Aptamer Library Research Articles

Screening and selection of artificial riboswitches

Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher percentage of potential ligand binders. The study was designed to produce structure switching aptamers, a necessary feature for riboswitch function and efficiently quantify this function using the dual color reporter system.

Imaging Agent for Redox‐Active Molecules

Redox‐active and cofactor molecules (such as glutathione, NAD+/NADH, and NADP+/NADPH) are involved in a wide array of important biological reactions. Their cellular concentrations are maintained within a range of values, and an imbalance is often linked to decreased cellular defense capability against oxidative stress. Currently, the most common techniques for quantifying these molecules require cell lysis. The goal of this project is to design sensor molecules that can be used in live cells. Our approach relies on the use of oligodeoxynucleotide strands (called aptamers) as the recognition moiety for the redox compound of interest. Aptamers, when selected for a specific target molecule, can display selectivity and affinity rivalling those of antibodies. Conjugation of the aptamer to a xenon‐containing organic molecule will yield an imaging agent that will report the concentration of the target as a measurable frequency value in the xenon nuclear magnetic resonance.Isolation of aptamers for a target molecule is achieved via an iterative in vitro selection technique called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Starting from a large pool of combinatorial library of aptamers (70‐base long strands containing 30 randomized bases), each iteration consists of: 1) incubation with the target molecule, 2) partitioning of the target‐binding and non‐binding aptamers, 3) amplification of the binding aptamers through polymerase chain reaction (PCR), and 4) separation of the amplicons into single‐stranded aptamers. This newly generated pool is then used as the starting library for the consecutive SELEX cycle. In the last round, the aptamer pool is cloned in E.coli and the sequence of each target binding strand is elucidated.The magnetic resonance frequency reporter is composed of 129Xe atom encapsulated in an organic ‘cage’ molecule. Starting from commercially available precursors, the synthesis of the Xe‐based component follows an established multi‐step organic synthesis procedure. In the final stage, conjugation of the aptamer to the organic ‘cage’ bearing the Xe atom is achieved via Huisgen 1,3‐dipolar cycloaddition or carboxylic acid‐amine condensation reaction.Toward this end, we have completed several rounds of SELEX and closely monitored the progress of each cycle. We started by incubating the aptamer pool with a high concentration of glutathione (10 mM), and observed aptamer binding to the target after several rounds. We have subsequently increased the selection pressure by gradually reducing the glutathione solution to 0.5 mM. At the same time, we have successfully completed several synthetic steps toward the Xe organic ‘cage’ molecule. The product of each step was fully characterized using standard organic chemistry methods.Upon completion of our project, we will have developed a magnetic resonance imaging (MRI) agent for glutathione which will be extended to other redox‐active and cofactor molecules.Support or Funding InformationFunding for the project came from the California State University East Bay (CSUEB) Faculty Support Grant. D.S. was a recipient of the CSUEB Center for Student Research Scholarship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P5-08-03: Poly-ligand profiling and target identification from formalin-fixed-paraffin embedded HER2+ breast cancer specimens

Abstract We have previously described the ADAPT Biotargeting System™as a novel platform for highly multiplexed poly-ligand profiling of complex phenotypes such as drug response. Here we report extended capabilities of the this platform for target identification directly from formalin-fixed-paraffin embedded (FFPE) tissues using aptamer libraries enriched toward HER2+ breast cancer. Standard mass spectrometry-based biomarker and drug target discovery from FFPE tissues can be challenging due to limited amounts of tissue, harsh conditions of fixation and extraction and the general problem of masking by highly abundant proteins. A single stranded-oligodeoxynucleotide aptamer library was enriched on HER2+ FFPE breast cancer specimens and conjugated with biotin as well as a label transfer reagent, Sulfo-NHS-SS-Diazirine (Sulfo-SDAD). The biotinylated-SDAD conjugated library (B-SDAD-EL) was applied to HER2+ FFPE tissues and photocrosslinked to cognate binding partners within the FFPE sample in order to preserve aptamer-protein interactions under harsh denaturing conditions required for protein extraction and sample preparation. Aptamer-protein complexes were affinity purified and the label was transferred from bound aptamers to their binding partners under reducing conditions that enable proteomic digestion and high resolution mass spectrometry detection. An open database search was performed where the precursor ion tolerance was set to ± 500 Da for database searching, which enabled identification of peptides containing the transferred label as well as additional unknown variable modifications induced by the tissue fixation process. We identified proteins with known roles in HER2+ breast cancer along with several potentially drugable targets not previously associated with HER2 positivity. Differential expression of candidate targets was orthogonally confirmed by immunohistochemistry. By nature of its extreme molecular complexity and its ability to be enriched or “trained,” toward phenotypes of interest, the ADAPT Biotargeting SystemTM can be deployed to advance precision medicine by identifying predictive biomarkers and drug targets with novel associations to complex interactomes. Citation Format: O'Neill HA, Tinder TT, Maher V, Rosenow M, Richards M, Santhanam R, Wei X, Domenyuk V, Miglarese MR, Spetzler D. Poly-ligand profiling and target identification from formalin-fixed-paraffin embedded HER2+ breast cancer specimens [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-08-03.

A peptide derived from enzymatic digestion of globulins from amaranth shows strong affinity binding to the replication origin of Tomato yellow leaf curl virus reducing viral replication in Nicotiana benthamiana

Tomato yellow leaf curl virus (TYLCV; genus Begomovirus; family Geminiviridae) infects mainly plants of the family Solanaceae, and the infection induces curling and chlorosis of leaves, dwarfing of the whole plant, and reduced fruit production. Alternatives for direct control of TYLCV and other geminiviruses have been reported, for example, the use of esterified whey proteins, peptide aptamer libraries or artificial zinc finger proteins. The two latter alternatives affect directly the replication of TYLCV as well as of other geminiviruses because the replication structures and sequences are highly conserved within this virus family. Because peptides and proteins offer a potential solution for virus replication control, in this study we show the isolation, biochemical characterization and antiviral activity of a peptide derived from globulins of amaranth seeds (Amaranthus hypochondriacus) that binds to the replication origin sequence (OriRep) of TYLCV and affects viral replication with a consequent reduction of disease symptoms in Nicotiana benthamiana. Aromatic peptides obtained from papain digests of extracted globulins and albumins of amaranth were tested by intrinsic fluorescent titration and localized surface resonance plasmon to analyze their binding affinity to OriRep of TYLCV. The peptide AmPep1 (molecular weight 2.076 KDa) showed the highest affinity value (Kd = 1.8 nM) for OriRep. This peptide shares a high amino acid similarity with a part of an amaranth 11S globulin, and the strong affinity of AmPep1 could be explained by the presence of tryptophan and lysine facilitating interaction with the secondary structure of OriRep. In order to evaluate the effect of the peptide on in vitro DNA synthesis, rolling circle amplification (RCA) was performed using as template DNA from plants infected with TYLCV or another begomovirus, pepper huasteco yellow vein virus (PHYVV), and adding AmPep1 peptide at different concentrations. The results showed a decrease in DNA synthesis of both viruses at increasing concentrations of AmPep1. To further confirm the antiviral activity of the peptide in vivo, AmPep1 was infiltrated into leaves of N. benthamiana plants previously infected with TYLCV. Plants treated with AmPep1 showed a significant decrease in virus titer compared with untreated N. benthamiana plants as well as reduced symptom progression due to the effect of AmPep1 curtailing TYLCV replication in the plant. The peptide also showed antiviral activity in plants infected with PHYVV. This is the first report, in which a peptide is directly used for DNA virus control in plants, supplied as exogenous application and without generation of transgenic lines.

Aptamers as biomarkers for neurological disorders. Proof of concept in transgenic mice.

The act of selecting aptamers against blood serum leads to deep libraries of oligonucleotide sequences that bind to a range of epitopes in blood. In this study we developed an enriched aptamer library by performing positive selection against a pool of blood serum samples from transgenic mice (P301S) carrying the human tau gene and counter selecting against pooled blood serum from negative segregant (wild type) mice. We demonstrated that a large proportion of the aptamer sequences observed with next generation sequence (NGS) analysis were the same from selection round 5 and selection round 6. As a second step, we applied aliquots of the selection round 5 enriched library to blood serum from 16 individual mice for a single round of selection. Each of these individual libraries were characterized through NGS analysis and the changes in relative frequency in aptamers from transgenic mice versus wild type were used to construct a diagnostic fingerprint of the effect of the action of the transgene on the composition of blood serum. This study serves as a model for similar applications with human subjects.

Targeting Inhibition of SmpB by Peptide Aptamer Attenuates the Virulence to Protect Zebrafish against Aeromonas veronii Infection.

Aeromonas veronii is an important pathogen of aquatic animals, wherein Small protein B (SmpB) is required for pathogenesis by functioning as both a component in stalled-ribosome rescue and a transcription factor in upregulation of virulence gene bvgS expression. Here a specific peptide aptamer PA-1 was selected from peptide aptamer library by bacterial two-hybrid system employing pBT-SmpB as bait. The binding affinity between SmpB and PA-1 was evaluated using enzyme-linked immunosorbent assay. The key amino acids of SmpB that interact with PA-1 were identified. After PA-1 was introduced into A. veronii, the engineered strain designated as A. veronii (pN-PA-1) was more sensitive and grew slower under salt stress in comparison with wild type, as the disruption of SmpB by PA-1 resulted in significant transcription reductions of virulence-related genes. Consistent with these observations, A. veronii (pN-PA-1) was severely attenuated in model organism zebrafish, and vaccination of zebrafish with A. veronii (pN-PA-1) induced a strong antibody response. The vaccinated zebrafish were well protected against subsequent lethal challenges with virulent parental strain. Collectively, we propose that targeting inhibition of SmpB by peptide aptamer PA-1 possesses the desired qualities for a live attenuated vaccine against pathogenic A. veronii.

Abstract 2754: A novel liquid biopsy method for development of aptamer libraries that bind blood plasma exosomes from breast cancer patients

Abstract Improved technologies capable of characterizing system-wide changes associated with complex diseases will be required to be able to detect millions of proteins and their isoforms as well as multi-molecular complexes. We present a method for developing aptamer libraries using blood plasma exosomes that provides unprecedented system-wide coverage of native exosomal complexes. To train a naïve aptamer library toward cancer samples (positive selection), the library (~1013 biotinylated ssODN species) was incubated with plasma from individual cancer patients and aptamer-bound exosomes were isolated using polymer-based precipitation. Negative selection was performed by contacting the aptamer library with exosomes from donors without breast cancer and recovering unbound aptamers from the supernatant. In all, 12 libraries trained toward 12 individual breast cancer patients were used to probe additional samples. Exosome-bound aptamers were identified and quantified by Next Generation Sequencing (NGS) to build highly accurate signatures for cancer/healthy donor classification. Using these signatures, cancer patients’ binding profiles were easily distinguishable from controls. Interestingly, cancer-trained libraries did not distinguish any of the negative control samples from each other, indicating that the selection pressure for cancer was high and noise due to inherent inter-healthy donor heterogeneity was minimal. Full validation studies are ongoing. Aptamer libraries may ultimately be deployed as a minimally-invasive diagnostic adjunct in breast and other cancers. Citation Format: Valeriy Domenyuk, Symon Levenberg, Adam Stark, Mark Miglarese, David Spetzler. A novel liquid biopsy method for development of aptamer libraries that bind blood plasma exosomes from breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2754. doi:10.1158/1538-7445.AM2017-2754

Selection and characterization of a DNA aptamer inhibiting coagulation factor XIa

Factor XIa (FXIa) is a serine protease that catalyzes the activation of Factor IX (FIX) in the blood coagulation cascade. FXIa and its precursor FXI are emergent therapeutic targets for the development of safer anticoagulant agents. Here, we sought a novel DNA-based agent to inhibit FXIa. Towards this goal, an 80 base, single-stranded DNA aptamer library (containing a 40 base randomized core) was screened for FXIa-binding candidates, using ten rounds of positive and negative selection. After selection, 6 of 89 different sequences inhibited FXIa-mediated chromogenic substrate S2366 cleavage. The most active anti-FXIa aptamer had a hypervariable central sequence 5′-AACCTATCGGACTATTGTTAGTGATTTTTATAGTGT-3′ and was designated Factor ELeven Inhibitory APtamer (FELIAP). FELIAP, but not a scrambled aptamer control (SCRAPT), competitively inhibited FXIa-catalyzed S2366 cleavage, FIX activation, and complex formation with antithrombin. No effect of FELIAP on FXI activation was observed. FELIAP inhibited plasma clotting and thrombin generation assays to a significantly greater extent than SCRAPT. Immobilized FELIAP bound FXIa with strong affinity and an equilibrium binding constant (KD) in the low nanomolar range determined using surface plasmon resonance. FELIAP is the first FXIa-inhibitory aptamer to be described and constitutes a lead compound to develop related aptamers for in vivo use.

Abstract P4-12-08: Use of an aptamer library based next generation omics platform for the development of a novel trastuzumab predictive assay

Abstract Introduction: Previous attempts to use individual aptamers as diagnostic reagents have failed to consistently achieve performance comparable to antibodies. Here we report a novel systems biology approach using poly-ligand aptamer libraries to identify responders and non-responders to traztuzumab-based regimens in metastatic breast cancer. Methods: To overcome the fundamental limitation of the individual aptamer binding affinities, large libraries (106 species) were created so that potentially thousands of aptamers could bind to each of a multitude of targets related to the whole cellular changes in response to trastuzumab therapy. A set of breast cancer patients, which received trastuzumab mono- or combined therapy for at least 7 months were classified as “Responders” (R); cases with particular regimen discontinued in the period not exceeding 5 months were classified as “Non-Responders”(NR). A library of 2x1012 unique 90-mer ssDNA oligodeoxynucleotides (ssODN) was trained on FFPE tissue of both R and NR patients. Partitioning of aptamer libraries was done by microdissection of the tumor tissue, after incubation of aptamer library with the entire tissue section, to drive selection pressure toward cancer cells. A total of 10 cases of R and NR, 6 Her2+ cases each, were used to train separate aptamer libraries, with 1 positive and 2 counter selection cases per enrichment. Enriched libraries were screened on 20 R and 20 NR cases (11 Her2+ cases each) by adopting modified immunohistochemistry protocol. Each library was used as an independent reagent (similar to an antibody in IHC) across all 40 cases to evaluate the efficacy of the aptamer library to distinguish differences between the R and NR groups. Staining (DAB chromogen) profiles were scored from 0 to 3+ (nuclear and cytoplasmic staining) by a pathologist without any knowledge of the clinical outcomes. Initial validation was done by t-test using raw histological scores. Four libraries showed significant p-values between groups of responders and non-responders, a classification algorithm was constructed and evaluated using area under the receiver-operator characteristic curve (AUC). The datasets of two best-performing libraries were combined into one model using logistic regression to further improved the classifier performance. Results: Of seventeen trained libraries, eight were evaluated and four showed significant correlation to clinical benefit with a minimum accuracy of 75% for each library when evaluated independently. Furthermore, two libraries showed exceptional performance (ROC curve AUC of 0.86 and 0.77). Combination of the profiling data from these two libraries using logistic regression resulted in an AUC of 0.985. A prospective validation of aptamer histochemical theranostic testing has been initiated. Summary: Enriched aptamer libraries appear to distinguish trastuzumab responsiveness in metastatic breast cancer. This technology could be used as an additional technique beyond FISH testing to determine sensitivity to anti-HER2 agents. The demonstrated platform is applicable to virtually any disease where the safe and effective use of corresponding drug is yet to be improved. Citation Format: Spetzler D, Domenyuk V, Santhanam R, Wei X, Stark A, Wang J, Gatalica Z, Miglarese M, Vidal G, Schwartzberg LS. Use of an aptamer library based next generation omics platform for the development of a novel trastuzumab predictive assay [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-08.

Cell-SELEX Identifies a "Sticky" RNA Aptamer Sequence.

Cell-SELEX is performed to select for cell binding aptamers. We employed an additional selection pressure by using RNAse to remove surface-binding aptamers and select for cell-internalizing aptamers. A common RNA sequence was identified from independent cell-SELEX procedures against two different pancreatic cancer cell lines, indicating a strong selection pressure towards this sequence from the large pool of other available sequences present in the aptamer library. The aptamer is not specific for the pancreatic cancer cell lines, and a similar sequence motif is present in previously published internalizing aptamers. The identified sequence forms a structural motif that binds to a surface protein, which either is highly abundant or has strong affinity for the selected aptamer sequence. Deselecting (removing) this sequence during cell-SELEX may increase the probability of identifying aptamers against cell type-specific targets on the cell surface.

Rapid Selection of RNA Aptamers that Activate Fluorescence of Small Molecules.

RNA aptamers can serve as valuable tools for studying and manipulating live cells. Fluorescent aptamers are the ones that bind to and turn on fluorescence of small-molecule dyes (fluorogens). Similarly to fluorescent proteins, fluorescent RNA aptamers can be used to image spatial and temporal RNA dynamics in live cells. Additionally, these aptamers can serve as a basis for engineering genetically encoded fluorescent biosensors. This chapter presents a protocol for rapid and efficient screening of RNA aptamer libraries to isolate fluorescent aptamers. The protocol describes how to design, clone, and express RNA aptamer library in bacterial cells and how to screen the bacteria to find aptamers with the desired fluorescent properties.

Novel IL-2Ralpha (CD25)-Binding RNA Aptamer to Target T Regulatory Cells

Background:RNA aptamers are short RNA molecules that bind to antigens and ligands in a manner analogous to antibodies. RNA aptamers are being evaluated as clinical therapeutic agents based on their advantages and flexibility as cell targeting agents. Here, we report development and evaluation of a novel human IL-2Ralpha (CD25)-binding RNA aptamer that can be used to target T regulatory (Treg) cells.Methods:A. RNA aptamers:A whole cell SELEX strategy was designed to enrich for RNA aptamers that specifically bind to CD4+CD25high Tregs cells, but not to lineage-related CD4+CD25-T effector (Teff) cells. Selection began with a library of random RNA aptamers (SEL2-N20) with an expected structural diversity of ~1012. The aptamer pool was pre-cleared of aptamers that bound to common T cell antigens using Teff cells before aptamers were positively selected that bound to Tregs from the same donor. This process was repeated for eight successive enrichment rounds, each round using T cells from different donor. Enriched aptamers were sequenced and the top enriched aptamer (Tr-1) was characterized for its antigen specificity.B. Target antigen identification:The specificity of Tr-1 aptamer and its structural mutants was evaluated by testing its binding to recombinant CD25 by RT-qPCR.C. Targeting Tregs with chimeric Tr-1:Chimeric Treg-targeting reagents were created by linking Tr-1 aptamers with cytotoxic Saporin (Tr-1-Sap) or with Treg-inhibiting Foxp3 silencing RNA (Tr-1-Foxp3 siRNA). Unfractionated CD4+ T cells or Tregs were treated with Tr-1-Sap or Tr-1-Foxp3 siRNA. Reduction in Treg percentage (for Tr-1-Sap treatment) or Foxp3 mRNA levels (for Tr-1-siRNA treatment) was determined by flow cytometry or RT-qPCR, respectively.Results:A. Treg-binding RNA aptamers:A large panel of Treg-binding RNA aptamers was identified using whole cell SELEX and were synthesized. Most of the selected aptamers, particularly Tr-1, bound to CD25high Treg cells to a greater degree than to CD25low Teff cells (Figure 1).B. Target antigen identification:Tr-1 bound to human CD25 (IL-2Ralpha) protein while a control aptamer (C-248) did not. Structural mutants of Tr-1 that had lost Treg binding ability showed significantly reduced binding to CD25 protein (Figure 2).C. Treg targeting with chimeric Tr-1:Treg-targeting agents created with Tr-1 aptamers successfully delivered toxic Saporin and Foxp3 siRNA into Tregs.a. CD4+ T cells treated with Tr-1-Sap had a decrease in the percentage of CD25+ Treg population as determined by flow cytometry.b. Enriched human Tregs treated with Tr-1-Foxp3 siRNA chimera showed reduction in their Foxp3 mRNA levels.Conclusion and significance:RNA aptamers that target human Tregs were identified. The most predominant Treg-binding aptamer, Tr-1, binds to human IL-2Ralpha/CD25, a clinically-targeted molecule expressed by Tregs. Chimeric reagents based on Tr-1 aptamer effectively targeted Tregs signifying their potential use as novel immunomodulators. Ongoing studies are further exploring the significance of Tr-1 aptamer as a diagnostic agent and as a therapeutic modulator of Treg activity. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Morph-X-Select: Morphology-based tissue aptamer selection for ovarian cancer biomarker discovery.

High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering an aptamer affinity reagent and its target biomarker protein. Here, we demonstrate a morphology-based tissue aptamer selection method that enables us to use tissue sections from individual patients and identify high-affinity aptamers and their associated target proteins in a systematic and accurate way. We created a combinatorial DNA aptamer library that has been modified with thiophosphate substitutions of the phosphate ester backbone at selected 5´dA positions for enhanced nuclease resistance and targeting. Based on morphological assessment, we used image-directed laser microdissection (LMD) to dissect regions of interest bound with the thioaptamer (TA) library and further identified target proteins for the selected TAs. We have successfully identified and characterized the lead candidate TA, V5, as a vimentin-specific sequence that has shown specific binding to tumor vasculature of human ovarian tissue and human microvascular endothelial cells. This new Morph-X-Select method allows us to select high-affinity aptamers and their associated target proteins in a specific and accurate way, and could be used for personalized biomarker discovery to improve medical decision-making and to facilitate the development of targeted therapies to achieve more favorable outcomes.

Genetic Selection of Peptide Aptamers That Interact and Inhibit Both Small Protein B and Alternative Ribosome-Rescue Factor A of Aeromonas veronii C4.

Aeromonas veronii is a pathogenic gram-negative bacterium, which infects a variety of animals and results in mass mortality. The stalled-ribosome rescues are reported to ensure viability and virulence under stress conditions, of which primarily include trans-translation and alternative ribosome-rescue factor A (ArfA) in A. veronii. For identification of specific peptides that interact and inhibit the stalled-ribosome rescues, peptide aptamer library (pTRG-SN-peptides) was constructed using pTRG as vector and Staphylococcus aureus nuclease (SN) as scaffold protein, in which 16 random amino acids were introduced to form an exposed surface loop. In the meantime both Small Protein B (SmpB) which acts as one of the key components in trans-translation, and ArfA were inserted to pBT to constitute pBT-SmpB and pBT-ArfA, respectively. The peptide aptamer PA-2 was selected from pTRG-SN-peptides by bacterial two-hybrid system (B2H) employing pBT-SmpB or pBT-ArfA as baits. The conserved sites G133K134 and D138K139R140 of C-terminal SmpB were identified by interacting with N-terminal SN, and concurrently the residue K62 of ArfA was recognized by interacting with the surface loop of the specific peptide aptamer PA-2. The expression plasmids pN-SN or pN-PA-2, which combined the duplication origin of pRE112 with the neokanamycin promoter expressing SN or PA-2, were created and transformed into A. veronii C4, separately. The engineered A. veronii C4 which endowing SN or PA-2 expression impaired growth capabilities under stress conditions including temperatures, sucrose, glucose, potassium chloride (KCl) and antibiotics, and the stress-related genes rpoS and nhaP were down-regulated significantly by Quantitative Real-time PCR (qRT-PCR) when treating in 2.0% KCl. Thus, the engineered A. veronii C4 conferring PA-2 expression might be potentially attenuated vaccine, and also the peptide aptamer PA-2 could develop as anti-microbial drugs targeted to the ribosome rescued factors in A. veronii.

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes.

In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques.

Abstract 3913: Conjugate-SELEX, a novel screening method, identifies aptamers that deliver payload to the cytosol of target cells

Abstract Introduction: Delivery of a nanoparticle payload to a specific cellular compartment is a prized goal of cancer nanomedicine. The well-known SELEX (systematic evolution of ligands by exponential enrichment) process is often used to identify aptamers that localize to target cellular compartments, but such aptamers cannot be guaranteed to traffic an attached payload nanoparticle to the target as well. We hypothesized that to find an aptamer capable of trafficking a nanoparticle to a specific cellular compartment, the screened library must consist of aptamer-nanoparticle conjugates. We therefore designed a screening procedure we call “Conjugate-SELEX”. In this process, the aptamer library is conjugated to liposomal nanoparticles, creating a nanoparticle-aptamer conjugate library for screening. At each round of Conjugate-SELEX, aptamers recovered from the desired cellular compartment are amplified, and used to create the conjugate library for the next round of Conjugate-SELEX. Negative screens against off-target cells (e.g. hepatocytes, unwanted uptake in which, causes many of the toxicities associated with nanoparticle therapeutics) facilitate the identification of an aptamer that efficiently transports payload into target cells while minimizing the uptake in off-target cells. Using fluorescently tagged particles, imaging the cells at each stage verifies the successful transport of the payload particles into the cytosol. Methods: We used conjugate-SELEX to screen for aptamers that carried an attached nanoparticle payload to the cytosol of UM-SCC-22A oral cancer cell line, while not being taken up by THLE-3 hepatocytes. IonTorrent sequencing of the remaining aptamers after each round demonstrated convergence to a small family of sequences with only one base difference between members. Mass spectrometric proteomics identified the surface receptor bound by these sequences as the neuroblast differentiation-associated protein AHNAK (desmoyokin) a ubiquitous intracellular protein with surface expression exclusively in certain epithelial cell types. Uptake studies with concertedly synthesized hit aptamer sequences showed enhanced uptake of targeted nanoparticles over controls, and increased cytotoxicity of doxorubicin in the nanoparticles over controls. Conclusions: Conjugate-SELEX is an excellent means of identifying aptamers with the ability to transport attached nanoparticle payloads to targeted cellular compartments, while minimizing off target effects. The technique also lends itself to the identification of surface receptors mediating the trafficking.[D.G., A.A., and N.V. contributed equally to this work.] Citation Format: Qingshan Mu, Akshaya Annapragada, Mayank Srivastava, Varatharasa Thiviyanathan, Xin Li, David Gorenstein, Annanth Annapragada, Nadarajah Vigneswaran. Conjugate-SELEX, a novel screening method, identifies aptamers that deliver payload to the cytosol of target cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3913.

Abstract LB-135: Adaptive dynamic artificial poly-ligand targeting (ADAPT) enables plasma-based exosome profiling with potential diagnostic utility

Abstract Introduction: Single stranded DNA (ssDNA) libraries consisting of several trillion oligodeoxynucleotides (ODNs) can adopt a nearly infinite number of three-dimensional structures. These structures can potentially bind any biomolecule and can be screened for specificity toward important biomarkers by employing suitable enrichment schemes. Since no prior knowledge on the binding partner is required, massively parallel biomarker identification is possible even on complex matrices like biological fluids and across a wide range of biological conditions. Here we present Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT) as a platform for biomarker and target discovery. We employed ADAPT for the molecular profiling of exosome-associated proteins in small volume plasma samples from women with breast cancer and healthy donors. Results: Random ssDNA-libraries of 1011 unique ODNs were subjected to a number of selection and counter-selection steps on pooled blood plasma of breast cancer and healthy women. Several positive and negative enrichment schemes were employed, and exosome isolation and ODNs library partitioning were performed by ultracentrifugation and/or PEG precipitation. After library enrichment reduction of complexity to 106-107), ODN libraries were used to probe an independent set of individual plasma samples from women with or without breast cancer. Two thousand differentially-binding aptamers with significant p-values were re-synthesized and combined in equimolar amounts to create a profiling library (L2000). The L2000 library was used to probe plasma samples from 323 individuals (206 from breast cancer patients and 117 from healthy donors) in triplicate. Using Next Generation Sequencing, we quantitated bound ODN from each plasma sample. ANOVA revealed 350 aptamers with significant p-values in distinguishing plasma samples from cancer patients and healthy donors, far in excess of the number of ODNs that would have achieved statistical significance by random sampling of the 2000 ODNs. Generalized linear model showed an AUC in a ROC curve of 0.94 for the training set. Random forest modelling was used to assess classification performance and revealed an AUC of 0.73 (p<0.002 in a permutation test) in Out-of-bag validation. A more strict 10-fold cross-validation yielded an AUC of 0.63 (p<0.01 in a permutation test). Conclusions: We have demonstrated the feasibility of aptamer library enrichment directly on blood plasma and have identified a set of 2000 DNA aptamers that distinguish plasma from women with breast cancer from women without breast cancer. This liquid biopsy approach requires only 200 microliters of plasma and is amenable to high-throughput processing. By employing a number of statistical approaches including rigorous cross-validation, we consistently achieve ROC AUC values >0.6. Further optimization of the aptamer library and testing on additional samples is ongoing. Upon complete validation, an ADAPTTM - derived breast cancer test may serve as a vital diagnostic adjunct that can be easily incorporated into standard clinical practice. Citation Format: Valeriy Domenyuk, Zhenyu Zhong, Jie Wang, Adam Stark, Nianqing Xiao, Mark Miglarese, George Poste, Michael Famulok, Günter Mayer, David Spetzler. Adaptive dynamic artificial poly-ligand targeting (ADAPT) enables plasma-based exosome profiling with potential diagnostic utility. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-135.

Applications and modifications of aptamers: potential tool for medical microbiology

Aptamers are ligands made of single-stranded DNA, oligonucleotide RNA or short peptides which bind specifically to their target molecules with high affinity. They resemble different types of monoclonal, polyclonal and recombinant antibodies in their mode of attachment. Aptamers optimally bind their targets in a range of μmol to ρmol. Targets vary from small molecules to macromolecules to whole cells. Systematic evolution of ligands by exponential enrichment is an affinity-based screening method for aptamers. Examples of aptamer applications include structural analysis of molecules, affinity/specificity testing, epitope mapping, making aptamer libraries by systematic evolution of ligands by exponential enrichment, pathogenic targeting, specific molecular targeting, live cell targeting, analytical applications, therapeutic potentials and drug delivery. Staphylococcal enterotoxin B is one of the most important bacterial toxins in different disorders. Aptamers offer a fast, feasible, reliable and affordable method for detecting this toxin.

Abstract P2-01-08: Adaptive dynamic artificial poly-ligand targeting: Aptamer-based profiling of liquid biopsies to improve the accuracy of breast cancer diagnoses in women with dense breast tissue

Abstract Introduction: Breast cancer screening relies upon mammography, but for women with dense breast tissue this method is often uninformative. Routine screening identifies suspicious breast lesions in some women, but the pain and risk associated with follow-up biopsies along with the poor accuracy of traditional histopathology urgently call for improved approaches to breast cancer screening. This is especially important for those high-risk patients for whom mammography is of limited value. We describe a non-invasive liquid biopsy method of profiling plasma exosome preps designed to improve the accuracy and safety of breast cancer screening for women with dense breast tissue. Results: We incubated plasma samples (300 microliters per sample) from breast cancer patients (n=60) and a control cohort (n=60) with a high-complexity DNA aptamer library using a modified SELEX scheme, termed “adaptive dynamic artificial poly-ligand targeting (ADAPTTM)”. Differentially bound (cancer vs. non-cancer) aptamers were recovered from precipitated exosomes and were identified by deep sequencing. Two thousand aptamer sequences were resynthesized and used to probe a larger set of 500 plasma samples from a patient cohort (n=206) and a control cohort comprised of self-reported healthy volunteers (n=117) and patients whose biopsies led to a diagnosis of non-cancer (n=177). We employed several statistical models to build a cancer/non-cancer predictor, including a Random Generalized Linear Model (RGLM) and a Random Forest Model (RFM). Both models yielded an equivalent classification performance with areas under the receiver-operator characteristic curve (ROC AUC) of 0.7. Testing the prediction performance by 100 Out-of-Bag permutations or by pre-filtered (read cutoff and estimated sample size) cross-validation (CV) resulted in ROC AUC values of 0.66 and 0.62, respectively. When samples were randomly assigned to groups, the aptamers were no longer able to distinguish the groups (ROC AUC = 0.54), indicating that the underlying information driving the model is truly specific to cancer. Importantly, incorporation of BIRAD results as a clinical covariate did not influence model performance, signifying that predictions by ADAPTTM were independent of breast tissue density. Conclusions: We have identified a set of 2000 DNA aptamers that distinguish women with breast cancer from women without breast cancer. Our liquid biopsy approach requires only 300 microliters of plasma and is amenable to high-throughput processing. By employing a number of statistical approaches including rigorous cross-validation, we consistently achieve cross validation ROC AUC values approaching 0.7. The performance of the predictor was not affected by BIRAD scores, supporting its potential utility in difficult cases where imaging is insufficient, such as in women with dense breast tissue. Further optimization of the aptamer library and testing on additional samples should improve performance. Upon complete validation, an ADAPTTM – derived breast cancer test may serve as a vital diagnostic adjunct that can be easily incorporated into standard clinical practice. Citation Format: Domenyuk V, Zhong Z, Wang J, Stark A, Chen W, Xiao N, Miglarese MR, Famulok M, Mayer G, Spetzler DB. Adaptive dynamic artificial poly-ligand targeting: Aptamer-based profiling of liquid biopsies to improve the accuracy of breast cancer diagnoses in women with dense breast tissue. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-01-08.

Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.

A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX.