1,721,017 research outputs found
Enzymatic PEGylation of oligonucleotides
No full textThe gene and RNAi therapies as well as therapeutic aptamers attracted a great interest owing to the potential high selectivity and the theoretical possibility to treat a wide range of diseases, even those that have no other therapies. Unfortunately, this promise was partially reconsidered in face of the many difficulties related to the in vivo application of these molecules and the issues associated with their stability and delivery. Unmodified DNAs are rapidly degraded by 3’ exonucleases [1] and, furthermore, they suffer a fast kidney clearance and scarce cell internalization.
To address these limitations, two main approaches of nucleic acids delivery have been proposed i.e. viral vectors and lipidic or polymeric non-viral vectors. PEGylation, the covalent attachment of polyethylene glycol (PEG) [2], has been proposed for prolonging the pharmacokinetic profile of nucleic acid materials: PEGylated oligonucleotides and siRNAs have demonstrated an improved cell internalization and stability with respect to the free nucleic acids [3, 4].
A method for the direct selective conjugation of PEG to nucleic acid would be desired to overcome the constrains of the nucleic acids PEGylation based on the covalent attachment of a properly activated PEG to modified DNA or RNA oligonucleotides. In the field of protein PEGylation, the attention has been recently directed towards the development of enzymatic methods of conjugation [2, 5]. These new perspectives in protein PEGylation inspired us to develop a method consisting of an enzymatic PEGylation of oligonucleotides.
T4 DNA ligase enzyme was used to ligate double stranded oligonucleotides to a 20kDa PEG bearing a short oligonucleotide sequence. The chemical synthesis of the oligo-PEG conjugate was characterized and standardized. Hence, the enzymatic ligation of the oligo-PEG conjugate to a dsDNA was performed using a set of appositely designed oligonucleotides as model system. The ligation protocol was optimized allowing the complete achievement of a stable PEGylated dsDNA system. This early study on enzymatic PEGylation of oligonucleotides can potentially be applied to every DNA or RNA sequence and can stimulate new researches in this field
Aptamer Microarray Development for Human Thrombin Detection: Interaction Analysis in Solution and in Solid Phase
No full textEnzymatic Formation of PEGylated Oligonucleotides
No full textGene therapy, siRNA, and therapeutic aptamers attract great interest owing to their versatility to treat a wide range of diseases and their potential high selectivity. Unfortunately, oligonucleotide-based therapeutics suffer rapid degradation by nucleases, scarce cell internalization, and fast kidney clearance. To address these limitations, the covalent attachment by mild chemical reactions of an activated polyethylene glycol (PEG) is widely used to obtain PEGylated nucleic acids showing a more favorable pharmacokinetic profile. We describe here a method for the enzymatic formation of PEGylated nucleic acids employing T4 DNA ligase: the ligation protocol was set up and optimized allowing the complete achievement of PEGylated oligonucleotides amenable to further enzymatic reactions. The feasibility of this approach for bioconjugation was demonstrated employing a set of PEG-donors and oligonucleotide acceptors, differing in the chemical link between PEG and the oligonucleotide donor, and in the length, sequence, and structure of the oligonucleotides employed. The ligase reaction allowed us to obtain double-stranded as well as single-stranded oligonucleotides, thus demonstrating the applicability of the method to a variety of substrates suitable for diagnostic and therapeutic applications
Synthesis and evaluation of a bis-3-chloropiperidine derivative incorporating an anthraquinone pharmacophore
No full textWith the aim to attain an alkylating agent with enhanced DNA-affinity, we have successfully synthesised lysine-linked bis-3-chloropiperidine 1 bearing an anthraquinone moiety known to bind double-stranded DNA. Consistent with our expectations, compound 1 appears to intercalate into the DNA double helix, which can be observed by conformational changes of plasmid DNA suggesting alkylation and intercalation-induced DNA unwinding. The results of this work can provide a meaningful starting point for investigating the molecular mechanism of action of this novel DNA alkylating conjugate 1 with improved affinity for DNA
Aptamer based-microarraysfor protein detection: a potential tool for diagnostic applications
No full textAptamers are an emerging class of nucleic acid molecules with applications in several fields. They are RNA or single stranded DNA molecules selected for their folding in complex tridimensional structures: their conformation, encoded in their nucleotide sequence, determines the specific interaction with diverse target molecules in solution. Aptamers bind to specific protein with high specificity and affinity, making them attractive alternatives to the commonly used monoclonal antibodies both for therapeutic and diagnostic applications. Besides lower production costs, added advantages over antibodies are their relative ease of isolation and modifications, tailored binding affinity and reversible denaturation, making them suitable candidates for use in detection systems.
We have developed a Sandwich Aptamer Microarray (SAM) for human thrombin detection immobilizing one aptamer on a glass slide (capture aptamer) and using a secondary aptamer for the detection of the analyte. This aptasensor represents a potential diagnostic system for thrombin and exploits two non-overlapping DNA antithrombin aptamers recognizing different exosites of the target protein, TBA1 and TBA2. The 15-mer aptamer TBA1 binds the fibrinogen binding site, whereas the 29-mer aptamer TBA2 binds the heparin binding domain of human thrombin. This distinct recognition pattern allows their use in tandem, since a ternary complex could possibly be formed by simultaneous recognition of thrombin.
To adapt aptamers to a defined solid phase and to a specific detection technique, appropriate chemical modifications must be introduced. In the case of the microarray, immobilization to the solid support and labeling for detection imply precise chemistries that could sensibly alter the aptamer structure. An extensive analysis on the binary complexes formation has been therefore performed in solution by Electrophoretic Mobility Shift Assay (EMSA) to verify the sandwich complex formation by a Supershift assay.
The system validated in solution was finally applied to the solid phase using an appropriate control and two different protocols for detection, resulting in an aptamer-based microarray for recognition of human thrombin with high specificity, using standard microarray slides and a fast and easy protocol. The aptamer-based biosensor could be used to analyze thrombin concentration in pathologic conditions, therefore representing a potential tool for diagnostic application
Bis-3-chloropiperidines containing bridging lysine linkers: Influence of side chain structure on DNA alkylating activity
No full textA series of bis-3-chloropiperidines containing lysine linkers was synthesised as DNA alkylating model compounds by using a bidirectional synthetic strategy. These novel piperidine mustard based agents have been evaluated for their alkylating properties towards nucleic acids and were shown to alkylate and cleave DNA with strong preference for guanine residues. Our studies reveal that the introduction of aromatic groups in the side chain of the lysine linker has an impact on DNA alkylating activity. Analysis by ESI mass spectrometry enabled the verification of the reactive aziridinium ion formation. Overall, the results confirm our recently proposed reaction mechanism of bis-3-chloropiperidines
B-CePs as cross-linking probes for the investigation of RNA higher-order structure
Full text linkElucidating the structure of RNA and RNA ensembles is essential to understand biological functions. In this work, we explored the previously uncharted reactivity of bis-chloropiperidines (B-CePs) towards RNA. We characterized at the molecular level the different adducts induced by the fast reacting compound B-CeP 1 with RNA. Following an approach based on solution thermal melting coupled with ESI mass spectrometry (STHEM-ESI), we proved the ability of B-CePs to induce inter-molecular cross-links between guanines in double stranded RNA. These results open the possibility of using B-CePs as structural probes for investigating higher-order structures, such as the kissing loop complex established by the dimerization initiation site (DIS) of the HIV-1 genome. We confirmed the potential of B-CePs to reveal the identity of RNA structures involved in long-range interactions, expecting to benefit the characterization of samples that are not readily amenable to traditional high-resolution techniques, and thus promoting the elucidation of pertinent RNA systems associated with old and new diseases
Human Thrombin Detection Through a Sandwich Aptamer Microarray: Interaction Analysis in Solution and in Solid Phase.
No full textWe have developed an aptamer-based microarray for human thrombin detection exploiting two non-overlapping DNA thrombin aptamers recognizing different exosites of the target protein. The 15-mer aptamer (TBA1) binds the fibrinogen-binding site, whereas the 29-mer aptamer (TBA2) binds the heparin binding domain. Extensive analysis on the complex formation between human thrombin and modified aptamers was performed by Electrophoresis Mobility Shift Assay (EMSA), in order to verify in solution whether the chemical modifications introduced would affect aptamers/protein recognition. The validated system was then applied to the aptamer microarray, using the solid phase system devised by the solution studies. Finally, the best procedure for Sandwich Aptamer Microarray (SAM) and the specificity of the sandwich formation for the developed aptasensor for human thrombin were optimized
Development and Optimization of a Thrombin Sandwich Aptamer Microarray
Full text linkA sandwich microarray employing two distinct aptamers for human thrombin has been optimized for the detection of subnanomolar concentrations of the protein. The aptamer microarray demonstrates high specificity for thrombin, proving that a two-site binding assay with the TBA1 aptamer as capture layer and the TBA2 aptamer as detection layer can ensure great specificity at times and conditions compatible with standard routine analysis of biological samples. Aptamer microarray sensitivity was evaluated directly by fluorescent analysis employing Cy5-labeled TBA2 and indirectly by the use of TBA2-biotin followed by detection with fluorescent streptavidin. Sub-nanomolar LODs were reached in all cases and in the presence of serum, demonstrating that the optimized aptamer microarray can identify thrombin by a low-cost, sensitive and specific method
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