Reporter-recruiting bifunctional aptasensor for bioluminescent analytical assays.

We report a novel bioluminescent aptasensor, which consists of 2'-F-RNA aptamer modules joined into a bi-specific aptamer construct. One aptamer module binds the analyte, then after structural rearrangement the second module recruits non-covalently Ca2+-dependent photoprotein obelin from the solution, thus providing a bioluminescent signal. This concept allows using free protein as a reporter, which brings such advantages as no need for aptamer-protein conjugation, a possibility of thermal re-folding of aptamer component with no harm to a protein, and simpler detection protocol. We developed the new 2'-F-RNA aptamer for obelin, and proposed the strategy for engineering structure-switching bi-modular aptamer constructs which bind the analyte and the obelin in a sequential manner. With the use of hemoglobin as a model analyte, we showed the feasibility of utilizing the aptasensor in a fast and straightforward bioluminescent microplate assay. With a proper design of a secondary structure, this strategy of aptasensor engineering might be further extended to bi-specific aptamer-based bioluminescent sensors for other analytes of interest.

DNA Binding to Gold Nanoparticles through the Prism of Molecular Selection: Sequence-Affinity Relation.

Native DNA strongly adsorbs to citrate-coated gold nanoparticles (AuNPs). The resulting composites (DNA/AuNPs) are valuable materials in many fields, especially in biomedicine. For this reason, the process of adsorption is a focus for intensive research. In this work, DNA adsorption to gold nanoparticles was studied using a molecular selection procedure followed by high-throughput DNA sequencing. The chemically synthesized DNA library containing a central N26 randomized fragment was sieved through four cycles of adsorption to AuNPs in a tree-like selection-amplification scheme (SELEX (Selective Evolution of Ligands by EXponential enrichment)). The frequencies of occurrence of specific oligomeric DNA motifs, k-mers ( k = 1-6), in the initial and selected pools were calculated. Distribution of secondary structures in the pools was analyzed. A large set of diverse A, T, and G enriched k-mers undergo a pronounced positive selection, and these sequences demonstrate faster and strong binding to the AuNPs. For facile binding, such structural motifs should be located in the loop regions of weak intramolecular complexes-hairpins with imperfect stem, or other portion of the structure, which is unpaired under selection conditions. Our data also show that, under the conditions employed in this study, cytosine is significantly depleted during the selection process, although guanine remains unchanged. These regularities were confirmed in a series of binding experiments with a set of synthetic DNA oligonucleotides. The detailed analysis of DNA binding to AuNPs shows that the sequence specificity of this interaction is low due to its nature, although the presence and the number of specific structural motifs in DNA affect both the rate of formation and the strength of the formed noncovalent associates with AuNPs.

Development and characterization of novel 2'-F-RNA aptamers specific to human total and glycated hemoglobins.

Aptamers are short DNA and RNA fragments which bind their molecular targets with affinity and specificity comparable to those of antibodies. Here, we describe the selection of novel 2'-F-RNA aptamers against total human hemoglobin or its glycated form HbA1c. After SELEX and high-throughput sequencing of the enriched libraries, affinities and specificities of candidate aptamers and their truncated variants were examined by the solid-phase bioluminescent assay. As a result, we identified aptamers specific to both hemoglobins or only glycated HbA1c. The developed 2'-F-RNA aptamers have shown their applicability for detection of total and glycated hemoglobin in one sample.

Key Aspects of Nucleic Acid Library Design for in Vitro Selection.

Nucleic acid aptamers capable of selectively recognizing their target molecules have nowadays been established as powerful and tunable tools for biospecific applications, be it therapeutics, drug delivery systems or biosensors. It is now generally acknowledged that in vitro selection enables one to generate aptamers to almost any target of interest. However, the success of selection and the affinity of the resulting aptamers depend to a large extent on the nature and design of an initial random nucleic acid library. In this review, we summarize and discuss the most important features of the design of nucleic acid libraries for in vitro selection such as the nature of the library (DNA, RNA or modified nucleotides), the length of a randomized region and the presence of fixed sequences. We also compare and contrast different randomization strategies and consider computer methods of library design and some other aspects.

Fast and Strong Adsorption of Native Oligonucleotides on Citrate-Coated Gold Nanoparticles.

The adsorption of oligonucleotides on citrate-coated gold nanoparticles (AuNPs) is studied under conditions "right after the synthesis", i.e., in a weak citrate solution at a pH value close to neutral (5.8 ± 0.2). We found that short-term elevation of reaction temperature under these conditions provides fast and strong adsorption of oligonucleotides on the surface of AuNPs. The affinity of oligonucleotides to AuNPs depends on the length of the oligonucleotide and its nucleotide composition. The shortest oligonucleotide in this study, T6, is the most affine, having the equilibrium binding constant KD = 0.10 ± 0.04 nM and the highest surface density-up to 200 molecules per one particle. Olygothymidylates are at least as affine to AuNPs as oligoadenylates, while oligocytidilates show the lowest affinity. We also studied the interaction of resulting DNA/AuNPs with a series of low- and high-molecular thiols, which provide a variety of operations with adsorbed oligonucleotides: displacement (complete or partial) and encapsulation in a secondary shell. These experiments imitate someway the conditions in a living cell or serum, and show that DNA/AuNPs obtained by this method can be applied in a number of bionanotechnological applications, including delivery of nucleic acid therapeutics and theranostics.

Multivalent Aptamers: Versatile Tools for Diagnostic and Therapeutic Applications.

Nucleic acid aptamers generated through an in vitro selection are currently extensively applied as very valuable biomolecular tools thanks to their prominent advantages. Diversity of spatial structures, ease of production through chemical synthesis and a large variety of chemical modifications make aptamers convenient building blocks for the generation of multifunctional constructs. An opportunity to combine different aptamer functionalities with other molecules of interest such as reporter groups, nanoparticles, chemotherapeutic agents, siRNA or antisense oligonucleotides provides a widest range of applications of multivalent aptamers. The present review summarizes approaches to the design of multivalent aptamers, various examples of multifunctional constructs and the prospects of employing them as components of biosensors, probes for affinity capture, tools for cell research and potential therapeutic candidates.

Aptamers Against Immunologic Targets: Diagnostic and Therapeutic Prospects.

The concept of in vitro selection of nucleic acid aptamers emerged 25 years ago, and since then tremendous progress has been achieved in the development of different aptamers and their applications for various bioanalytical and therapeutic purposes. Among other protein targets of aptamers, immune system proteins are of particular interest both as diagnostic markers and therapeutic targets. The present review summarizes up-to-date articles concerning the selection and design of DNA and RNA aptamers against immunologic targets such as antibodies, cytokines, and T-cell and B-cell receptors. We also discuss the prospects of employing aptamers as recognizing modules of diagnostic aptasensors, potential therapeutic candidates for the treatment of autoimmune diseases and cancer, and specific tools for functional studies of immune system proteins.

Efficient cleavage of DS DNA by bleomycin conjugated via hexaethylene glycol linker to triplex-forming oligonucleotides.

New conjugates of bleomycin A5 with oligonucleotides are synthesized. The bleomycin residue was attached to the 3'- or 5'- terminus of hexadecathymidilate via a hexaethylene glycol phosphate linker. Newly designed conjugates were shown to cleave site-specifically both strands of a dsDNA fragment within a triplex. The maximum extent of cleavage for individual strand amounts to 61%.

Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates.

Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.