Challenges and opportunities for small molecule aptamer development

Aptamers are single-stranded oligonucleotides that bind to targets with high affinity and selectivity. Their use as molecular recognition elements has emerged as a viable approach for biosensing, diagnostics, and therapeutics. Despite this potential, relatively few aptamers exist that bind to small molecules. Small molecules are important targets for investigation due to their diverse biological functions as well as their clinical and commercial uses. Novel, effective molecular recognition probes for these compounds are therefore of great interest. This paper will highlight the technical challenges of aptamer development for small molecule targets, as well as the opportunities that exist for their application in biosensing and chemical biology

Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O 6 -methylguanine in vivo

© 2018, Springer-Verlag GmbH Germany, part of Springer Nature. N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O 6 -methylguanine (O 6 -MeG), which is repaired by O 6 -methylguanine-DNA methyltransferase (MGMT). Persistent O 6 -MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O 6 -MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) to monitor O 6 -MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O 6 -MeG in CRC cells. Using the highly sensitive and specific UPLC–MS/MS, TMZ-induced O 6 -MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O 6 -MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC–MS/MS measurements and dose–response modeling revealed a non-linear formation of hepatic and colonic O 6 -MeG adducts in WT, whereas linear O 6 -MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC–MS/MS analysis is highly sensitive and specific for O 6 -MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O 6 -methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures

PARP-1 improves leukemia outcomes by inducing parthanatos during chemotherapy.

Previous chemotherapy research has focused almost exclusively on apoptosis. Here, a standard frontline drug combination of cytarabine and idarubicin induces distinct features of caspase-independent, poly(ADP-ribose) polymerase 1 (PARP-1)-mediated programmed cell death "parthanatos" in acute myeloid leukemia (AML) cell lines (n = 3/10 tested), peripheral blood mononuclear cells from healthy human donors (n = 10/10 tested), and primary cell samples from patients with AML (n = 18/39 tested, French-American-British subtypes M4 and M5). A 3-fold improvement in survival rates is observed in the parthanatos-positive versus -negative patient groups (hazard ratio [HR] = 0.28-0.37, p = 0.002-0.046). Manipulation of PARP-1 activity in parthanatos-competent cells reveals higher drug sensitivity in cells that have basal PARP-1 levels as compared with those subjected to PARP-1 overexpression or suppression. The same trends are observed in RNA expression databases and support the conclusion that PARP-1 can have optimal levels for favorable chemotherapeutic responses

Development of a DNA aptamer for direct and selective homocysteine detection in human serum

l-Homocysteine has been an amino acid intermediate of interest for over 20 years due to its implication in various adverse health conditions, including cardiovascular disease. Here, we report the first in vitro selection and application of high affinity aptamers for the target l-homocysteine. Two novel aptamer sequences were selected following 8 rounds of selection that displayed high affinity binding and selectivity to homocysteine compared to other amino acids. One of the selected aptamers, Hcy 8 (KD = 600 ± 300 nM), was used to develop a gold-nanoparticle biosensor capable of sensitive and selective homocysteine detection in human serum, with a limit of detection of 0.5 μM and a linear range of 0.5-3.0 μM. This biosensor allows rapid detection of free homocysteine in human serum samples at low cost, with little preparation time and could be adapted to be part of a po

Determination of ochratoxin A in wheat after clean-up through a DNA aptamer-based solid phase extraction column

A DNA aptamer with high affinity and specificity to ochratoxin A (OTA) was conjugated to a coupling gel and used as sorbent for the prepa

Screening and Identification of DNA Aptamers to Tyramine Using in Vitro Selection and High-Throughput Sequencing

Aptamers are synthetic single-stranded DNA or RNA sequences that can fold into tertiary structures allowing them to interact with and bind to targets with high affinity and specificity. This paper describes the first selection and identification of DNA aptamers able to recognize the biogenic amine tyramine. To successfully isolate aptamers to this challenging small molecule target, the SELEX methodology was adapted by combining a systematic strategy to increase the selection stringency and monitor enrichment success. As the benefits of applying high-throughput sequencing (HTS) in SELEX experiments is becoming more clear, this method was employed in combination with bioinformatics analysis to evaluate the utility of the selection strategy and to uncover new potential high affinity sequences. On the basis of the presence of consensus regions (sequence families) and family similarities (clusters), 15 putative aptamers to tyramine were identified. A recently described workflow approach to perform a primary screening and characterization of the aptamer candidates by microequilibrium dialysis and by microscale thermophoresis was next leveraged. These candidate aptamers exhibited dissociation constant (Kd) values in the range of 0.2-152 μM with aptamer Tyr-10 as the most promising one followed by aptamer Tyr-14. These aptamers could be used as promising molecular recognition tools for the development of inexpensive, robust and innovative biosensor platforms for the detection of tyramine in food and beverages

Quantification and Mapping of Alkylation in the Human Genome Reveal Single Nucleotide Resolution Precursors of Mutational Signatures

Chemical modifications to DNA bases, including DNA adducts arising from reactions with electrophilic chemicals, are well-known to impact cell growth, miscode during replication, and influence disease etiology. However, knowledge of how genomic sequences and structures influence the accumulation of alkylated DNA bases is not broadly characterized with high resolution, nor have these patterns been linked with overall quantities of modified bases in the genome. For benzo(a) pyrene (BaP), a ubiquitous environmental carcinogen, we developed a single-nucleotide resolution damage sequencing method to map in a human lung cell line the main mutagenic adduct arising from BaP. Furthermore, we combined this analysis with quantitative mass spectrometry to evaluate the dose-response profile of adduct formation. By comparing damage abundance with DNase hypersensitive sites, transcription levels, and other genome annotation data, we found that although overall adduct levels rose with increasing chemical exposure concentration, genomic distribution patterns consistently correlated with chromatin state and transcriptional status. Moreover, due to the single nucleotide resolution characteristics of this DNA damage map, we could determine preferred DNA triad sequence contexts for alkylation accumulation, revealing a characteristic DNA damage signature. This new BaP damage signature had a profile highly similar to mutational signatures identified previously in lung cancer genomes from smokers. Thus, these data provide insight on how genomic features shape the accumulation of alkylation products in the genome and predictive strategies for linking single-nucleotide resolution in vitro damage maps with human cancer mutations.ISSN:2374-795

Comprehensive Analytical Comparison of Strategies Used for Small Molecule Aptamer Evaluation

Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high selectivity and affinity. The past few years have seen a dramatic increase in aptamer development and interest for diagnostic and therapeutic applications. As the applications for aptamers expand, the need for a more standardized, stringent, and informative characterization and validation methodology increases. Here we performed a comprehensive analysis of a panel of conventional affinity binding assays using a suite of aptamers for the small molecule target ochratoxin A (OTA). Our results highlight inconsistency between conventional affinity assays and the need for multiple characterization strategies. To mitigate some of the challenges revealed in our head-to-head comparison of aptamer binding assays, we further developed and evaluated a set of novel strategies that facilitate efficient screening and characterization of aptamers in solution. Finally, we provide a workflow that permits rapid and robust screening, characterization, and functional verification of aptamers thus improving their development and integration into novel applications

Analysis of In Vitro Aptamer Selection Parameters

Nucleic acid aptamers are novel molecular recognition tools that offer many advantages compared to their antibody and peptide-based counterparts. However, challenges associated with in vitro selection, characterization, and validation have limited their wide-spread use in the fields of diagnostics and therapeutics. Here, we extracted detailed information about aptamer selection experiments housed in the Aptamer Base, spanning over two decades, to perform the first parameter analysis of conditions used to identify and isolate aptamers de novo. We used information from 492 published SELEX experiments and studied the relationships between the nucleic acid library, target choice, selection methods, experimental conditions, and the affinity of the resulting aptamer candidates. Our findings highlight that the choice of target and selection template made the largest and most significant impact on the success of a de novo aptamer selection. Our results further emphasize the need for improved documentation and more thorough experimentation of SELEX criteria to determine their correlation with SELEX success

The minimum aptamer publication standards (MAPS guidelines) for de novo aptamer selection

Aptamers were first described in 1990 and since then many aptamers have been reported in the literature for numerous applications in both diagnostics and therapeutics. However, as with most fields, missing or unclear information presented in the publication makes it difficult to replicate some of the work described in the literature. To increase the reproducibility of the data and facilitate academic laboratories and industrial companies to develop reliable aptamer work, essential guidelines should be proposed and followed in any aptamer publication, especially in those that highlight de novo aptamer sequences. Here, we provide suggestions for authors, reviewers, and editors to follow when performing and reporting their aptamer work to ensure that we meet the minimum standards for publication of future aptamer sequences