UTexas Aptamer Database: the collection and long-term preservation of aptamer sequence information.

A growing interest in aptamer research, as evidenced by the increase in aptamer publications over the years, has led to calls for a go-to site for aptamer information. A comprehensive, publicly available aptamer dataset, which may be a repository for aptamer data, standardize aptamer reporting, and generate opportunities to expand current research in the field, could meet such a demand. There have been several attempts to create aptamer databases; however, most have been abandoned or removed entirely from public view. Inspired by previous efforts, we have published the UTexas Aptamer Database, https://sites.utexas.edu/aptamerdatabase, which includes a publicly available aptamer dataset and a searchable database containing a subset of all aptamer data collected to date (1990-2022). The dataset contains aptamer sequences, binding and selection information. The information is regularly reviewed internally to ensure accuracy and consistency across all entries. To support the continued curation and review of aptamer sequence information, we have implemented sustaining mechanisms, including researcher training protocols, an aptamer submission form, data stored separately from the database platform, and a growing team of researchers committed to updating the database. Currently, the UTexas Aptamer Database is the largest in terms of the number of aptamer sequences with 1,443 internally reviewed aptamer records.

Systematic Review of Aptamer Sequence Reporting in the Literature Reveals Widespread Unexplained Sequence Alterations.

Aptamers have been the subject of more than 144 000 papers to date. However, there has been a growing concern that discrepancies in the reporting of aptamer research limit the reliability of these reagents for research and other applications. These observations noting inconsistencies in the use of our RNA antilysozyme aptamer served as an impetus for our systematic review of the reporting of aptamer sequences in the literature. Our detailed examination of the literature citing the RNA antilysozyme aptamer revealed that 93% of the 61 publications reviewed reported unexplained altered sequences with 96% of those using DNA variants. The 10 most cited aptamers were examined using a standardized methodology in order to categorize the extent to which the sequences themselves and altered sequences were adequately described in the literature. Our review of 780 aptamer publications spanned decades, multiple journals, and research groups and revealed that 41% of the papers reported unexplained sequence alterations or omitted sequences. We identified 10 common categories of sequence alterations including deletions, substitutions, and additions, among others. Overall, our findings can be used as a starting point for building better practices in author submissions and publication standards, elevating the rigor and reproducibility of aptamer research.

Aptamers in Education: Undergraduates Make Aptamers and Acquire 21st Century Skills Along the Way.

Aptamers have a well-earned place in therapeutic, diagnostic, and sensor applications, and we now show that they provide an excellent foundation for education, as well. Within the context of the Freshman Research Initiative (FRI) at The University of Texas at Austin, students have used aptamer selection and development technologies in a teaching laboratory to build technical and 21st century skills appropriate for research scientists. One of the unique aspects of this course-based undergraduate research experience is that students develop and execute their own projects, taking ownership of their experience in what would otherwise be a traditional teaching lab setting. Of the many successes, this work includes the isolation and characterization of novel calf intestinal alkaline phosphatase (anti-CIAP) RNA aptamers by an undergraduate researcher. Further, preliminary survey data suggest that students who participate in the aptamer research experience express significant gains in their self-efficacy to conduct research, and their perceived ability to communicate scientific results, as well as organize and interpret data. This work describes, for the first time, the use of aptamers in an educational setting, highlights the positive student outcomes of the aptamer research experience, and presents the research findings relative to the novel anti-CIAP aptamer.

Science Educational Outreach Programs That Benefit Students and Scientists.

Both scientists and the public would benefit from improved communication of basic scientific research and from integrating scientists into education outreach, but opportunities to support these efforts are limited. We have developed two low-cost programs--"Present Your PhD Thesis to a 12-Year-Old" and "Shadow a Scientist"--that combine training in science communication with outreach to area middle schools. We assessed the outcomes of these programs and found a 2-fold benefit: scientists improve their communication skills by explaining basic science research to a general audience, and students' enthusiasm for science and their scientific knowledge are increased. Here we present details about both programs, along with our assessment of them, and discuss the feasibility of exporting these programs to other universities.

In vitro selection using modified or unnatural nucleotides.

Incorporation of modified nucleotides into in vitro RNA or DNA selections offers many potential advantages, such as the increased stability of selected nucleic acids against nuclease degradation, improved affinities, expanded chemical functionality, and increased library diversity. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for evaluating and optimizing transcription reactions, as well as confirming the incorporation of the modified nucleotides; protocols for evaluating modified nucleotide transcripts as template in reverse transcription reactions; protocols for the evaluation of the fidelity of modified nucleotides in the replication and the regeneration of the pool; and a protocol to compare modified nucleotide pools and selection conditions.

Continuous in vitro evolution of a ribozyme ligase: a model experiment for the evolution of a biomolecule.

Evolution is a defining criterion of life and is central to understanding biological systems. However, the timescale of evolutionary shifts in phenotype limits most classroom evolution experiments to simple probability simulations. In vitro directed evolution (IVDE) frequently serves as a model system for the study of Darwinian evolution but produces noticeable phenotypic shifts in a matter of hours. An IVDE demonstration lab would serve to both directly demonstrate how Darwinian selection can act on a pool of variants and introduce students to an essential method of modern molecular biology. To produce an IVDE demonstration lab, continuous IVDE of a T500 ribozyme ligase population has been paired with a fluorescent strand displacement reporter system to visualize the selection of improved catalytic function. A ribozyme population is taken through rounds of isothermal amplification dependent on the self-ligation of a T7 promoter. As the population is selectively enriched with better ligase activity, the strand displacement system allows for the monitoring of the population's ligation rate. The strand displacement reporter system permits the detection of ligated ribozyme. Once ligated with the T7 promoter, the 5' end of the ribozyme displaces paired fluorophore-quencher oligonucleotides, in turn, generating visible signal upon UV light excitation. As the ligation rate of the population increases, due to the selection for faster ligating species, the fluorescent signal develops more rapidly. The pairing of the continuous isothermal system with the fluorescent reporting scheme allows any user, provided with minimal materials, to model the continuous directed evolution of a biomolecule.

Widespread reorganization of metabolic enzymes into reversible assemblies upon nutrient starvation.

Proteins are likely to organize into complexes that assemble and disassemble depending on cellular needs. When approximately 800 yeast strains expressing GFP-tagged proteins were grown to stationary phase, a surprising number of proteins involved in intermediary metabolism and stress response were observed to form punctate cytoplasmic foci. The formation of these discrete physical structures was confirmed by immunofluorescence and mass spectrometry of untagged proteins. The purine biosynthetic enzyme Ade4-GFP formed foci in the absence of adenine, and cycling between punctate and diffuse phenotypes could be controlled by adenine subtraction and addition. Similarly, glutamine synthetase (Gln1-GFP) foci cycled reversibly in the absence and presence of glucose. The structures were neither targeted for vacuolar or autophagosome degradation nor colocalized with P bodies or major organelles. Thus, upon nutrient depletion we observe widespread protein assemblies displaying nutrient-specific formation and dissolution.

Technical and biological issues relevant to cell typing with aptamers.

A number of aptamers have been selected against cell surface biomarkers or against eukaryotic tissue culture cells themselves. To determine the general utility of aptamers for assessing the cell surface proteome, we developed a standardized flow cytometry assay and carried out a comprehensive study with 7 different aptamers and 14 different cell lines. By examining how aptamers performed with a variety of cell lines, we identified difficulties in using aptamers for cell typing. While there are some aptamers that show excellent correlation between cell surface binding and the expression of a biomarker on the cell surface, other aptamers showed nonspecific binding by flow cytometry. For example, it has recently been claimed that an anti-PTK7 (protein tyrosine kinase 7) aptamer identified a new biomarker for leukemia cells, but data with the additional cell lines shows that it is possible that the aptamer instead identifies a propensity for adherence. Better understanding and controlling for the role of background and nonspecific binding to cells should open the way to using arrays of aptamers for describing and quantifying the cell surface proteome.

Aptamer therapeutics advance.

Aptamers are selected nucleic acid binding species with affinities and specificities for protein targets that rival those of monoclonal antibodies. Furthermore, aptamers have definite advantages over antibodies, in that they can be chemically synthesized and modifications can be introduced that improve their stabilities and pharmacokinetic properties. A number of aptamers against therapeutically important targets have shown efficacy in cell and animal models, and a handful of aptamers are now in clinical trials or are being used as drugs. Recent advances in selection technologies and a more thorough exploration of how to deliver nucleic acids to target cells and tissues should further speed the process of drug development.