The design of probes for the selective recognition of biopolymers (nucleic acids and proteins) is a fundamental task for studying, diagnosing, and treating diseases. Traditional methods utilize a single component (small molecule or oligonucleotide) that binds directly to the target biopolymer. However, many biopolymers are unable to be targeted with this approach. The overarching goal of this dissertation is to explore a new, binary approach for designing probes. The binary approach requires two components that cooperatively bind to the target, triggering a recognition event. The requisite binding of two-components allows the probes to have excellent selectivity and modularity. The binary approach was applied to design a new sensor, called operating cooperatively (OC) sensor, for recognition of nucleic acids, including selectively differentiating between single nucleotide polymorphisms (SNPs). An OC sensor contains two oligonucleotide probe strands, called O and C, each with two domains. The first domain contains a target recognition sequence, whereas the second domain is complementary to a molecular beacon (MB) probe. Binding of both probe strands to the fully matched analyte generates a full MB probe recognition site, allowing a MB to bind and report the presence of the target analyte. Importantly, we show that the OC sensor selectively discriminates between single nucleotide polymorphisms (SNPs) in DNA and RNA targets at room temperature, including those with stable secondary structures. Furthermore, the combinatorial use of OC sensors to create a DNA logic gate capable of analyzing DNA sequences of Mycobacterium tuberculosis is described. The binary approach was also applied to design covalent inhibitors for HIV-1 reverse transcriptase (RT). In this application, two separate pre-reactive groups were attached to a natural RT ligand, deoxythymidine triphosphate (dTTP). Upon binding of both dTTP analogs in the RT active site, the pre-reactive groups are brought into the proper proximity and react with each other forming an intermediate that subsequently reacts with an amino acid side chain from the RT. This leads to covalent modification of RT, and inhibition of its DNA polymerase activity. This concept was tested in vitro using dTTP analogs containing pre-reactive groups derived from ?-lactamase inhibitors clavulanic acid (CA) and sulbactam (SB). Importantly, our in vitro assays show that CA based inhibitors are more potent than zidovudine (AZT), a representative of the dominant class of RT inhibitors currently used in anti-HIV therapy. Furthermore, molecular dynamics simulations predict that complexes of RT with these analogs are stable, and point to possible reaction mechanisms. The inhibitors described in this work may serve as the basis for the development of the first covalent inhibitors for RT. Moreover, the pre-reactive groups used in this study can be used to design covalent inhibitors for other targets by attaching them to different ligands. Overall, the work presented herein establishes the binary approach as a straightforward way to develop new probes to selectively recognize nucleic acids and proteins.

Nucleic acids are fundamental molecules of living organisms functioning essentially as the molecular information carriers of life. From how an organism is built to how it responds to external conditions, all of it, can be found in the form of nucleic acid sequences inside every single cell of every life form on earth. Therefore, accessing these sequences provides key information regarding the molecular identity and functional state of any living organism, this is very useful for areas like biomedicine, where accessing and understanding these molecular signatures is the key to develop strategies to understand, treat and diagnose diseases. Decades of research and technological advancements have led to the development of a number of molecular tools and engineering technologies that allow accessing the information contained in the nucleic acids. This thesis provides a general overview of the tools and technologies available for nucleic acid analysis, and proposes an illustrative concept on how molecular tools and emergent technologies can be combined in a modular fashion to design methods for addressing different biomedical questions. The studies included in this thesis, are focused on the particular use of the molecular tools named: padlock and selector probes, rolling circle amplification, and fluorescence detection of single molecules in combination with microfluidics and portable microscopy. By using this combination, it became possible to design and demonstrate novel approaches for integrated nucleic acid analysis, inexpensive digital quantification, mobile-phone based diagnostics and the description of viral infections. These studies represent a step forward towards the adoption of the selected group of tools and technologies, for the design and building of methods that can be used as powerful alternatives to conventional tools used in molecular diagnostics and virology. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 1: Manuscript.</p>