2 research outputs found
Direct TAMRA-dUTP labeling of M. tuberculosis genes using loop-mediated isothermal amplification (LAMP)
Abstract Fluorescent molecule-based direct labeling of amplified DNA is a sensitive method employed across diverse DNA detection and diagnostics systems. However, using pre-labeled primers only allows for the attachment of a single fluorophore to each DNA strand and any modifications of the system are less flexible, requiring new sets of primers. As an alternative, direct labeling of amplified products with modified nucleotides is available, but still poorly characterized. To address these limitations, we sought a direct and adaptable approach to label amplicons produced through Loop-mediated isothermal amplification (LAMP), using labeled nucleotides (dUTPs) rather than primers. The focus of this study was the development and examination of a direct labeling technique of specific genes, including those associated with drug resistance in Mycobacterium tuberculosis. We used 5-(3-Aminoallyl)-2′-deoxyuridine-5′triphosphate, tagged with 5/6-TAMRA (TAMRA-dUTP) for labeling LAMP amplicons during the amplification process and characterized amplification and incorporation efficiency. The optimal TAMRA-dUTP concentration was first determined based on amplification efficiency (0.5% to total dNTPs). Higher concentrations of modified nucleotides reduced or completely inhibited the amplification yield. Target size also showed to be determinant to the success of amplification, as longer sequences showed lower amplification rates, thus less TAMRA incorporated amplicons. Finally, we were able to successfully amplify all four M. tuberculosis target genes using LAMP and TAMRA-modified dUTPs
Prefusion-specific antibody- derived peptides trivalently presented on DNA- nanoscaffolds as an innovative strategy against RSV entr
Human respiratory syncytial virus (RSV) is the primary cause of acute lower
respiratory tract infections in children and the elderly worldwide, for which
neither a vaccine nor an effective therapy is approved. The entry of RSV into the
host cell is mediated by stepwise structural changes in the surface RSV fusion
(RSV-F) glycoprotein. Recent progress in structural and functional studies of
RSV-F glycoprotein revealed conformation-dependent neutralizing epitopes
which have become attractive targets for vaccine and therapeutic
development. As RSV-F is present on viral surface in a trimeric form, a
trivalent binding interaction between a candidate fusion inhibitor and the
respective epitopes on each of the three monomers is expected to prevent
viral infection at higher potency than a monovalent or bivalent inhibitor. Here
we demonstrate a novel RSV entry inhibitory approach by implementing a
trimeric DNA nanostructure as a template to display up to three linear peptide
moieties that simultaneously target an epitope on the surface of the prefusion
RSV-F protein. In order to design synthetic binding peptides that can be
coupled to the DNA nanostructure, the prefusion RSV-F-specific monoclonal
antibody (D25) was selected. Complementarity-determining region 3 (CDR3)
derived peptides underwent truncation and alanine-scanning mutagenesis
analysis, followed by systematic sequence modifications using non-canonical
amino acids. The most effective peptide candidate was used as a binding
moiety to functionalize the DNA nanostructure. The designed DNA-peptide
construct was able to block RSV infection on cells more efficiently than the
monomeric peptides, however a more moderate reduction of viral load was
observed in the lungs of infected mice upon intranasal application, likely due to
dissociation or absorption of the underlying DNA structure by cells in the lungs.Taken together, our results point towards the inhibitory potential of a novel
trimeric DNA-peptide based approach against RSV and open the possibility to
apply this platform to target other viral infections