Letter Ruling 14-3: Application of Massachusetts sales tax to portable medical device under G.L. c. 64H, s. 6(l)

ISSN:0002-7863ISSN:1520-512

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

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

Selection and Characterization of a Novel DNA Aptamer for Label-Free Fluorescence Biosensing of Ochratoxin A

Nucleic acid aptamers are emerging as useful molecular recognition tools for food safety monitoring. However, practical and technical challenges limit the number and diversity of available aptamer probes that can be incorporated into novel sensing schemes. This work describes the selection of novel DNA aptamers that bind to the important food contaminant ochratoxin A (OTA). Following 15 rounds of in vitro selection, sequences were analyzed for OTA binding. Two of the isolated aptamers demonstrated high affinity binding and selectivity to this mycotoxin compared to similar food adulterants. These sequences, as well as a truncated aptamer (minimal sequence required for binding), were incorporated into a SYBR® Green I fluorescence-based OTA biosensing scheme. This label-free detection platform is capable of rapid, selective, and sensitive OTA quantification with a limit of detection of 9 nM and linear quantification up to 100 nM

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

Aptamer base: a collaborative knowledge base to describe aptamers and SELEX experiments

Over the past several decades, rapid developments in both molecular and information technology have collectively increased our ability to understand molecular recognition. One emerging area of interest in molecular recognition research includes the isolation of aptamers. Aptamers are single-stranded nucleic acid or amino acid polymers that recognize and bind to targets with high affinity and selectivity. While research has focused on collecting aptamers and their interactions, most of the information regarding experimental methods remains in the unstructured and textual format of peer reviewed publications. To address this, we present the Aptamer Base, a database that provides detailed, structured information about the experimental conditions under which aptamers were selected and their binding affinity quantified. The open collaborative nature of the Aptamer Base provides the community with a unique resource that can be updated and curated in a decentralized manner, thereby accommodating the ever evolving field of aptamer research

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

Screening and Initial Binding Assessment of Fumonisin B1 Aptamers

Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B1. Six unique sequences were obtained, each showing improved binding to fumonisin B1 compared to controls. Sequence FB1 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns