Enhancing aptamer function and stability via in vitro selection using modified nucleic acids.

Nucleic acid aptamers have emerged as a promising alternative to antibodies for use as recognition elements in therapeutics, bioimaging, and analytical applications. A key benefit that aptamers possess relative to antibodies is their ability to be chemically synthesized. This advantage, coupled with the broad range of modified nucleotide building blocks that can be constructed using chemical synthesis, has enabled the discovery and development of modified aptamers having extraordinary affinity, specificity, and biostability. Early efforts to generate modified aptamers focused on selection of a native DNA or RNA aptamer, followed by post-selection trial-and-error testing of modifications. However, recent advances in polymerase engineering and templated nucleic acid synthesis have enabled the direct selection of aptamers having modified backbones and nucleobases. This review will discuss these technological advances and highlight the improvements in aptamer function that have been realized through in vitro selection of non-natural nucleic acids.

Synthesis and polymerase incorporation of ß,γ-modified α-l-threofuranosyl thymine triphosphate mimics.

Three ß,γ-modified α-l-threofuranosyl nucleoside triphosphates were synthesized. The ß,γ-modified tTTPs undergo a single incorporation event with HIV RT but undergo multiple incorporations to form full-length product with engineered thermophilic polymerases.

Detection of large rearrangements in a hereditary pan-cancer panel using next-generation sequencing.

BACKGROUND: Healthcare providers increasingly use information about pathogenic variants in cancer predisposition genes, including sequence variants and large rearrangements (LRs), in medical management decisions. While sequence variant detection is typically robust, LRs can be difficult to detect and characterize and may be underreported as a cause for hereditary cancer risk. This report describes the outcomes of hereditary cancer genetic testing using a comprehensive strategy that employs next-generation sequencing (NGS) for LR detection, coupled with LR confirmation using repeat hybrid capture NGS, microarray comparative genomic hybridization (microarray-CGH), and/or multiplex ligation-dependent probe amplification (MLPA). METHODS: Sequencing and LR analysis were conducted in a consecutive series of 376,159 individuals who received clinical testing with a hereditary pan-cancer gene panel from September 2013 through May 2017. NGS dosage analysis was used to evaluate potential deletions or duplications, with controls in place to exclude pseudogene reads. Samples positive for a putative LR based on NGS were confirmed using a comprehensive approach that included targeted microarray-CGH and/or MLPA analysis, with further examination as needed to ascertain the nature of the LR. RESULTS: A total of 3461 LRs were identified and classified as a deleterious mutation (DM), suspected deleterious mutation (SDM) or variant of uncertain significance. Pathogenic LRs (DM/SDM) accounted for the majority of LRs (67.7%), the largest proportion of which were deletions (86.1%), followed by duplications (11.3%), insertions (1.8%), triplications (0.5%), and inversions (0.3%). Several cases presented illustrate that the laboratory approach employed here can ensure consistent identification and accurate characterization of LRs. In the absence of this comprehensive testing strategy, 9% of LRs identified in this testing population might have been missed, potentially leading to inappropriate medical management in as many as 210 individuals referred for hereditary cancer testing. CONCLUSIONS: These data show that copy number analysis using NGS coupled with confirmatory testing reliably detects and characterizes LRs. Further, LRs comprise a substantial proportion (7.2%) of pathogenic variants identified by the test. A robust and accurate LR identification strategy is an essential component of a high-quality genetic testing program, enabling clinicians to optimize patient medical management decisions.

Fluorescent RNA labeling using self-alkylating ribozymes.

The ability to fluorescently label specific RNA sequences is of significant utility for both in vitro and live cell applications. Currently, most RNA labeling methods utilize RNA-nucleic acid or RNA-protein molecular recognition. However, in the search for improved RNA labeling methods, harnessing the small-molecule recognition capabilities of RNA is rapidly emerging as a promising alternative. Along these lines, we propose a novel strategy in which a ribozyme acts to promote self-alkylation with a fluorophore, providing a robust, covalent linkage between the RNA and the fluorophore. Here we describe the selection and characterization of ribozymes that promote self-labeling with fluorescein iodoacetamide (FIA). Kinetic studies reveal a second-order rate constant that is on par with those of other reactions used for biomolecular labeling. Additionally, we demonstrate that labeling is specific to the ribozyme sequences, as FIA does not react nonspecifically with RNA.