Peptide-oligonucleotide Conjugates Research Articles

Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment.

Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD.

Peptide-mediated Cell and In Vivo Delivery of Antisense Oligonucleotides and siRNA

Antisense oligonucleotides and short interfering RNAs (siRNAs) are nucleic acids-targeting reagents for gene expression modulation that are being developed as drugs for many applications. A number of useful synthetic nucleic acids analogues have been introduced recently to greatly improve their properties for use as therapeutics. However, their full effectiveness in cells and in vivo has often only been realized through development of suitable nonviral delivery systems. Among these, a range of natural and synthetic peptides have been found useful for enhancing cellular uptake and/or cell targeting of oligonucleotide analogues and siRNA. Such peptides are synthetically conjugated, used as noncovalent complexes, or used in combination with polymer, liposomal or exosome formulation techniques. This review begins by describing the modes of action of antisense reagents and siRNA and goes on to focus on recent advances in their peptide-mediated cell and in vivo delivery and how peptide use has influenced drug development. The review discusses the challenges associated with understanding the physiological and toxicological aspects of peptide-mediated delivery. Developments towards clinical use are also highlighted, with particular emphasis on peptide conjugates of oligonucleotide analogues used for treatment of neuromuscular diseases.

An activated triple bond linker enables 'click' attachment of peptides to oligonucleotides on solid support.

A general procedure, based on a new activated alkyne linker, for the preparation of peptide–oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker enhances the Cu(I) catalyzed 1,3-dipolar cycloaddition (‘click’ reaction). The preferred scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of (i) an H-phosphonate-based aminolinker; (ii) the triple bond donor p-(N-propynoylamino)toluic acid (PATA); and (iii) azido-functionalized peptides. The method gives conversion of oligonucleotide to the POC on solid support, and only involves a single purification step after complete assembly. The synthesis is flexible and can be carried out without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. Methodology for the ready conversion of peptides into ‘clickable’ azidopeptides with the possibility of selecting either N-terminus or C-terminus connection also adds to the flexibility and usability of the method. Examples of synthesis of POCs include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals.

Coupling Strategies for the Synthesis of Peptide-Oligonucleotide Conjugates for Patterned Synthetic Biomineralization

This work describes preparation strategies for peptide-oligonucleotide conjugates that combine the self-assembling behavior of DNA oligonucleotides with the molecular recognition capabilities of peptides. The syntheses include a solution-phase fragment coupling reaction and a solid-phase fragment coupling strategy where the oligonucleotide has been immobilized on DEAE Sepharose. The yield of four coupling reagents is evaluated, two reagents in water, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and DMTMM (4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methyl-morpholinium chloride), and two in dimethylformamide (DMF), PyBOP ((Benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate) and HBTU (O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate), while the oligonucleotide fragment is either in solution or immobilized on DEAE. These coupling strategies rely on an unprotected 5′ amino linker on the oligonucleotide reacting with the peptide C-terminus. The peptide, selected from a combinatorial library for its gold-binding behavior, was 12 amino acids long with an N-terminus acetyl cap. Formation of the conjugates was confirmed by gel electrophoresis and mass spectrometry while molecular recognition functionality of the peptide portion was verified using atomic force microscopy. Solution-phase yields were superior to their solid-phase counterparts. EDC resulted in the highest yield for both solution-phase (95%) and solid-phase strategies (24%), while the DMF-based reagents, PyBOP and HBTU, resulted in low yields with reduced recovery. All recoverable conjugates demonstrated gold nanoparticle templating capability.

Synthesis of peptide-oligonucleotide conjugates using a heterobifunctional crosslinker.

Peptide-oligonucleotide conjugates (POCs) are molecular chimeras composed of a nucleic acid moiety covalently attached to a polypeptide moiety. POCs have been used in numerous applications from therapeutics to nanotechnology, and most recently as combinatorial agents in the assembly of bivalent protein affinity reagents. This unit describes the synthesis and purification of POC molecules using the heterobifunctional crosslinking reagent succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), which enables amine-modified oligonucleotides to become covalently linked to cysteine-modified polypeptides. This solution-based protocol consists of a two-step synthesis followed by a single purification step.

Versatile Phosphoramidation Reactions for Nucleic Acid Conjugations with Peptides, Proteins, Chromophores, and Biotin Derivatives

Chemical conjugations of nucleic acids with macromolecules or small molecules are common approaches to study nucleic acids in chemistry and biology and to exploit nucleic acids for medical applications. The conjugation of nucleic acids such as oligonucleotides with peptides is especially useful to circumvent cell delivery and specificity problems of oligonucleotides as therapeutic agents. However, current approaches are limited and inefficient in their ability to afford peptide-oligonucleotide conjugates (POCs). Here, we report an effective and reproducible approach to prepare POCs and other nucleic acid conjugates based on a newly developed nucleic acid phosphoramidation method. The development of a new nucleic acid phosphoramidation reaction was achieved by our successful synthesis of a novel amine-containing biotin derivative used to systematically optimize the reactions. The improved phosphoramidation reactions dramatically increased yields of nucleic acid-biotin conjugates up to 80% after 3 h reaction. Any nucleic acids with a terminal phosphate group are suitable reactants in phosphoramidation reactions to conjugate with amine-containing molecules such as biotin and fluorescein derivatives, proteins, and, most importantly, peptides to enable the synthesis of POCs for therapeutic applications. Polymerase chain reactions (PCRs) to study incorporation of biotin or fluorescein-tagged DNA primers into the reaction products demonstrated that appropriate controls of nucleic acid phosphoramidation reactions incur minimum adverse effects on inherited base-pairing characteristics of nucleotides in nucleic acids. The phosphoramidation approach preserves the integrity of hybridization specificity in nucleic acids when preparing POCs. By retaining integrity of the nucleic acids, their effectiveness as therapeutic reagents for gene silencing, gene therapy, and RNA interference is ensured. The potential for POC use was demonstrated by two-step phosphoramidation reactions to successfully synthesize nucleic acid-tetraglycine conjugates. In addition, phosphoramidation reactions provided a facile approach to prepare nucleic acid-BSA conjugates with good yields. In summary, the new approach to phosphoramidation reactions offers a universal method to prepare POCs and other nucleic acid conjugates with high yields in aqueous solutions. The methods can be easily adapted to typical chemistry or biology laboratory setups which will expedite the applications of POCs for basic research and medicine.

Stepwise synthesis of oligonucleotide–peptide conjugates containing guanidinium and lipophilic groups in their 3′-termini

Two different series of oligonucleotide–peptide conjugates have been efficiently synthesized by stepwise solid-phase synthesis. First, oligonucleotides and oligonucleotide phosphorothioates containing polar groups at the 3′-termini, such as amine and guanidinium groups were prepared. ODNs conjugates carrying several lysine residues were obtained directly from Fmoc deprotection whereas ODN conjugates with guanidinium groups were obtained by post-synthetic guanidinylation. The second family contains different urea moieties that were achieved by standard protocols. All products were fully characterized by reversed phase HPLC and MALDI-TOF mass spectrometry yielding satisfactory results. Oligonucleotide–phosphorothioate conjugates were evaluated as potential antisense oligonucleotides in the inhibition of the luciferase gene.

Native chemical ligation in the synthesis of internally modified oligonucleotide–peptide conjugates

Peptide-oligonucleotide conjugates have frequently been synthesized to improve cellular delivery of antisense or antigene compounds, to allow the immobilization of peptide and protein conjugates on DNA arrays, or to decorate nucleic acid architectures with peptide functions. In such applications, the site of conjugation is of little importance, and peptides have predominantly been appended to one of the terminal ends of the oligonucleotide by using an oxime-, thioether-, or disulfide-linkage or native chemical ligation. We, herein, demonstrate the first coupling of peptides to sequence internal sites. This attachment mode provides better control of the spatial arrangement of peptides presented by self-assembled nucleic acid scaffolds. Internal modification requires special phosphoramidite building blocks that can be used in automated DNA synthesis. For this purpose, Fmoc/StBu-protected cysteine was attached via an aminopropargyl linker to the C5-position of uridine. The rigid triple bond conferred a high reactivity in native chemical ligation reactions of 5-6mer peptide thioesters with up to 15 nucleotides long oligonucleotides. The desired peptide-oligonucleotide conjugates were obtained in high yields after purification. UV melt experiments revealed that the peptide modification does not hamper nucleic acid hybridization. This finding marked an important step in our research program devoted to studies of multivalent presentation of peptides via modular assembly of nucleic acid complexes.

Chemical Strategies for the Synthesis of Peptide−Oligonucleotide Conjugates

The use of synthetic oligonucleotides and their mimics to inhibit gene expression by hybridizing with their target sequences has been hindered by their poor cellular uptake and inability to reach the nucleus. Covalent postsynthesis or solid-phase conjugation of peptides to oligonucleotides offers a possible solution to these problems. As feasible chemistry is a prerequisite for biological studies, development of efficient and reproducible approaches for convenient preparation of peptide-oligonucleotide conjugates has become a subject of considerable importance. The present review gives an account of the main synthetic methods available to prepare covalent conjugation of peptides to oligonucleotides.

Development of peptide-oligonucleotide conjugates for regulation of small RNA function

Recently, various microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified and play important roles in gene regulatory networks. However it is a little known about their biological functions. These small RNAs exhibit their function to form a ribonucleoprotein complex, the RISC (RNA-induced silencing complex), which modulates gene expression by translational repression. In this study, we developed a novel peptide antagonist to inhibit the RISC function. The peptide was conjugated to 2'-O-methyl oligoribonucleotides, which have a complementary sequence to the guide strand of siRNA, and regulatory effects of the peptide on RISC activity were examined. It was revealed that the peptide drastically enhanced inhibitory effects of the oligonucleotide on RISC activity. Here we demonstrate our peptide-oligonucleotide conjugate that can provide a powerful and specific way to regulate the small RNA function.

Organic Synthesis and Antisense Effects of Oligonucleotide-Peptide Conjugates

Organic Synthesis and Antisense Effects of Oligonucleotide-Peptide Conjugates

A Novel Approach to the Preparation of Peptide-Oligonucleotide Conjugates

A novel approach to the synthesis of peptide-oligonucleotide conjugates (POC) based on the oxathiaphospholane chemistry has been developed. Peptide and oligonucleotide fragments, which were separately prepared, were linked postsynthetically by the functionalization of the peptide either by attachment of the oxathiaphospholane residue directly at the N-terminus or at the hydroxy group of the linker connected to N-terminus. The conjugation reaction, based on DBU-assisted nucleophilic attack of hydroxy group of oligonucleotide on phosphorus atom in the oxathiaphospholane derivatives of peptide, furnished the desired POC.

A Novel Conformationally Constrained Parallel G Quadruplex

Guanine-rich DNA sequences are known to form highly ordered structures called G quadruplexes. These structures play an important role in many relevant biological processes, such as telomere stabilization, oncogene activation, and the regulation of the immunoglobulin switch region. The G-quadruplex motif is based on the association of planar G quartets of four guanine residues that are held together by eight Hoogsteen-type hydrogen bonds (Figure 1A). The G-quadruplex motif requires monovalent cations, such as Na and K , for stabilization. A wide variety of topologies can be adopted depending on the number of strands involved in the structure, the strand direction, as well as variations in loop size and sequence (Figure 1). The structure of parallel-stranded as well as antiparallel-stranded quadruplexes have been extensively studied by using different methods, such as NMR spectroscopy, X-ray diffraction and circular dichroism, but the exact conformation present in vivo is still under discussion. The design of small molecules that can bind to G quadruplexes has thus received attention because these nucleic acid motifs represent valuable pharmaceutical targets. For this purpose, a large number of small molecules has been evaluated for their binding with these particular DNA structures. However, as mentioned, the G quadruplex can adopt different topologies that can confuse the study of recognition phenomena. The design of a system that is able to mimic a well-defined conformation of G quadruplex is thus of great interest to precisely study the molecular interactions that can occur with small organic molecules. In 1985, Mutter proposed the TASP concept (template-assembled synthetic proteins) for the design of folded proteins. These pioneering works described the use of a cyclodecapeptide that allows the preparation of artificial proteins with a predetermined three-dimensional structure. Despite a large number of examples that use this template, to our knowledge, it has not been applied for the design of a specific folded structure of nucleic acid. With this in mind, we investigated the use of a peptidic scaffold as a topological template that directs the intramolecular assembly of covalently attached oligonucleotides into a single characteristic folding topology of G quadruplex. We anticipated that the scaffold should permit the preorganization of the DNA strands and the stabilization of the quadruplex structure. We report herein the synthesis and characterization of the novel water-soluble peptidic scaffold–oligonucleotide conjugate 1 that mimics the parallel-stranded conformation of G quadruplex (Scheme 1). We demonstrate that the use of the scaffold allows the precise control of the conformation of the quadruplex and dramatically increases the stability of the motif—all the more so as the formation of the quadruplex motif is possible even without the addition of any monovalent cations, such as K . We also show that mimic 1 can be used for surface functionalization, and this permits the study of the molecular interaction with G-quadruplex ligands by using surface plasmon resonance (SPR). The scaffold used for the synthesis of mimic 1 is a cyclic decapeptide with two independently functionalizable faces, which are due to the orientation of the lysine side-chains. On one side, the four oligonucleotides derived from the human telomeric sequence d(TTAGGGT) were anchored by using oxime bond formation, and a biotin residue was incorporated on the other side for attachment to streptavidin-immobilized surfaces. Earlier work from our laboratory has demonstrated that the oxime coupling strategy allows the efficient preparation of peptide–oligonucleotide conjugates. Figure 1. G-quartet motif and possible folded structures of the G quadruplex. A) G quartet; B) intermolecular parallel form; C) intramolecular parallel form; D) intramolecular antiparallel form.

Design and Synthesis of Peptide-Oligonucleotide Conjugates as Potential Artificial Ribonucleases

A series of peptide-oligonucleotide conjugates was synthesized. The peptide fragment was attached to the middle part of DNA sequence. For this purpose the new amidophosphite dC with linker at C5-position was synthesized.

Antisense Inhibition of Human Telomerase by Phosphorothioate Oligonucleotide-Peptide Conjugates

Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by SPFC. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed peptides with amphipathic character. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugates-RNA hybrids could activate RNase H as effective as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into cell nucleus, (f) the conjugates with NES could be localized in cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells much more strongly than phosphorothioate oligonucleotides.

Peptide−Oligonucleotide Conjugates Form Stable and Selective Complexes with Antibody and DNA

The present work demonstrates that the relatively low molecular weight synthetic peptide-oligonucleotide conjugates are capable of stable and selective three-component complex formation with complementary 72-100mer DNA oligonucleotides and a cardiac troponin I monoclonal antibody. Neither the Watson-Crick-type interaction between peptide-oligonucleotide conjugate and DNA nor the conjugate-antibody interaction dramatically hampers the other. These interactions remain selective and specific in the presence of several other conjugates not specific to cardiac troponin I monoclonal antibody as well as in the presence of control 100mer DNA oligonucleotides. The data herein demonstrate the feasibility of the synthetic peptide-oligonucleotide conjugates as convenient molecular tools, e.g., for antibody epitope mapping.

Oligonucleotide conjugation to a cell-penetrating (TAT) peptide by Diels–Alder cycloaddition

Modifed oligonucleotides are routinely employed as analytical probes for use in diagnostics, e.g. in the examination of specific RNA sequences for infectious diseases, however, a major limiting factor in oligonucleotide-based diagnostics is poor cellular uptake of naked oligonucleotides. This problem can be overcome by covalent attachment of a so-called 'cell-penetrating peptide' to form an oligonucleotide peptide conjugate. Stepwise solid phase synthesis of such a conjugate is difficult and expensive due to the conflicting chemistries of oligonucleotides and peptides. A simple approach to overcome this is post-synthetic conjugation. Diels-Alder cycloaddition is an attractive methodology for oligonucleotide peptide conjugation; the reaction is fast, chemoselective and the reaction rate is greatly enhanced in aqueous media - ideal conditions for biological moieties. An oligodeoxyribonucleotide sequence has been derivatised with a series of dienes at the 5'-terminus, using a series of unique dienyl-modified phosphoramidites, and investigation into the effect of diene type on the efficiency of conjugation, using Diels-Alder cycloaddition with a maleimido-derivatised cell-penetrating (TAT) peptide, has been performed. This led to the observation that the optimal diene for conjugation was cyclohexadiene, allowing conjugation of oligodeoxyribonucleotides to a cell-penetrating peptide by Diels-Alder cycloaddition for the first time.

A High-Complexity, Multiplexed Solution-Phase Assay for Profiling Protease Activity on Microarrays

We have developed a miniaturized and multiplexed solution assay for the measurement of protease activity in complex samples. This technology can accelerate research in functional proteomics and enable biologists to carry out multiplexed protease inhibitor screens on a large scale. The assay readout is based on Illumina's universal Sentrix BeadArrays. The peptide sequences that serve as protease substrates are conjugated to oligonucleotide sequences complementary to the oligo tags on randomly assembled and decoded bead arrays. The peptide portion is C-terminally labeled with a biotin residue and contains a sequence of five histidine residues on the amino terminus. The unique oligonucleotide part of each oligonucleotide-peptide conjugate is attached to amino terminus of the peptide sequence. Upon protease cleavage, the biotin residue is cleaved from the oligonucleotide-peptide conjugate. Following the reaction, all biotin-containing species are captured and removed by incubation with streptavidin beads. The cleaved conjugates that remain in solution are captured by hybridization of their oligo sequence to Sentrix BeadArrays and detected using a labeled antibody against pentahistidine tag of the conjugate or by an antibody sandwich assay. We have generated multiple sets of oligonucleotide tagged peptide substrates of varying complexity (100 to 1000 substrates in a mixture) and show that the response of individual substrate is independent of the complexity of the mixture. Our initial results demonstrate the feasibility of assaying proteases in a multiplexed environment with high sensitivity.

Controlling the activity of peptides and proteins with smart nucleic acid–protein hybrids

Oligonucleotide-peptide conjugates have frequently been used to control the localisation of the conjugate molecule. For example, the oligonucleotide segment has allowed spatially addressed immobilization of peptides and proteins on DNA-arrays via hybridisation while the peptide part has most frequently been used to confer transfer of oligonucleotide cargo into live cells. The regulation of functional properties such as the affinity of these bioconjugates for protein targets has rarely been addressed. This review article describes the current developments in the application of smart oligonucleotide-peptide hybrids. The mutual recognition between nucleic acid segments is used to constrain the structure or control the distance between peptide and protein segments. Application of these new type of oligonucleotide-peptide hybrids allowed not only the regulation of binding affinity of peptide ligands but also control of enzymatic and optical activity of proteins.

Synthesis of Oligonucleotide Derivatives Using ChemMatrix Supports

The synthesis of oligonucleotides on poly(ethylene glycol)-based (ChemMatrix) supports was studied. Results show that oligonucleotides can be indeed prepared in good yields using slightly modified synthesis cycles and automated DNA synthesizers. The use of these supports for the synthesis of oligonucleotide-peptide conjugates and for the ligation of oligonucleotides using Cu(+)-catalyzed cycloadition reactions is reported. Moreover, these supports can be used for the preparation of oligonucleotides in anhydrous solvents, followed by hybridization of the complementary sequences in aqueous buffers.