, no significant perturbation of the lipid bilayer and its stability was observed. However, the non-lipidated end of the oligonucleotide is exposed to the aqueous environment, is relatively mobile, and is free to interact with complementary DNA strands. Binding of the complementary single-stranded DNA molecules is fast and accomplished by the formation of Watson-Crick base pairs, which was confirmed by 1H NMR chemical shift analysis and fluorescence resonance energy transfer. The molecular structure of the membrane bound DNA double helix is very similar to the free double-stranded DNA. Further, the membrane bound DNA double strands also undergo regular melting. Finally, in raft-like membrane mixtures, the lipophilic oligonucleotide was shown to preferentially sequester into liquid-disordered membrane domains.     

Synthesis of neoglycolipids for the development of non-viral gene delivery systems

Synthesis of lipid conjugates with galactose as a targeting ligand intended for the development of non-viral systems for the targeted delivery of nucleic acids into hepatocytes is described. 3,4-Diethoxycyclobut-3-ene-1,2-dione (diethyl squarate) was used to bind the galactose moiety to the lipid component.     

Spherical Nucleic Acid Nanoparticle Conjugates Enhance G‐Quadruplex Formation and Increase Serum Protein Interactions

To understand the effect of three‐dimensional oligonucleotide structure on protein corona formation, we studied the identity and quantity of human serum proteins that bind to spherical nucleic acid (SNA) nanoparticle conjugates. SNAs exhibit cellular uptake properties that are remarkably different from those of linear nucleic acids, which have been related to their interaction with certain classes of proteins. Through a proteomic analysis, this work shows that the protein binding properties of SNAs are sequence‐specific and supports the conclusion that the oligonucleotide tertiary structure can significantly alter the chemical composition of the SNA protein corona. This knowledge will impact our understanding of how nucleic acid‐based nanostructures, and SNAs in particular, function in complex biological milieu.     

Calixarenes in a membrane environment: a monolayer study on the miscibility of three p-tert-butylcalix[4]arene beta-lactam derivatives with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine

Literature data indicate that some calixarene derivatives with antimicrobial activities may be useful as drugs; one of the aspects of the biological activity of different classes of antibiotics concerns interactions with lipid membranes. Here, the possibility of incorporation and/or translocation of three amphiphilic p-tert-butylcalix[4]arene derivatives across membranes was studied using lipid monolayers. The derivatives used have 6-aminopenicillanic acid or benzylpenicillin moieties grafted in alternate positions at the calixarene lower rim; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), a model bacterial membrane lipid, was used to prepare the monolayers. The miscibility of calixarene-antibiotic conjugates with lipid films was studied using surface pressure and surface potential measurements, as well as Brewster angle microscopy. The results obtained show that the miscibility is significantly different for the 6-aminopenicillanic acid and the two benzylpenicillin derivatives. Molecular modeling allowed the assessment of the lowest energy conformations of the calixarene derivatives and gave more insight into the interactions with the DMPE films.     

Semisynthetic DNA–Protein Conjugates for Biosensing and Nanofabrication

Conjugation with artificial nucleic acids allows proteins to be modified with a synthetically accessible, robust tag. This attachment is addressable in a highly specific manner by means of molecular recognition events, such as Watson–Crick hybridization. Such DNA–protein conjugates, with their combined properties, have a broad range of applications, such as in high‐performance biomedical diagnostic assays, fundamental research on molecular recognition, and the synthesis of DNA nanostructures. This Review surveys current approaches to generate DNA–protein conjugates as well as recent advances in their applications. For example, DNA–protein conjugates have been assembled into model systems for the investigation of catalytic cascade reactions and light‐harvesting devices. Such hybrid conjugates are also used for the biofunctionalization of planar surfaces for micro‐ and nanoarrays, and for decorating inorganic nanoparticles to enable applications in sensing, materials science, and catalysis.     

A calorimetric evaluation of the interaction of amphiphilic prodrugs of idebenone with a biomembrane model

Lipoamino acids (LAA) are useful promoieties to modify physicochemical properties of drugs, namely lipophilicity and amphiphilicity. The resulting membrane-like character of drug-LAA conjugates can increase the absorption profile of drugs through cell membranes and biological barriers. To show the role of amphiphilicity with respect to lipophilicity in the interaction of drugs with biomembranes, in the present study we evaluated the mode of such an interaction of lipophilic conjugates of LAA with the antioxidant drug idebenone (IDE). DSC analysis and transfer kinetic studies were carried out using dimyristoylphosphatidylcholine (DMPC) multilamellar liposomes (MLVs) as a model. For comparison, two esters of IDE with alkanoic acids were synthesized and included in the analysis. The experimental results indicate that based on their different structure, IDE-LAA conjugates interacted at different levels with respect to pure IDE with DMPC bilayers. In particular, a progressive penetration inside the vesicles was observed upon incubation of IDE-LAA compounds with empty liposomes. The enhanced amphiphilicity of the drug due to the LAA moieties caused more complex interactions with DMPC bilayers, compared to those registered with the native drug or IDE alkanoate esters.     

In Situ Vesicle Formation by Native Chemical Ligation

Phospholipid vesicles are of intense fundamental and practical interest, yet methods for their de novo generation from reactive precursors are limited. A non‐enzymatic and chemoselective method to spontaneously generate phospholipid membranes from water‐soluble starting materials would be a powerful tool for generating vesicles and studying lipid membranes. Here we describe the use of native chemical ligation (NCL) to rapidly prepare phospholipids spontaneously from thioesters. While NCL is one of the most popular tools for synthesizing proteins and nucleic acids, to our knowledge this is the first example of using NCL to generate phospholipids de novo. The lipids are capable of in situ synthesis and self‐assembly into vesicles that can grow to several microns in diameter. The selectivity of the NCL reaction makes in situ membrane formation compatible with biological materials such as proteins. This work expands the application of NCL to the formation of phospholipid membranes.     

PHOTODYNAMIC LIPID PEROXIDATION BY THE PHOTOSENSITIZING NONSTEROIDAL ANTIINFLAMMATORY DRUGS SUPROFEN AND TIAPROFENIC ACID

Abstract The photochemistry of the photosensitizing nonsteroidal antiinflammatory drugs tiaprofenic acid and suprofen involves the intermediacy of short-lived species ( i.e. radicals). The data obtained in the present work strongly suggest that such intermediates may be responsible for the phototoxicity of 2-arylpropionic acids by inducing photodynamic lipid peroxidation at drug concentrations likely to be reached in the skin. This has been investigated using linoleic acid as a model lipid and determining the amount of hydroperoxides by measuring the spectrophotometric absorption at 233 nm, associated with the formation of dienic hydroperoxides. The major photoproducts of tiaprofenic acid and suprofen are derivatives bearing an ethyl side chain. Photoproducts of this type, due to the lack of polar moieties, are highly lipophilic and likely to accumulate in the lipid bilayer of cell membranes. Taking into account their ability to induce photodynamic lipid peroxidation and their marked photostability, it is conceivable that such photoproducts can participate in many catalytic cycles, playing a significant role in the mechanism of photosensitizatinn by tiprofenic acid and suprofen.     

Surface enhanced Raman scattering detection of water-soluble derivatives of fullerene C<Subscript>60</Subscript> and their covalent conjugates with dyes in biological model systems

The potential of surface enhanced Raman spectroscopy (SERS) for the detection of water-soluble fullerene derivatives and their covalent conjugates with xanthene dyes was investigated in model biological liposome membranes and in the albumin protein structure. It was shown that in liposomes and in albumin, fullerene derivatives and their covalent conjugates with dyes show characteristic SERS spectra, which allows detection of water-soluble fullerene derivatives in phosphatidylcholine liposomes at the lipid/fullerene derivative ratio of 100 as well as fullerene–dye conjugates in liposomes and albumin.     

Determination of monomethylarsonic acid and dimethylarsinic acid by derivatization with thioglycolic acid methylester and gas—liquid chromatographic

An elegant method for the determination of monomethylarsonic acid and dimethylarsinic acid in biological materials is described. The methylarsenic acids are derivatized with thioglycolic acid methylester to yield lipophilic species, which can be determined by gas chromatography using a flame ionization detector or the more selective thermionic specific detector. The method permits the quick determination of methylarsenic acids in concentrations as low as 10 ng ml^-1 in urine or blood.     

PNA: Synthetic Polyamide Nucleic Acids with Unusual Binding Properties

The astonishing discovery that peptide nucleic acids (PNAs, B=nucleobase), in spite of their drastic structural difference to natural DNA, are better nucleic acid mimetics than many other oligonucleotides has resulted in an explosion of research into this class of compounds. The synthesis, physical properties, and biological interactions of PNAs as well as their chimeras with DNA and RNA are summarized here.     

Point of attachment and sequence of immobilized peptide-acridine conjugates control affinity for nucleic acids

Screening of combinatorial libraries by spatial arraying strategies requires library members to be solid-phase immobilized. However, for nucleic acid ligands that bind via intercalation, immobilization may inhibit binding if the tethering functionality is present at the edge of the heterocyle that approaches the duplex during the binding reaction. We report here a method for immobilizing peptide-acridine conjugates (PACs) via either their C- or their N-terminus, corresponding to functionalization at either the 4- or the 9-position of acridine, respectively, and for assaying the nucleic acid binding properties of the resulting resins. We find that both the amino acid sequence of the PAC as well as its point of attachment to the solid support are important in determining affinity for duplex nucleic acids. These results have implications for the design of future on-bead and microarray-based selections and in understanding the nucleic acid binding of functionalized intercalators.     

Design and synthesis of sphingomyelin-cholesterol conjugates and their formation of ordered membranes

A lipid raft is a cholesterol (Chol)-rich microdomain floating in a sea of lipid bilayers. Although Chol is thought to interact preferentially with sphingolipids such as sphingomyelin (SM), rather than with glycerophospholipids, the origin of the specific interaction has remained unresolved, primarily because of the high mobility of lipid molecules and weak intermolecular interactions. In this study, we synthesized SM-Chol conjugates with functionally designed linker portions to restrain Chol mobility and examined their formation of ordered membranes by a detergent insolubility assay, fluorescence anisotropy experiments, and fluorescence-quenching assay. In all of the tests, membranes prepared from the conjugates showed properties of ordered domains comparable to a SM-Chol (1:1) membrane. To gain insight into the structure of bilayers composed from the conjugates, we performed molecular dynamics simulations with 64 molecules of the conjugates, which suggested that the conjugates form a stable bilayer structure by bending at the linker portion and, mostly, reproduce the hydrogen bonds between the SM and Chol portions. These results imply that the molecular recognition between SM and Chol in an ordered domain is essentially reproduced by the conjugated molecules and, thus, demonstrates that these conjugate molecules could potentially serve as molecular probes for understanding molecular recognition in lipid rafts.     

The synthesis and properties of oligoribonucleotide-spermine conjugates

Polyamines stabilise nucleic acids against chemical and enzymatic degradation, facilitate the formation of secondary and tertiary structures and enhance cellular uptake. Therefore methods for the syntheses of polyamine-nucleic acid conjugates are of interest. A route for the syntheses of RNA-spermine conjugates has been developed. The polyamine was introduced to the C-5 position of uridine via an ethyl tether and the molecule elaborated into a synthon suitable for oligoribonucleotide assembly. The resultant oligomers were components of the hairpin ribozyme. Characterisation of the spermine-conjugated catalytic RNA revealed that attachment of the polyamine was well tolerated in three of four positions, namely U41, U37 and U34, suggesting that conjugation to C-5 brings about minimal structural perturbation.     

Sensitivity by combination: immuno-PCR and related technologies

The versatility of immunoassays for the detection of antigens can be combined with the signal amplification power of nucleic acid amplification techniques in a broad range of innovative detection strategies. This review summarizes the spectrum of both, DNA-modification techniques used for assay enhancement and the resulting key applications. In particular, it focuses on the highly sensitive immuno-PCR (IPCR) method. This technique is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR or related techniques for signal generation and read-out. Various strategies for the combination of antigen detection and nucleic acid amplification are discussed with regard to their laboratory analytic performance, including novel approaches to the conjugation of antibodies with DNA, and alternative pathways for signal amplification and detection. A critical assessment of advantages and drawbacks of these methods for a number of applications in clinical diagnostics and research is conducted. The examples include the detection of viral and bacterial antigens, tumor markers, toxins, pathogens, cytokines and other targets in different biological sample materials.