Boron-containing aptamers to ATP

Boron neutron capture therapy (BNCT), an experimental treatment for certain cancers, destroys only cells near the boron; however, there is a need to develop highly specific delivery agents. As nucleic acid aptamers recognize specific molecular targets, we investigated the influence of boronated nucleotide analogs on RNA function and on the systematic evolution of ligands by exponential enrichment (SELEX) process. Substitution of guanosine 5′-(α-P-borano) triphosphate (bG) for GTP or uridine 5′-(α-P-borano) triphosphate (bU) for UTP in several known aptamers diminished or eliminated target recognition by those RNAs. Specifically, ATP-binding aptamers containing the ζ-fold, which appears in several selections for adenosine aptamers, became inactive upon bG substitution but were only moderately affected by bU substitution. Selections were carried out using the bG or bU analogs with C8-linked ATP agarose as the binding target. The selections with bU and normal NTP yielded some ζ-fold aptamers, while the bG selection yielded none of this type. Non-ζ aptamers from bU and bG populations tolerated the borano substitution and many required it. The borano nucleotide requirement is specific; bU could not be used in bG-dependent aptamers nor vice versa. The borano group plays an essential role, as yet undefined, in target recognition or RNA structure. We conclude that the bG and bU nucleotides are fully compatible with SELEX, and that these analogs could be used to make boronated aptamers as therapeutics for BNCT.     

The selection of aptamers specific for membrane molecular targets

A growing number of RNA aptamers have been selected experimentally using the SELEX combinatorial approach, and these aptamers have several advantages over monoclonal protein antibodies or peptides with respect to their applications in medicine and nanobiotechnology. Relatively few successful selections have been reported for membrane molecular targets, in contrast to the situation with non-membrane molecular targets. This review compares the procedures and techniques used in selections against membrane proteins and membrane lipids. In the case of membrane proteins, the selections were performed against soluble protein fragments, detergent-membrane protein mixed micelles, whole cells, vesicles derived from cellular membranes, and enveloped viruses. Liposomes were used as an experimental system for the selection of aptamers against membrane lipids. RNA structure-dependent aptamer binding for rafts in lipid vesicles was reported. Based on the selected aptamers against DOPC and the amino acid tryptophan, a specific passive membrane transporter composed of RNA was constructed. The determination of the selectivity of aptamers appears to be a crucial step in a selection, but has rarely been fully investigated. The selections, which use whole cells or vesicles derived from membranes, can yield aptamers not only against proteins but also against membrane lipids.     

Analyses of SELEX-derived ZAP-binding RNA aptamers suggest that the binding specificity is determined by both structure and sequence of the RNA

The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including murine leukemia virus, Sindbis virus and Ebola virus, by targeting the viral mRNAs for degradation. ZAP directly binds to the target viral mRNA and recruits the cellular RNA degradation machinery to degrade the RNA. No significant sequence similarity or obvious common motifs have been found in the so far identified target viral mRNAs. The minimum length of the target sequence is about 500nt long. Short workable ZAP-binding RNAs should facilitate further studies on the ZAP-RNA interaction and characterization of such RNAs may provide some insights into the underlying mechanism. In this study, we used the SELEX method to isolate ZAP-binding RNA aptamers. After 21 rounds of selection, ZAP-binding aptamers were isolated. Sequence analysis revealed that they are G-rich RNAs with predicted stem-loop structures containing conserved “GGGUGG” and “GAGGG” motifs in the loop region. Insertion of the aptamer sequence into a luciferase reporter failed to render the reporter sensitive to ZAP. However, overexpression of the aptamers modestly but significantly reduced ZAP’s antiviral activity. Substitution of the conserved motifs of the aptamers significantly impaired their ZAP-binding ability and ZAP-antagonizing activity, suggesting that the RNA sequence is important for specific interaction between ZAP and the target RNA. The aptamers identified in this report should provide useful tools to further investigate the details of the interaction between ZAP and the target RNAs.     

Comparison of different strategies to select aptamers against a transmembrane protein target

Binding of aptamers is dependent on their target conformation, which in turn is conditioned by the target's environment. Therefore, selection of aptamers against the active forms of membrane proteins could require their correct membrane insertion in order to maintain their native conformation. Here, we compare different SELEX strategies to identify aptamers against the mutated form of the membrane receptor tyrosine kinase RET(C634Y). (1) selections S1 and S2 against living cells transformed to express the protein yielded a minority of RET-targeted aptamers while the bulk of aptamers recognized more abundant membrane proteins, suggesting that a high level of expression of the target protein is crucial to allow the isolation of aptamers at cell surface; (2) selection S3 against the purified extracellular moiety of RET yielded aptamers unable to recognize RET expressed at the cell membrane; (3) crossover selections S4 and S5 alternating cells and recombinant RET enhanced the enrichment of the aptamers directed against RET; however, these aptamers displayed a weaker affinity for Ret than those obtained with S1 and S2. In our case, using transformed cell lines as the partitioning matrix during SELEX appears to be essential in order to obtain aptamers able to recognize the RET receptor tyrosine kinase in its physiologic environment.     

Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers

Aptamers are small nucleic acid ligands that bind to their targets with specificity and high affinity. They are generated by a combinatorial technology, known as SELEX. This in vitro approach uses iterative cycles of enrichment and amplification to select binders from nucleic acid libraries of high complexity. Here we combine SELEX with the yeast three-hybrid system in order to select for RNA aptamers with in vivo binding activity. As a target molecule, we chose the RNA recognition motif-containing RNA-binding protein Rrm4 from the corn pathogen Ustilago maydis. Rrm4 is an ELAV-like protein containing three N-terminal RNA recognition motifs (RRMs). It has been implicated in microtubule-dependent RNA transport during pathogenic development. After 11 SELEX cycles, four aptamer classes were identified. These sequences were further screened for their in vivo binding activity applying the yeast three-hybrid system. Of the initial aptamer classes only members of two classes were capable of binding in vivo. Testing representatives of both classes against Rrm4 variants mutated in one of the three RRM domains revealed that these aptamers interacted with the third RRM. Thus, the yeast three-hybrid system is a useful extension to the SELEX protocol for the identification and characterization of aptamers with in vivo binding activity.     

Characterisation of aptamer–target interactions by branched selection and high-throughput sequencing of SELEX pools

Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. Typically, aptamers are identified from libraries containing up to 10¹⁶ different RNA or DNA sequences by 5–10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic changes in the pool composition during the SELEX process and by facilitating large-scale post-SELEX characterisation. In the present study, we demonstrate how high-throughput sequencing of SELEX pools, before and after a single round of branched selection for binding to different target variants, can provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2′-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard selection. The results demonstrate that it is possible to perform large-scale detailed characterisation of aptamer sequences directly in the complex pools obtained from library selection methods, thus without the need to produce individual aptamers.     

A sol-gel-based microfluidics system enhances the efficiency of RNA aptamer selection

RNA and DNA aptamers that bind to target molecules with high specificity and affinity have been a focus of diagnostics and therapeutic research. These aptamers are obtained by SELEX often requiring many rounds of selection and amplification. Recently, we have shown the efficient binding and elution of RNA aptamers against target proteins using a microfluidic chip that incorporates 5 sol-gel binding droplets within which specific target proteins are imbedded. Here, we demonstrate that our microfluidic chip in a SELEX experiment greatly improved selection efficiency of RNA aptamers to TATA-binding protein, reducing the number of selection cycles needed to produce high affinity aptamers by about 80%. Many aptamers were identical or homologous to those isolated previously by conventional filter-binding SELEX. The microfluidic chip SELEX is readily scalable using a sol-gel microarray-based target multiplexing. Additionally, we show that sol-gel embedded protein arrays can be used as a high-throughput assay for quantifying binding affinities of aptamers.     

一种结合SELEX与二代测序技术筛选RNA结合蛋白特异识别序列新方法的建立

RNA结合蛋白通过特异识别RNA底物发挥重要的生物学作用。指数富集的配体系统进化(Systematic evolution of ligands by exponential enrichment,SELEX)技术是一种体外筛选核酸底物的基本方法,SELEX技术通过重复多轮筛选从随机核酸序列库中筛选出特异性与靶物质高度亲和的核酸底物,本研究将利用该技术与二代高通量测序(NGS)相结合,体外合成含有20个随机碱基的RNA文库,将所要研究的蛋白构建到带有可被链亲和酶素磁珠捕获的SBP标记的载体上去,显著提高筛选效率,仅需1轮筛选即可获得所需RNA底物motif。通过该方法获得了人的hn RNP A1的UP1结构域特异识别AGG和AG二种RNA序列,并通过EMSA实验证实其可以与获得的RNA motif结合。这一方法的建立对于研究RNA结合蛋白识别底物的序列特异性,并进一步了解其在生物体内的调控机制有重要意义。     

Monitoring Genomic Sequences during SELEX Using High-Throughput Sequencing: Neutral SELEX

Background

SELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA sequences binding a ligand of interest, called genomic aptamers. Concerns have been raised regarding requirements imposed on RNA sequences undergoing SELEX selection.

Methodology/Principal Findings

To evaluate SELEX and assess the extent of these effects, we designed and performed a Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of SELEX''s amplification steps. Using high-throughput sequencing, we obtained thousands of full-length sequences from the initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtained from a Genomic SELEX experiment deriving from the same initial library, but screening for RNAs binding with high affinity to the E. coli regulator protein Hfq. With each round of Neutral SELEX, sequences became less stable and changed in nucleotide content, but no sequences were enriched. In contrast, we detected substantial enrichment in the Hfq-selected set with enriched sequences having structural stability similar to the neutral sequences but with significantly different nucleotide selection.

Conclusions/Significance

Our data indicate that positive selection in SELEX acts independently of the neutral selective requirements imposed on the sequences. We conclude that Genomic SELEX, when combined with high-throughput sequencing of positively and neutrally selected pools, as well as the gnomic library, is a powerful method to identify genomic aptamers.     

RNA aptamers specific for bovine thrombin

Bovine thrombin is widely used in clinical wound healing after surgery. There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin. Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported. RNA ligands (aptamers) have been used to bind to target molecules with sometimes higher specificity than antibodies. Here we report the isolation of aptamers specific for bovine thrombin by systematic evolution of ligands by exponential enrichment (SELEX) from an RNA pool containing a 25-nucleotide randomized region. After seven rounds of selection, two aptamers specific for bovine thrombin were identified with a K(d) of 164 and 240 nM, respectively. Significantly, these aptamers do not bind to human thrombin. Secondary structure prediction revealed potential stem-loop structures for these RNAs. Both RNA aptamers inhibit only bovine thrombin-catalyzed fibrin clot formation in vitro. Competition assay results suggested that the RNA aptamers might bind to the electropositive domain of bovine thrombin, that is, heparin-binding site, instead of fibrinogen-recognition exosite. The resulting bovine-specific thrombin inhibitor might be used in some clinical applications when bovine thrombin activity needs to be contained or in research where human and bovine thrombin need to be distinguished.     

A novel artificial intelligence-based approach for identification of deoxynucleotide aptamers

The selection of a DNA aptamer through the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method involves multiple binding steps, in which a target and a library of randomized DNA sequences are mixed for selection of a single, nucleotide-specific molecule. Usually, 10 to 20 steps are required for SELEX to be completed. Throughout this process it is necessary to discriminate between true DNA aptamers and unspecified DNA-binding sequences. Thus, a novel machine learning-based approach was developed to support and simplify the early steps of the SELEX process, to help discriminate binding between DNA aptamers from those unspecified targets of DNA-binding sequences. An Artificial Intelligence (AI) approach to identify aptamers were implemented based on Natural Language Processing (NLP) and Machine Learning (ML). NLP method (CountVectorizer) was used to extract information from the nucleotide sequences. Four ML algorithms (Logistic Regression, Decision Tree, Gaussian Naïve Bayes, Support Vector Machines) were trained using data from the NLP method along with sequence information. The best performing model was Support Vector Machines because it had the best ability to discriminate between positive and negative classes. In our model, an Accuracy (A) of 0.995, the fraction of samples that the model correctly classified, and an Area Under the Receiving Operating Curve (AUROC) of 0.998, the degree by which a model is capable of distinguishing between classes, were observed. The developed AI approach is useful to identify potential DNA aptamers to reduce the amount of rounds in a SELEX selection. This new approach could be applied in the design of DNA libraries and result in a more efficient and faster process for DNA aptamers to be chosen during SELEX.     

Screening and Characterization of a Novel RNA Aptamer That Specifically Binds to Human Prostatic Acid Phosphatase and Human Prostate Cancer Cells

Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2′-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2′-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer.     

Selection of RNA aptamers against recombinant transforming growth factor-beta type III receptor displayed on cell surface

In most cases, anti-protein aptamers are selected by systematic evolution of ligands by exponential enrichment (SELEX) using purified recombinant protein targets. Cell surface proteins, however, are not easy targets for SELEX due to the difficulties associated with their purification. Here, we developed a novel SELEX procedure (referred to as TECS-SELEX) in which cell-surface displayed recombinant protein is directly used as the selection target. Using this method, we isolated RNA aptamers against transforming growth factor-beta type III receptor expressed on Chinese hamster ovary (CHO) cells. One of the RNA aptamers has a dissociation constant in the 1 nM range and competed with transforming growth factor-beta to bind to the cell surface receptor in vitro.     

Minimal primer and primer-free SELEX protocols for selection of aptamers from random DNA libraries

Standard systematic evolution of ligands by exponential enrichment (SELEX) protocols require libraries that contain two primers, one on each side of a central random domain, which allow amplification of target-bound sequences via PCR or RT-PCR. However, these primer sequences cause nonspecific binding by their nature (generally adding about 20 nt on each end of the random sequence of about 30-40 nt), and can result in large numbers of false-positive binding sequences and/or interfere with good binding random sequences. Here, we have developed two DNA-based methods that reduce and/or eliminate the primer sequences from the target-binding step, thus reducing or eliminating the interference caused by the primer sequences. In these methods, the starting selection libraries contain a central random sequence that is: (i) flanked by only 2 nt on each side (minimal primer); or (ii) flanked only by either a 2- or 0-nt overhand on the 3' end (primer-free). These methods allow primer regeneration and re-elimination after and before selection, are fast and simple, and don't require any chemical modifications for selection in a variety of conditions. Further, the selection rounds are performed with DNA oligomers, which are generally employed as end product aptamers.     

Analytical applications of aptamers

So far, several bio-analytical methods have used nucleic acid probes to detect specific sequences in RNA or DNA targets through hybridisation. More recently, specific nucleic acids, aptamers, selected from random sequence pools, have been shown to bind non-nucleic acid targets, such as small molecules or proteins. The development of in vitro selection and amplification techniques has allowed the identification of specific aptamers, which bind to the target molecules with high affinity. Many small organic molecules with molecular weights from 100 to 10,000 Da have been shown to be good targets for selection. Moreover, aptamers can be selected against difficult target haptens, such as toxins or prions. The selected aptamers can bind to their targets with high affinity and even discriminate between closely related targets.

Aptamers can thus be considered as a valid alternative to antibodies or other bio-mimetic receptors, for the development of biosensors and other analytical methods. The production of aptamers is commonly performed by the SELEX (systematic evolution of ligands by exponential enrichment) process, which, starting from large libraries of oligonucleotides, allows the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction.

Aptamers are suitable for applications based on molecular recognition as analytical, diagnostic and therapeutic tools. In this review, the main analytical methods, which have been developed using aptamers, will be discussed together with an overview on the aptamer selection process.     

Isolation and Characterization of 2′-amino-modified RNA Aptamersfor Human TNFα

Human tumor necrosis factor α (hTNFα), a pleiotropic cytokine with activities ranging from host defense mechanisms in infection and injury to severe toxicity in septic shock or other related diseases, is a promising target for drug screening. Using the SELEX (systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotide ligands (aptamers) with high affinities for hTNFα.Aptamers were selected from a starting pool of 40 randomized sequences composed of about 10^15 RNA molecules. Representative aptamers were truncated to the minimal length with high affinity for hTNFα and were further modified by replacement of 2′-OH with 2′-F and 2′-NH2 at all ribopurine positions. These modified RNA aptamers were resistant to nuclease. The specificity of these aptamers for hTNFα was confirmed, and their activity to inhibit the cytotoxicity of hTNFα on mouse L929 cells was determined. Results demonstrated that four 2′-NH2-modified aptamers bound to hTNFα with high affinity and blocked the binding of hTNFα to its receptor, thus protecting the L929 cells from the cytotoxicity of hTNFα. Oligonucleotide aptamers described here are potential therapeutics and diagnostics for hTNFα-related diseases.     

In vitro selection using a dual RNA library that allows primerless selection

High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC₅₀ of 4.5 nM at 37°C.     

Isolation and Characterization of 2'-amino-modified RNA Aptamers for Human TNFα

Human tumor necrosis factor a (hTNFa), a pleiotropic cytokine with activities ranging from host defense mechanisms in infection and injury to severe toxicity in septic shock or other related diseases, is a promising target for drug screening. Using the SELEX (systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotide ligands (aptamers) with high affinities for hTNFa. Aptamers were selected from a starting pool of 40 randomized sequences composed of about 1015 RNA molecules. Representative aptamers were truncated to the minimal length with high affinity for hTNFa and were further modified by replacement of 2'-OH with 2'-F and 2'-NH2 at all ribopurine positions. These modified RNA aptamers were resistant to nuclease. The specificity of these aptamers for hTNFa was confirmed, and their activity to inhibit the cytotoxicity of hTNFa on mouse L929 cells was determined. Results demonstrated that four 2'-NH2-modified aptamers bound to hTNFa with high affinity and blocked the