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Maffucci, I.

Publications and source records attributed to Maffucci, I..

3 recordsLinked to original sources

Design of DNA Aptamers for Lyme disease Diagnosis Combining experimental and numerical approaches

Aptamers are single stranded DNA or RNA molecules selected for their high affinity and specificity to bind target molecules, similar to antibodies. They are commonly selected through the SELEX process, which involves the iterative exposure of a random sequence library to a target and retaining the sequences showing good binding properties. To improve Lyme disease detection, we propose designing aptamers that specifically bind to the CspZ protein on the surface of Borrelia burgdorferi, the bacterium responsible for the disease. Starting with a SELEX process consisting of thirteen rounds, from which selected in vitro sequence candidates have emerged, we aim to propose a holistic process that selects in silico new sequence candidates that are further validated experimentally. Our approach relies on 1) using Machine Learning (ML) techniques, specifically a Restricted Boltzmann Machine (RBM), to digitally replicate the last round of the SELEX process, 2) integrating insights from text analysis methods, such as word2vec and n-grams, into the RBM model trained on the final-round SELEX dataset to represent and compare newly generated sequences with in vitro candidates, 3) selecting in silico sequences with strong potential to bind to CspZ protein, 4) experimentally validating the selected in silico sequences of step 3. Our holistic approach combines biological insights with statistical models to improve the efficiency and outcome of the SELEX process. We enhance the RBM model, designed to replicate the distribution of the final SELEX round, by integrating geometric representations of sequences, which is especially advantageous when dealing with limited datasets relative to the vast sequence space. In addition, it provides in silico sequence candidates with strong binding properties.

bioinformatics↗

Selection and characterization of DNA aptamers targeting the surface borrelial protein CspZ with high-throughput cross-over SELEX

Lyme borreliosis (LB) is the most prevalent tick-borne illness, with an estimated 700 000 cases annually in the United States and Europe. LB diagnosis based on two-tiered serology remains controversial due to its indirect nature and low sensitivity during the early stage of the disease. Aptamers are single-stranded DNA or RNA oligonucleotides that exhibit high selectivity and specificity for their target due to their unique three-dimensional structure. By applying cross-over-SELEX process, an enrichment of DNA oligonucleotide sequences against a surface protein of Borrelia, named CspZ, has been performed and monitored using absorbance at 260 nm, melting curves and NGS analyses. Beyond sequence enrichment, oligonucleotides binding to CspZ were observed during the selection rounds by Dot Blot and beads assays. Thirteen unique and highly redundant oligonucleotide sequences have been further characterized using multiple approaches such as Dot Blot, BioLayer Interferometry and Surface Plasmon Resonance. The selected aptamers showed KD values from tens of nanomolar to the micromolar range by BLI and SPR. Two aptamers, characterized by flow cytometry and epifluorescence microscopy, were able to specifically recognize Borrelia burgdorferi sensu stricto. This novel strategy holds promise for the development of an improved diagnosis assay. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/632687v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@3a2697org.highwire.dtl.DTLVardef@1bba993org.highwire.dtl.DTLVardef@17a0a60org.highwire.dtl.DTLVardef@13f4a4f_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIDNA aptamers were selected by cross-over SELEX. C_LIO_LIAptamers KDs ranged from tens of nanomolar to the micromolar range. C_LIO_LIAptamer characterization was performed by Dot Blot, beads assay, BLI, and SPR. C_LIO_LIInteraction with Borrelia was tested by flow cytometry and epifluorescence microscopy. C_LIO_LIAptamers bind to both the recombinant and borrelial surface CspZ protein. C_LI

biochemistry↗

AptaMat: a matrix-based algorithm to compare single-stranded oligonucleotides secondary structures

Comparing single-stranded nucleic acids (ssNAs) secondary structures is fundamental when investigating their function and evolution and predicting the effect of mutations on the ssNAs structures. Many comparison metrics exist, although they are either too elaborate or not enough sensitive to distinguish close ssNAs structures. In this context, we developed AptaMat, a simple and sensitive algorithm for ssNAs secondary structures comparison based on matrices representing the ssNAs secondary structures and a metric built upon the Manhattan distance in the plane. We applied AptaMat to several examples and compared the results to those obtained by the most frequently used metrics, namely the Hamming distance and the RNAdistance, and by a recently developed image-based approach. We showed that AptaMat is able to discriminate between similar sequences, outperforming all the other here considered metrics.

bioinformatics↗