DNA Display II. Genetic Manipulation of Combinatorial Chemistry Libraries for Small-Molecule Evolution

Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA “genes,” making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This “chemical translation” provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment (iterated chemical translation, selection, and amplification) on a library of 10(6) nonnatural peptides. The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science “biology.

DNA Display III. Solid-Phase Organic Synthesis on Unprotected DNA

DNA-directed synthesis represents a powerful new tool for molecular discovery. Its ultimate utility, however, hinges upon the diversity of chemical reactions that can be executed in the presence of unprotected DNA. We present a solid-phase reaction format that makes possible the use of standard organic reaction conditions and common reagents to facilitate chemical transformations on unprotected DNA supports. We demonstrate the feasibility of this strategy by comprehensively adapting solid-phase 9-fluorenylmethyoxycarbonyl–based peptide synthesis to be DNA-compatible, and we describe a set of tools for the adaptation of other chemistries. Efficient peptide coupling to DNA was observed for all 33 amino acids tested, and polypeptides as long as 12 amino acids were synthesized on DNA supports. Beyond the direct implications for synthesis of peptide–DNA conjugates, the methods described offer a general strategy for organic synthesis on unprotected DNA. Their employment can facilitate the generation of chemically diverse DNA-encoded molecular populations amenable to in vitro evolution and genetic manipulation

Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells

Template-Directed Ligation of Peptides to Oligonucleotides

Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template

Nucleic Acids Conjugates for Biotechnological Applications

Life in biological systems is maintained by the cooperative actions of various biomolecules. With the development of chemical and biological technologies related to nucleic acids, the details of the mechanisms of such cooperative actions between nucleic acids and other biomolecules have been elucidated and further applied in various applications. In the papers published in this Special Issue, advanced research works involved in nucleic acid conjugates are reported in wide application fields, such as artificial gene regulation, biomolecular sensing, and therapeutics from leading scientists in nucleic acids chemistry and engineering

Diels-Alder cycloadditions in water for the straightforward preparation of peptide–oligonucleotide conjugates

The Diels-Alder reaction between diene-modified oligonucleotides and maleimide-derivatized peptides afforded peptide–oligonucleotide conjugates with high purity and yield. Synthesis of the reagents was easily accomplished by on-column derivatization of the corresponding peptides and oligonucleotides. The cycloaddition reaction was carried out in mild conditions, in aqueous solution at 37°C. The speed of the reaction was found to vary depending on the size of the reagents, but it can be completed in 8–10 h by reacting the diene-oligonucleotide with a small excess of maleimide-peptide

DNA-Directed Assembly of a Cell-Responsive Biohybrid Interface for Cargo Release

The development of a DNA-based cell-responsive biohybrid interface that can be used for spatially confined release of molecular cargo is reported. To this end, tailored DNA–protein conjugates are designed as gatekeepers that can be specifically cleaved by matrix metalloproteases (MMPs), which are secreted by many cancer cells. These gatekeepers can be installed by DNA hybridization on the surface of mesoporous silica nanoparticles (MSNs). The MSNs display another orthogonal DNA oligonucleotide that can be exploited for site-selective immobilization on solid glass surfaces to yield micropatterned substrates for cell adhesion. Using the human fibrosarcoma cell line HT1080 that secretes MMPs, it is demonstrated that the biohybrid surface is specifically modified by the cells to release both MSN-bound gatekeeper proteins and the encapsulated cargo peptide KLA. In view of the enormously high modularity of the system presented here, this approach promising for applications in drug delivery, tissue engineering, or other areas of nanobiotechnology is considered

PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor.

A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100-1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids

Cancer theranostics: multifunctional gold nanoparticles for diagnostics and therapy

Doctorate in Biology, Specialty in BiotechnologyThe use of gold nanoparticles (AuNPs) has been gaining momentum in molecular diagnostics due to their unique physico-chemical properties these systems present huge advantages, such as increased sensitivity, reduced cost and potential for single-molecule characterisation. Because of their versatility and easy of functionalisation, multifunctional AuNPs have also been proposed as optimal delivery systems for therapy (nanovectors). Being able to produce such systems would mean the dawn of a new age in theranostics (diagnostics and therapy)driven by nanotechnology vehicles. Nanotechnology can be exploit for cancer theranostics via the development of diagnostics systems such as colorimetric and imunoassays, and in therapy approaches through gene therapy, drug delivery and tumour targeting systems. The unique characteristics of nanoparticles in the nanometre range, such as high surface-tovolume ratio or shape/size-dependent optical properties, are drastically different from those of their bulk materials and hold pledge in the clinical field for disease therapeutics This PhD project intends to optimise a gold-nanoparticle based technique for the detection of oncogenes’ transcripts (c-Myc and BCR-ABL) that can be used for the evaluation of the expression profile in cancer cells, while simultaneously developing an innovative platform of multifunctional gold nanoparticles (tumour markers, cell penetrating peptides, fluorescent dyes) loaded with siRNA capable of silencing the selected proto-oncogenes, which can be used to evaluate the level of expression and determine the efficiency of silencing. This work is a part of an ongoing collaboration between Research Centre for Human Molecular Genetics, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal and Biofunctional Nanoparticles and Surfaces Group, Instituto de Nanociencia de Aragón, Spain within a European project [NanoScieE+ - NANOTRUCK]. In order to achieve this goal we developed effective conjugation strategies to combine, in a highly controlled way, biomolecules to the surface of AuNPs with specific functions such as: ssDNA oligos to detect specific sequences and for mRNA quantification; Biofunctional spacers: Poly(ethylene glycol) (PEG) spacers used to increase solubility and biocompatibility and confer chemical functionality; Cell penetrating peptides: to overcome the lipophilic barrier of the cellular membranes and deliver molecules into cells using TAT peptide to achieve cytoplasm and nucleus; Quaternary ammonium: to introduce stable positively charged in gold nanoparticles surface; and RNA interference: siRNA complementary to a master regulator gene, the proto-oncogene c-Myc, that is implicated in cell growth, proliferation, loss of differentiation, and cell death. In order to establish that they are viable alternatives to the available methods, these innovative nanoparticles were extensively characterized on their chemical functionalization, ease of uptake, cellular toxicity and inflammation, and knockdown of MYC protein expression in several cancer cell lines and in in vivo models.Fundação para a Ciência e Tecnologia - (SFRH/BD/62957/2009); PTDC/BIO/66514/2006; NANOLIGHT-PTDC/QUI-QUI/112597/2009; Silencing the silencers via multifunctional gold nanoconjugates towards cancer therapy - PTDC/BBB-NAN/1812/201

Highly Parallel Translation of DNA Sequences into Small Molecules

A large body of in vitro evolution work establishes the utility of biopolymer libraries comprising 1010 to 1015 distinct molecules for the discovery of nanomolar-affinity ligands to proteins.[1], [2], [3], [4], [5] Small-molecule libraries of comparable complexity will likely provide nanomolar-affinity small-molecule ligands.[6], [7] Unlike biopolymers, small molecules can offer the advantages of cell permeability, low immunogenicity, metabolic stability, rapid diffusion and inexpensive mass production. It is thought that such desirable in vivo behavior is correlated with the physical properties of small molecules, specifically a limited number of hydrogen bond donors and acceptors, a defined range of hydrophobicity, and most importantly, molecular weights less than 500 Daltons.[8] Creating a collection of 1010 to 1015 small molecules that meet these criteria requires the use of hundreds to thousands of diversity elements per step in a combinatorial synthesis of three to five steps. With this goal in mind, we have reported a set of mesofluidic devices that enable DNA-programmed combinatorial chemistry in a highly parallel 384-well plate format. Here, we demonstrate that these devices can translate DNA genes encoding 384 diversity elements per coding position into corresponding small-molecule gene products. This robust and efficient procedure yields small molecule-DNA conjugates suitable for in vitro evolution experiments