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Anti-Peptide Aptamers Recognize Amino Acid Sequence and Bind a Protein Epitope
Abstract
In vitro selection of nucleic acid binding species (aptamers) is superficially similar to the immune response. Both processes produce biopolymers that can recognize targets with high affinity and specificity. While antibodies are known to recognize the sequence and conformation of protein surface features (epitopes), very little is known about the precise interactions between aptamers and their epitopes. Therefore, aptamers that could recognize a particular epitope, a peptide fragment of human immunodeficiency virus type I Rev, were selected from a random sequence RNA pool. Several of the selected RNAs could bind the free peptide more tightly than a natural RNA ligand, the Rev-binding element. In accord with the hypothesis that protein and nucleic acid binding cusps are functionally similar, interactions between aptamers and the peptide target could be disrupted by sequence substitutions. Moreover, the aptamers appeared to be able to bind peptides with different solution conformations, implying an induced fit mechanism for binding. Just as anti-peptide antibodies can sometimes recognize the corresponding epitope when presented in a protein, the anti-peptide aptamers were found to specifically bind to Rev.
... 37,38 As illustrated in Scheme 1A and B, proteinaceous cargo is tagged with an arginine-rich rev peptide tag that recognizes and binds to an RNA aptamer upstream of the ribosome binding site. 39 An RNA hairpin downstream of the stop codon binds to the interior of the CP monomers directing the revtagged protein in the interior of VLP. Conceptually, this expression system uses the mRNA as a rope that electrostatically ties cargo to the CP. ...
Virus-like particles (VLPs) are engineered nanoparticles that mimic the properties of viruses-like high tolerance to heat and proteases-but lack a viral genome, making them non-infectious. They are easily modified chemically and genetically, making them useful in drug delivery, enhancing vaccine efficacy, gene delivery, and cancer immunotherapy. One such VLP is Qβ, which has an affinity towards an RNA hairpin structure found in its viral RNA that drives the self-assembly of the capsid. It is possible to usurp the native way infectious Qβ self-assembles to encapsidate its RNA to place enzymes inside the VLP's lumen as a protease-resistant cage. Further, using RNA templates that mimic the natural self-assembly of the native capsid, fluorescent proteins (FPs) have been placed inside VLPs in a "one pot" expression system. Autofluorescence in tissues can lead to misinterpretation of results and unreliable science, so we created a single-pot expression system that uses the fluorescent protein smURFP, which avoids autofluorescence and has spectral properties compatible with standard commercial filter sets on confocal microscopes. In this work, we were able to simplify the existing "one-pot" expression system while creating high-yielding fluorescent VLP nanoparticles that could easily be imaged inside lung epithelial tissue.
... Aptamers, a class related to functional molecules, are short artificial oligonucleotides (RNA or DNA) in an approximate range of 10-100 molecules containing a phosphate and a nucleoside, which have particularly significant tendencies toward different targets, including adenosine triphosphate (ATP) [6], proteins [7], and amino acids [8]. They are being widely used in protein sensors as recognition elements [9]. ...
Thrombin is considered an adaptable enzyme, and it has a major role in the process of injured vessel hemostasis. Hence, the detection of thrombin is crucial in the course of any illness. Aptamers, a class related to functional molecules, are short artificial oligonucleotides that are being widely used in protein sensors as recognition elements. Tracking and detection of different biomolecules is crucial not just in the medical diagnostics field but in biotechnology and biological research, biodefense, drug discovery, drug design, and environmental monitoring as well. Direct detection methods, including surface plasmon resonance spectroscopy and mass spectrometry, are also extensively utilized for the detection of thrombin-aptamer interactions. We summarize the studies on spectroscopy and the thrombin-aptamer interaction in this review.
... Aptamers, a category of functional molecules, are single-stranded artificial oligonucleotides (DNA or RNA) in the range of 10-100 nucleotides, which selectively have remarkable affinities to various targets, such as amino acids [6], adenosine triphosphate (ATP) [7], proteins [8] and even cells [9]. Aptamers for specific targets can be artificially isolated from combinatorial oligonucleotides libraries by the systematic evolution of ligands by the exponential enrichment (SELEX) technique [10]. ...
Thrombin facilitates the aggregation of platelet in hemostatic processes and participates in the regulation of cell signaling. Therefore, the development of thrombin sensors is conducive to comprehending the role of thrombin in the course of a disease. Biosensors based on aptamers screened by SELEX have exhibited superiority for thrombin detection. In this review, we summarized the aptamer-based sensors for thrombin detection which rely on the specific recognitions between thrombin and aptamer. Meanwhile, the unique advantages of different sensors including optical and electrochemical sensors were also highlighted. Especially, these sensors based on electrochemistry have the potential to be miniaturized, and thus have gained comprehensive attention. Furthermore, concerns about aptamer-based sensors for thrombin detection, prospects of the future and promising avenues in this field were also presented.
... They can be customized to incorporate chemical modifications and can be joined with affinity tags or labels. [1][2][3][4][5]. Aptamers are a rising technology applied for detecting targets using random nucleotides with specific binding affinity. ...
Antibodies are the most used technological tool in histochemistry. However, even with monoclonal antibodies, their standardization is difficult due to variation of biological systems as well as to variability due to the affinity and amplification of the signal arising from secondary peroxidase detection systems. In this article we combined two synthetic molecules to facilitate the standardization of a detection protocol of protein markers in histological sections. The first molecule was an aptamer, a 50-base single-stranded DNA fragment, which recognizes a PTEN tumor suppressor. The second molecule used was also another single stranded 18-base aptamer DNA fragment, which forms a quadruplex structure guanine box. This G-quadruplex recognizes and attaches a molecule of hemin, increasing the catalytic capacity for the hydrogen peroxide. Our results show how the correct structural design of DNA combining an aptamer together with the peroxidase-like DNAzyme allows to detect proteins in histological sections. This tool offers the standardization of the detection of prognostic markers in cancer, in quality and quantity, due to its synthetic nature and its 1:1 antigen:enzyme ratio. This is the first time that reproducible results have been presented in histological sections staining a cancer marker using a single-stranded DNA molecule with dual function.
... Insulin is a polypeptide hormone made up of 51 amino acids (molecular weight 5.8 kDa), 31,32 and the affinity of ins24 is typical for aptamers to polypeptide targets, which generally falls within the high nM to low μM range. 33,34 However, it remains notable that this aptamer achieves such affinity in the context of human serum. These results therefore confirm our capacity to efficiently identify base-modified aptamers that exhibit both excellent affinity and specificity for their target in complex biological specimens. ...
Aptamers incorporating chemically modified bases can achieve superior affinity and specificity compared to natural aptamers, but their discovery remains a labor-intensive, low-throughput task. Here we describe the non-natural aptamer array (N2A2) system, which enables fully automated, high-throughput screening of base-modified aptamers using a minimally modified Illumina MiSeq instrument. We demonstrate the capability to screen multiple different base modifications to identify the optimal choice for high-affinity target binding. We further use N2A2 to generate aptamers that specifically recognize protein posttranslational modifications, and which maintain strong target affinity in serum. Finally, we demonstrate comprehensive profiling of single- and double-base aptamer mutations to rapidly identify key sequence motifs responsible for binding activity in a single run. N2A2 requires only minor mechanical modifications to the MiSeq and a software suite for automation that we have made freely available. As such, we believe our platform offers a broadly accessible and user-friendly tool for generating custom reagents on-demand.
... Aptamers are single-stranded DNA or RNA oligonucleotides selected from large oligonucleotide sequence libraries using an in vitro technique known as 'systematic evolution of ligands by exponential enrichment' (SELEX). [1,2] As each aptamer assumes distinct secondary and tertiary conformations, they possess high binding affinity and specificity to a wide variety of ligands, including metal ions, [3][4][5] small molecules, [6][7][8] peptides, [9,10] proteins, [11][12][13] and even microorganisms. [14][15][16] Compared with monoclonal antibodies, aptamers offer similar binding profiles with many additional advantages such as inexpensive synthesis, better chemical and thermal stability, reusability, improved tissue penetration, and low immunogenicity. ...
Fabrication of low-cost biosensing platforms with high selectivity and sensitivity is important for constructing portable devices for personal health monitoring. Herein, we report a simple biosensing strategy based on the combination of a cationic AIEgen (aggregation-induced emission fluorogen), TPE-2+, with an aptamer for specific protein detection. The target protein can displace the dye molecules on the dye–aptamer complex, resulting in changes in the fluorescence signal. Selectivity towards different targets can be achieved by simply changing the aptamer sequence. The working mechanism is also investigated.
... Systematic Evolution of Ligands by Exponential enrichment (SELEX) is now a common technique by which DNA aptamers, a kind of short single-stranded oligodeoxynucleotide molecule, can be selected from a DNA library [13,14]. The ability to fold into secondary and tertiary structures gives aptamers their ability to recognize such target molecules as metal ions [15,16], organic molecules [17][18][19], peptides [20] and protein biomacromolecules [21][22][23][24] with dissociation constants (Kds) down to picomolar values. In particular, aptamers can be selected to specifically recognize target molecules in their native conformations on the cell surface without prior knowledge of the molecular signatures of target cells when a SELEX procedure is directed against live cells (cell-SELEX) [25]. ...
The elegant properties of deoxyribonucleic acid (DNA), such as accurate recognition, programmability and addressability, make it a well-defined and promising material to develop various molecular probes, drug delivery carriers and theranostic systems for cancer diagnosis and therapy. In addition, supramolecular chemistry, also termed "chemistry beyond the molecule", is a promising research field that aims to develop functional chemical systems by bringing discrete molecular components together in a manner that invokes noncovalent intermolecular forces, such as hydrophobic interaction, hydrogen bonding, metal coordination, and shape or size matching. Thus, DNA-supramolecule conjugates (DSCs) combine accurate recognition, programmability and addressability of DNA with the greater toolbox of supramolecular chemistry. This review discusses the applications of DSCs in sensing, protein activity regulation, cell behavior manipulation, and biomedicine.
... DNA or RNA aptamers are single-stranded oligonucleotides with high affinity for specific targets such as ions, 3,4 small molecules, [5][6][7][8] peptides, [9][10][11][12][13] proteins, [14][15][16] and entire cells. [17][18][19] Compared with antibodies, aptamers offer several specific advantages because of their low cost of production, ease of chemical modification, relatively small size, and lower immunogenicity. ...
... DNA or RNA aptamers are single-stranded oligonucleotides with high affinity for specific targets such as ions, 3,4 small molecules, [5][6][7][8] peptides, [9][10][11][12][13] proteins, [14][15][16] and entire cells. [17][18][19] Compared with antibodies, aptamers offer several specific advantages because of their low cost of production, ease of chemical modification, relatively small size, and lower immunogenicity. ...
We present a rapidly neutralizable and highly anticoagulant thrombin-binding aptamer with a short toehold sequence, originally discovered by systematic evolution of ligands by exponential enrichment (SELEX) with microbead-assisted capillary electrophoresis (MACE). MACE is a novel CE-partitioning method for SELEX and able to separate aptamers from a library of unbound nucleic acids, where the aptamer and target complexes can be detected reliably and partitioned with high purity even in the first selection cycle. Three selection rounds of MACE-SELEX discovered several TBAs with a nanomolar affinity (K d = 4.5–8.2 nM) that surpasses previously reported TBAs such as HD1, HD22, and NU172 (K d = 118, 13, and 12 nM, respectively). One of the obtained aptamers, M08, showed a 10- to 20-fold longer prolonged clotting time than other anticoagulant TBAs, such as HD1, NU172, RE31, and RA36. Analyses of the aptamer and thrombin complexes using both bare and coated capillaries suggested that a large number of efficient aptamers are missed in conventional CE-SELEX because of increased interaction between the complex and the capillary. In addition, the toehold-mediated rapid antidote was designed for safe administration. The efficient aptamer and antidote system developed in the present study could serve as a new candidate for anticoagulant therapy.
... Although aptamers as riboswitches are common in nature, introduction of SELEX protocol made it feasible to select aptamers in vitro. Earlier various peptides were used for aptamer selection such as substance P [40], Rev peptide [41], K Ras-derived peptide [42], Amyloid peptide [43], and GSH [39]. Since hTERT is a 120 kDa protein, it would use the entire protein as bait for selecting cognate aptamer against it will yield numerous binders with little or no effect on telomerase activity. ...
Human telomerase reverse transcriptase is an
essential rate-limiting component of telomerase complex.
hTERT protein in association with other proteins and the
human telomerase RNA (hTR) shows telomerase activity,
essential for maintaining genomic integrity in proliferating
cells. hTERT binds hTR through a decapeptide located in
the RID2 (RNA interactive domain 2) domain of N-terminal
region. Since hTERT is essential for telomerase
activity, inhibitors of hTERT are of great interest as
potential anti-cancer agent. We have selected RNA aptamers
against a synthetic peptide from the RID2 domain of
hTERT by employing in vitro selection protocol (SELEX).
The selected RNAs could bind the free peptide, as CD
spectra suggested conformational change in aptamer upon
RID2 binding. Extracts of cultured breast cancer cells
(MCF7) expressing this aptamer showed lower telomerase
activity as estimated by TRAP assay. hTERT-binding RNA
aptamers hold promise as probable anti-cancer therapeutic
agent
... (52) Conformational flexibility has been observed in protein-aptamer interactions, for example, an induced fit in the target polypeptide following the binding of aptamers raised against the HIV-1 regulatory protein. (53) Conversely, the conformation of the nucleic acid was seen to change upon binding of an RNA aptamer to its amino acid target. (54) ...
Aptamer is an artificial nucleic acid ligand, the so-called artificial antibody, can be generated in vitro from the synthetic nucleic acid library by the process called ‘systematic evolution of ligands by exponential enrichment’ (SELEX). This process involves iterative rounds complex formation, separation, and amplification. At the end of this process, predominant aptamer will be found and it has high affinity to the cognate target. There are several modifications that have been demonstrated in the selection process with the generation of high-affinity aptamers. The aptamer undergoes proper folding to interact with their target and fulfil the desired function. The aptamer behaves similar to the antibody and in several instances attested to be better than antibody. The aptamer and antibody complementation has also been shown with high-performance assays. Currently, several aptamers are undergoing different clinical phase trials toward their applications in theranostics.
... To simultaneously induce gene activation and repression, we generated an alternative sgRNA design in which stem loops 2 and 3 of the sgRNA were replaced by the MS2 or Rev aptamer 25 (Supplementary Table 2). We speculated that this alternative design may enable simultaneous activation and repression of endogenous genes when the MS2-dCas9 and Rev-dCas9-VP64 were coexpressed within a single cell (Fig. 5a). ...
The complex phenotypes of eukaryotic cells are controlled by decision-making circuits and signaling pathways. A key obstacle to implementing artificial connections in signaling networks has been the lack of synthetic devices for efficient sensing, processing and control of biological signals. By extending sgRNAs to include modified riboswitches that recognize specific signals, we can create CRISPR-Cas9-based 'signal conductors' that regulate transcription of endogenous genes in response to external or internal signals of interest. These devices can be used to construct all the basic types of Boolean logic gates that perform logical signal operations in mammalian cells without needing the layering of multiple genetic circuits. They can also be used to rewire cellular signaling events by constructing synthetic links that couple different signaling pathways. Moreover, this approach can be applied to redirect oncogenic signal transduction by controlling simultaneous bidirectional (ON-OFF) gene transcriptions, thus enabling reprogramming of the fate of cancer cells.
... [109] With the aid of systematic evolution of ligands by exponential enrichment or SELEX, nucleic acid sequences that bind specific ligands have been identified already more than 20 years ago. [110][111][112][113][114][115] Among the small molecules able to be recognized are various aromatic ligands such as theophylline [116], amino acids like citrulline and arginine [117], oligosaccharides like tobramycin [118] and neomycin [119], peptides (REVbinding elements) [120][121][122] and proteins (phage MS2 coat protein) [123]. ...
... Aptamers promise to be such additional candidates for prophylaxis and treatment of infectious diseases, as they can be directed against a wide variety of target molecules like toxins or even complete microorganisms (Xu and Ellington et al., 1996;Hamm, 1996). Like the antibodies, aptamers bind to their target by their three-dimensional structure with high affinity and specificity. ...
Aptamers are a new class of therapeutic and diagnostic reagents identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX).This method is an in vitro method of selection of DNA or RNA sequences, involving several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. These range from inhibition of receptors and enzymes to the identification of small molecules in sensor applications, and from the development of targeted therapeutic to the design of novel diagnostic and imaging agents. Furthermore, aptamers have been designed for targets that cover a wide range of diseases, from HIV to tropical diseases, cancer and inflammation. Their easy development and flexibility of use and manipulation, offers further potential as effective diagnostic tools.
... The ability of these aptamers to recognize amino acid residues in CD44 exon v10 would be particularly valuable where antibodies cannot recognize. Indeed, previous studies demonstrated the efficacy of mutation analysis of target proteins to identify specific aptamer binding sites [44,45]. It is expected that the identification of Apt#4 and Apt#7 would provide significant information to characterize the nature of the interaction between CD44 exon v10 and EphA2. ...
Triple negative (TN) breast cancers are defined by a lack of expression of estrogen, progesterone, and her-2/neu receptors. It is widely recognized that TN breast cancers have a poorer prognosis than other subtypes of breast caner. Given the lack of effective targeted therapies for TN breast cancer patients, understanding of the mechanisms of growth and invasion of these tumors will provide insight into developing novel approaches to lower the mortality from TN breast cancer.
We utilized three TN breast cancer cell lines (HCC38, HCC1806, HCC1937) as model systems to characterize mechanisms of growth, migration, and invasion. FACS analyses demonstrated that these TN cell lines showed a phenotype of CD44+CD24-/lowepithelial specific antigen (ESA)+. An inhibitory antibody against exon v10 of CD44 significantly inhibited three-dimensional (3D) growth of tumor cells. Importantly, this antibody also inhibited alpha2beta1/alpha3beta1 integrins-mediated migration and invasion into matrigel. These results suggest that CD44 harboring exon 10 facilitates progression and metastasis of TN breast cancer cells in vivo by enhancing growth, migration, and invasion. We also demonstrated that this exon interacts with a cell surface tyrosine kinase,
EphA2. Thus, we hypothesize that CD44 would form a molecular complex with EphA2 through exon v10 on TN breast cancer cells that plays a key role in facilitating invasion and metastasis.
In order to develop novel reagents to inhibit TN breast cancer invasion and metastasis, we have utilized SELEX (systematic evolution of ligands by exponential enrichment) to isolate DNA aptamers that specifically recognize exon v10 of CD44. DNA aptamers are functional molecules with the appropriate sequence and structure to form a complex with a target molecule. Typically, dissociation constants for these aptamer-target complexes are in the high pico-molar to low nano-molar range, which is comparable to antibodies. Given the fact of the ease of chemical synthesis and modifications of oligonucleotides, DNA aptamers that recognize cancer cells will be easier to develop and have a great impact for the next generation of strategies for cancer detection, diagnosis, and therapy. We describe the development of “function-blocking” DNA aptamers specific for exon v10 of CD44 that inhibits TN breast cancer invasion and metastasis.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-08.
... The immobilization and labeling tag effects we have observed for gli1 aptamer with regard to 33-mer and gliadin binding are summarized in Table 2. Gli1 was obtained using a short peptide (the 33-mer immunotoxic peptide) as a target, and it binds two related ligands, 33-mer peptide and PWG-protein, with similar affinities. The possibility of generating aptamers from specific short amino acid sequences to bind that epitope in the protein was previously shown with a peptide from Rev protein but this strategy was never generalized [39] and led to up to 16 times decrease in affinity when challenged against the whole protein [40]. In this work we have shown that it is possible to obtain aptamers with similar affinity for the full-length protein. ...
... As a result, the requirements of the biosensor platform are limited to the detection and differentiation of RNA sequences. At the same time, specific characteristics of RNA molecules including hybridization [8], charge detection [9], peptide/ protein interactions [10], and novel nanotechnology interfaces such as molecular electronics [11,12] can be used to improve biosensor characteristics. Additionally, molecular linkages between BREs and DNA templates can be exchanged for different targets and target types in a straightforward manner. ...
Bio-nanotransduction is a biological detection scheme that is based on biological recognition and the production of biological nano-signals (Bio) which allows the change of recognition events into universal signal molecules (nanotransduction). A single sample can be converted into a pool of nano-signals specific for each target. Detection and differentiation of the nano-signals are correlated to the presence of targets in the original sample. It is possible to link various biological recognition events to platforms (such as biosensors) capable of detecting nano-signals. This allows the application of new target recognition while using a universal platform previously optimized for nano-signal detection. In this paper we explored the utility of the Bio-nanotransduction for the detection of biomolecular targets in a single sample using three detection platforms for nano-signal profile recording. We also demonstrated the flexibility of the system adapting to the detection of new targets.
... The ability of these aptamers to recognize amino acid residues in CD44 exon v10 would be particularly valuable where antibodies cannot recognize. Indeed, previous studies demonstrated the efficacy of mutation analysis of target proteins to identify specific aptamer binding sites [44,45]. It is expected that the identification of Apt#4 and Apt#7 would provide significant information to characterize the nature of the interaction between CD44 exon v10 and EphA2. ...
CD44 adhesion molecules are expressed in many breast cancer cells and have been demonstrated to play a key role in regulating malignant phenotypes such as growth, migration, and invasion. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. The diversity of the biological functions of CD44 is the result of the various splicing variants of these exons. Previous studies suggest that exon v10 of CD44 plays a key role in promoting cancer invasion and metastasis, however, the molecular mechanisms are not clear. Given the fact that exon v10 is in the ectodomain of CD44, we hypothesized that CD44 forms a molecular complex with other cell surface molecules through exon v10 in order to promote migration of breast cancer cells. In order to test this hypothesis, we selected DNA aptamers that specifically bound to CD44 exon v10 using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). We selected aptamers that inhibited migration of breast cancer cells. Co-immunoprecipitation studies demonstrated that EphA2 was co-precipitated with CD44. Pull-down studies demonstrated that recombinant CD44 exon v10 bound to EphA2 and more importantly aptamers that inhibited migration also prevented the binding of EphA2 to exon v10. These results suggest that CD44 forms a molecular complex with EphA2 on the breast cancer cell surface and this complex plays a key role in enhancing breast cancer migration. These results provide insight not only for characterizing mechanisms of breast cancer migration but also for developing target-specific therapy for breast cancers and possibly other cancer types expressing CD44 exon v10.
Controllable protein nanoarchitectonics refers to the process of manipulating and controlling the assembly of proteins at the nanoscale to achieve domain‐limited and accurate spatial arrangement. In nature, many proteins undergo precise self‐assembly with other structural domains to engage in synergistic physiological activities. Protein nanomaterials prepared through protein nanosizing have received considerable attention due to their excellent biocompatibility, low toxicity, modifiability, and versatility. This review focuses on the fundamental strategies used for controllable protein nanoarchitectinics, which include computational design, self‐assembly induction, template introduction, complexation induction, chemical modification, and in vivo assembly. Precise controlling of the nanosizing process has enabled the creation of protein nanostructures with different dimensions, including 0D spherical oligomers, 1D nanowires, nanorings, and nanotubes, as well as 2D nanofilms, and 3D protein nanocages. The unique biological properties of proteins hold promise for diverse applications of these protein nanomaterials, including in biomedicine, the food industry, agriculture, biosensing, environmental protection, biocatalysis, and artificial light harvesting. Protein nanosizing is a powerful tool for developing biomaterials with advanced structures and functions.
RNA molecules have emerged as increasingly attractive biomaterials with important applications such as RNA interference (RNAi) for cancer treatment and mRNA vaccines against infectious diseases. However, it remains challenging to engineer RNA biomaterials with sophisticated functions such as non‐covalent light‐switching ability. Herein, light‐responsive RNA‐protein nanowires are engineered to have such functions. It first demonstrates that the high affinity of RNA aptamer enables the formation of long RNA‐protein nanowires through designing a dimeric RNA aptamer and an engineered green fluorescence protein (GFP) that contains two TAT‐derived peptides at N‐ and C‐ termini. GFP is then replaced with an optogenetic protein pair system, LOV2 (light–oxygen–voltage) protein and its binding partner ZDK (Z subunit of protein A), to confer blue light‐controlled photo‐switching ability. The light‐responsive nanowires are long (>500 nm) in the dark, but small (20–30 nm) when exposed to light. Importantly, the co‐assembly of this RNA‐protein hybrid biomaterial does not rely on the photochemistry commonly used for light‐responsive biomaterials, such as bond formation, cleavage, and isomerization, and is thus reversible. These RNA‐protein structures can serve as a new class of light‐controlled biocompatible frameworks for incorporating versatile elements such as RNA, DNA, and enzymes.
Invasive and noninvasive methods for diagnosing Alzheimer's disease (AD) are discussed and efforts to test less invasively are presented. The literature for this review was searched using Google Scholar (http://scholar.google.com), PubMed (http://www.ncbi.nlm.nih.gov/pubmed) and Scopus (www.scopus.com) database. According to the method of acquiring samples required for AD diagnosis, they were divided into invasive and noninvasive methods. Sample acquisition sites and biomarkers recently proposed for diagnosis in a less invasive way are presented and the potential of using aptamers to replace conventional antibody-based AD diagnostic methods is briefly introduced.
Neuropsychological screening test is performed first to diagnose AD and to screen out patients with suspected dementia. On the other hand, brain imaging is one of the last test methods used to confirm AD. Immunoassays and mass spectrometry are invasive methods that require specific samples from a patient for analysis. Both methods are performed as a final step to confirm the diagnosis of AD. There are several studies on noninvasive diagnosis of AD with faster and more convenient tests. Existing AD biomarkers Aβ and tau and several biomarkers have been found in tear, saliva, and urine samples. If the usefulness of other biomarkers is evaluated through additional studies, samples can be obtained in a noninvasive manner and used in the diagnosis of AD. In addition, there are attempts to combine biosensors and nanomaterials to overcome limitations of existing diagnostic methods. A biosensor combining an aptamer and a nanomaterial has been reported. It appears to effectively detect AD-associated biomarkers. Identification of new biomarkers related to AD and research on the development of aptamer-based biosensors can serve as a stepping stone for minimally invasive diagnosis of AD.
There is a critical need for the development of more potent inhibitors for osteoarthritis (OA) therapy given the poor life quality of arthritis patients. Aggrecanase ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) is an established drug target identified for osteoarthritis. In this study, we evolved and characterised two new DNA aptamer inhibitors of ADAMTS-5, namely apt21 and apt25. The aptamers exhibited nanomolar binding affinity and high specificity against ADAMTS-5. KD values of apt21 and apt25 were determined by the Enzyme-linked Oligonucleotide Assay (ELONA) at 1.54 ± 0.16 nM and 1.79 ± 0.08 nM, respectively. Circular Dichroism (CD) analysis demonstrated that both aptamers formed monovalent cation dependent G-quadruplex structures. Calcium ions did not affect the binding of the aptamers to ADAMTS-5. The inhibitory effects of apt21 and apt25 on ADAMTS-5 were evaluated by the Förster Resonance Energy Transfer (FRET) assay, in which IC50 values of apt21 and apt25 were estimated at 52.76 ± 6.70 μM and 61.14 ± 9.67 μM, respectively. These two aptamers are the first DNA G-quadruplex aptamers demonstrated to inhibit ADAMTS-5 and could have value for OA therapy.
While the protein assemblies have been found widely existing and playing significant roles in biological systems, their imitation and re‐construction is further boosting more applications in biomedical research, such as enzymatic reaction regulation, sensing, and biomedicine. DNA nanotechnology provides a programmable strategy for the fabrication of nanostructures with unprecedented accuracy on the nanoscale. By linking the DNA nanotechnology with proteins of different functions, the precise construction of DNA‐guided protein assemblies can be achieved for various biomedical applications. This minireview summarizes the recent advances in the programmable protein assemblies on DNA nanoplatforms and discusses the outlook of DNA‐guided protein assemblies in the biomedical research.
The innovative discovery of aptamers was based on target-specific treatment in clinical diagnostics and therapeutics. Aptamers are synthetic, single-stranded oligonucleotides, simply described as chemical antibodies, which can bind to diverse targets with high specificity and affinity. Aptamers are synthesized by the SELEX technique, and possess distinctive properties as small size (10–50 kDa), higher stability, easy manufacture and less immunogenicity. These oligonucleotides are easily degraded by nucleases, so require some important modifications like capping and incorporation of modified nucleotides. RNA aptamers can be modified chemically on 2′ positions using -NH3, -F, -deoxy, or -OMe groups to enhance their nuclease resistance. Aptamers have been employed for multiple purposes, as direct drugs or aptamer–drug conjugates targeted against different diseased cells. Different aptamer-conjugated nanovehicles (e.g., micelles, liposomes, silica nano-shells) have been designed to transport diverse anticancer-drugs like doxorubicin and cisplatin in bulk to minimize systemic cytotoxicity. Some drug-loaded nanovehicles (up to 97% loading capacity) and conjugated with specific aptamer resulted in more than 60% tumor inhibition as compared to unconjugated drug-loaded nanovehicles which showed only 31% cancer inhibition. In addition, aptamers have been widely used in basic research, food safety, environmental monitoring, clinical diagnostics and therapeutics. Different FDA-approved RNA and DNA aptamers are now available in the market, used for the treatment of diverse diseases, especially cancer. These aptamers include Macugen, Pegaptanib, etc. Despite a good progress in aptamer use, the present-day chemotherapeutics and drug targeting systems still face great challenges. Here in this review article, we are discussing nucleic acid aptamers, preparation, role in the transportation of different nanoparticle vehicles and their applications as therapeutic agents.
This chapter concentrates on the application of systematic evolution of ligands by exponential enrichment (SELEX-derived aptamers). SELEX is a combinatorial ligand discovery technology that utilizes large libraries of random sequence oligonucleotides to isolate ligands with high affinity and specificity to molecular targets. These ligands are known as aptamers. SELEX has been used to isolate aptamers to a broad range of targets ranging from small organic molecules such as theophylline to proteins involved in pathological processes. Libraries are commonly composed of either deoxyribonucleic acid (DNA) or modified ribonucleic acid (RNA). An RNA modification conferring extensive in vivo stability relative to unmodified RNA is substitution of the 2'-OH with 2'-F on pyrimidine nucleotides. Such libraries are chemically stable upon prolonged incubation with a variety of biological sources including human plasma. Aptamers to selectins have been shown to recognize the carbohydrate domains and bind to selectins on cell surfaces. Methodology to identify specific contact positions between aptamers and targets has been developed utilizing both photochemistry and solution chemistry. In this chapter, interactions of specific aptamers with cognate targets as well as progress in defining higher order structural motifs of aptamer-target complexes are given.
Although RNA aptamers can show comparable or better specificity and affinity to antibodies and have the advantage of being able to access different live cell compartments, they are often much less stable in vivo. We report here the first aptamer that binds human retinoblastoma protein (RB) and is stable in live cells. RB is both a key protein in cell cycle control and also a tumour suppressor. The aptamer was selected from an RNA library against a unique 12-residue helical peptide derived from RB rather than the whole protein molecule. It binds RB with high affinity (Kd = 5.1 ± 0.1 nM) and is a putative RNA G-quadruplex structure formed by an 18-nucleotide sequence (18E16 - GGA GGG UGG AGG GAA GGG), which may account for its high stability. Confocal fluorescence microscopy of live cells transfected with the aptamer shows it is stable intracellularly and efficient in entering the nucleus where an analogous antibody was inaccessible. The findings demonstrate this aptamer is an advanced probe for RB in live cell applications.
This study reports the development of a novel label-free aptasensor which uses the orientation properties of nematic liquid crystals (LCs) to detect mucin 1 (MUC1) is developed. The sensor is made of immobilised amine-functionalised aptamers on the surface, with LCs serving as response elements. When MUC1 interacts with the immobilised aptamers, it forms a complex that disrupts the LC orientation, taking them from homeotropic to random alignment. This change in the orientation of LCs is easily observed as a shift in the optical LC image from dark to bright using a polarised light microscope. This sensing system is highly sensitive (detection limit of 5 fg mL⁻¹) and exhibits good specificity for detection of MUC1 without labelling or an additional amplification step.
Cancer is one of the leading causes of mortality worldwide, and worse still, the number of cancer cases is startlingly growing every year. The intrinsic complexity of tumors, including intratumor heterogeneity, dynamic evolution, and abnormal tumor microenvironments (TME), often leads to the failure of traditional chemotherapy. Cancer theranostics integrate molecular diagnosis and targeted cancer therapy, promoting the transition from conventional to personalized and precision medicine. Functional nucleic acids (FNAs), such as aptamers, DNAzymes, and DNA-based nanomachines (DNMs), are nucleic acids with functions beyond the well-known genetic roles. In addition to the basic advantages of nucleic acids-based materials, such as low cost, biocompatibility, low immunogenicity, and simplicity of chemical modification. FNAs also possess many superior properties, including high binding affinity and specificity of aptamers, efficient and specific gene editing ability of DNAzymes, and logic-controlled designability of DNA machines, all making FNAs an attractive material for applications in cancer theranostics in recent years. This review highlights the development and most recent applications of FNAs in cancer theranostics, as well as the challenges and opportunities in this research field.
Los aptámeros son secuencias de ADN o ARN de cadena sencilla que adoptan la forma de estructuras tridimensionales únicas, lo cual les permite reconocer un blanco específico con gran afinidad. Sus usos potenciales abarcan, entre otros, el diagnóstico de enfermedades, el desarrollo de nuevos agentes terapéuticos, la detección de riesgos alimentarios, la producción de biosensores, la detección de toxinas, el transporte de fármacos en el organismo y la señalización de nanopartículas.
El pegaptanib es el único aptámero aprobado para uso comercial por la Food and Drug Administration (FDA).
En parasitología, se destacan los estudios que se vienen realizando en Leishmania spp., con la obtención de aptámeros que reconocen la proteína de unión a poliA (LiPABP) y que pueden tener potencial utilidad en la investigación, el diagnóstico y el tratamiento de la leishmaniasis. En cuanto a la malaria, se han obtenido aptámeros que permiten identificar eritrocitos infectados e inhiben la formación de rosetas, y otros que prometen ser alternativas para el diagnóstico al detectar de forma específica la proteína lactato deshidrogenasa (PfLDH). Para Cryptosporidium parvuum se han seleccionado aptámeros que detectan ooquistes a partir de alimentos o aguas contaminadas. Para Entamoeba histolytica se han aislado dos aptámeros llamados C4 y C5, que inhiben la proliferación in vitro de los trofozoítos y tienen potencial terapéutico. Los aptámeros contra Trypanosoma cruzi inhiben la invasión de células LLC-MK2 (de riñón de mono) en un 50 a 70 % y aquellos contra T. brucei transportan moléculas tóxicas al lisosoma parasitario como una novedosa estrategia terapéutica.
Natural killer (NK) cells are a key component of the innate immune system as they can attack cancer cells without prior sensitization. However, due to lack of cell‐specific receptors, NK cells are not innately able to perform targeted cancer immunotherapy. Aptamers are short single‐stranded oligonucleotides that specifically recognize their targets with high affinity in a similar manner to antibodies. To render NK cells with target‐specificity, synthetic CD30‐specific aptamers are anchored on cell surfaces to produce aptamer‐engineered NK cells (ApEn‐NK) without genetic alteration or cell damage. Under surface‐anchored aptamer guidance, ApEn‐NK specifically bind to CD30‐expressing lymphoma cells but do not react to off‐target cells. The resulting specific cell binding of ApEn‐NK triggers higher apoptosis/death rates of lymphoma cells compared to parental NK cells. Additionally, experiments with primary human NK cells demonstrate the potential of ApEn‐NK to specifically target and kill lymphoma cells, thus presenting a potential new approach for targeted immunotherapy by NK cells.
Herein, a simple colorimetric aptasensor based on unmodified gold nanoparticles (AuNPs) has been developed for quantifying pancreatic polypeptide (PP), a peptide hormone associated with nonfunctional neuroendocrine pancreatic tumor (NF-PNET). First, several PP-specific aptamers have been screened by systematic evolution of ligands by exponential enrichment. The generated aptamers showed specificity and affinity higher than those reported so far. The sensing platform was constructed based on the optical properties of AuNPs as well as the adsorption of single-stranded DNA aptamers. The high sensitivity of the developed aptasensor was achieved by optimizing the sensing conditions, including salt tolerance, time of reaction, and optimal aptamer concentration. The developed aptasensor showed a wide linear dynamic range (1-60 nM) with a limit of detection of 521 pM. Furthermore, the performance of the aptasensor was evaluated for the detection of PP in human serum and showed recovery values ranging between 92.4 and 93.9%. Therefore, the presented colorimetric aptasensor has a great potential in the diagnosis of NF-pNETs.
Precision medicine has recently emerged as a promising strategy for cancer therapy because it not only specifically targets cancer cells but it also does not have adverse effects on normal cells. Oligonucleotide aptamers are a class of small molecule ligands that can specifically bind to their targets on cell surfaces with high affinity. Aptamers have great potential in precision cancer therapy due to their unique physical, chemical, and biological properties. Therefore, aptamer technology has been widely investigated for biomedical and clinical applications. This review focuses on the potential applications of aptamer technology as a new tool for precision treatment of hematological malignancies, including leukemia, lymphoma, and multiple myeloma.
The simultaneous expression in Escherichia coli cells of the Qβ virus-like particle (VLP) capsid protein and protein “cargo” tagged with a positively charged Rev peptide sequence leads to the spontaneous self-assembly of VLPs with multiple copies of the cargo inside. We report the packaging of four new enzymes with potential applications in medicine and chemical manufacturing. The captured enzymes are active while inside the nanoparticle shell and are protected from environmental conditions that lead to free-enzyme destruction. We also describe genetic modifications to the packaging scheme that shed light on the self-assembly mechanism of this system and allow indirect control over the internal packaging density of cargo. The technology was extended to create, via self-assembly, VLPs that simultaneously display protein ligands on the exterior and contain enzymes within. Inverse relationships were observed between the size of both the packaged and externally displayed protein or domains and nanoparticle yield. These results provide a general method for the rapid creation of robust protein nanoparticles with desired catalytic and targeting functionalities.
Ever since the notion of using DNA as a material was realized, it has been employed in the construction of complex structures that facilitate the assembly of nanoparticles or macromolecules with nanometer-scale precision. Specifically, tiles fashioned from DNA strands and DNA origami sheets have been shown to be suitable as scaffolds for immobilizing proteins with excellent control over their spatial positioning. Supramolecular assembly of proteins into periodic arrays in one or more dimensions is one of the most challenging aspects in the design of scaffolds for biomolecular investigations and macromolecular crystallization. This review provides a brief overview of how various biomolecular interactions with high degree of specificity such as streptavidin-biotin, antigen-antibody, and aptamer-protein interactions have been used to fabricate linear and multidimensional assemblies of structurally intact and functional proteins. The use of DNA-binding proteins as adaptors, polyamide recognition on DNA scaffolds and oligonucleotide linkers for protein assembly are also discussed.
The efficiency and complexity of natural biopolymers have long been a source of fascination to both biologists and chemists. Studies of molecular phylogeny have confirmed that the nanomachines of the cell are the products of evolution, just as earlier work in comparative physiology and zoology showed that macroscopic traits are under selective pressure. The understanding that molecules could evolve led inevitably to attempts to ‘breed’ better molecules, just as the understanding that genetics affected phenotype led to the startling diversity of dog, pigeon, and horse phenotypes originally commented on by Darwin. Starting with Spiegelman in the early 1970s [1], numerous researchers have carried out ‘test tube evolution’ experiments designed to produce novel molecular phenotypes.
Liver cancer is one of the most common and highly malignant cancers in the world. There are no effective therapeutic options if an early liver cancer diagnosis is not achieved. In this work, detection of HepG2 cells by label-free microcantilever array aptasensor was developed. The sensing microcantilevers were functionalized by HepG2 cells-specific aptamers. Meanwhile, to eliminate the interferences induced by the environment, the reference microcantilevers were modified with 6-mercapto-1-hexanol self-assembled monolayers. The aptasensor exhibits high specificity over not only human liver normal cells, but also other cancer cells of breast, bladder, and cervix tumors. The linear relation ranges from 1×10(3) to 1×10(5)cells/mL, with a detection limit of 300cells/mL (S/N=3). Our work provides a simple method for detection of liver cancer cells with advantages in terms of simplicity and stability.
Background: The systematic evolution of ligands by exponential enrichment (SELEX) technique is a combinatorial library approach in which DNA or RNA molecules (aptamers) are selected by their ability to bind their protein targets with high affinity and specificity, comparable to that of monoclonal antibodies. In contrast to antibodies conventionally selected in animals, aptamers are generated by an in vitro selection process, and can be directed against almost every target, including antigens like toxins or nonimmunogenic targets, against which conventional antibodies cannot be raised. Methods: Aptamers are ideal candidates for cytomics, as they can be attached to fluorescent reporters or nanoparticles in order to study biological function by fluorescence microscopy, by flow cytometry, or to quantify the concentration of their target in biological fluids or cells using ELISA, RIA, and Western blot assays. Results: We demonstrate the in vitro selection of anti-kinin B1 receptor aptamers that could be used to determine B1 receptor expression during inflammation processes. These aptamers specifically recognize their target in a Northern-Western blot assay, and bind to their target protein whenever they are exposed in the membrane. Conclusions: Currently, aptamers are linked to fluorescent reporters. We discuss here the present status and future directions concerning the use of the SELEX technique in cytomics.
The binding of human immunodeficiency virus Rev protein via its arginine-rich motif (ARM) to an internal loop in the Rev-response element region IIB (RRE IIB) is necessary for viral replication. Many variant RNAs and ARMs that bind Rev and RRE IIB have been found. Despite the essential role of Rev asparagine 40 in recognition, the Rev ARM double-mutant R35G-N40V functions well in a Rev-RRE IIB reporter assay, indicating R35G-N40V uses a distinct recognition strategy. To examine how RRE IIB may evolve specificity to wild-type Rev ARM and R35G-N40V, 10 RRE IIB libraries, each completely randomized in overlapping regions, were screened with wild-type Rev ARM and R35G-N40V using a reporter system based on bacteriophage λ N antitermination. Consistent with previous studies, a core element of RRE IIB did not vary, and substitutions occurred at conserved residues only in the presence of other substitutions. Notably, the groove-widening, non-canonical base-pair G48:G71 was mutable to U48:G71 without strong loss of binding to wild-type Rev ARM, suggesting U48:G71 performs the same role by adopting the nearly isosteric, reverse wobble base pair. Originating from RRE IIB, as few as one or two substitutions are sufficient to confer specificity to wild-type Rev or Rev R35G-N40. The diversity of RRE IIB mutants that maintain binding to wild-type Rev ARM and R35G-N40V supports neutral theories of evolution and illustrates paths by which viral RNA-protein interactions can evolve new specificities. Rev-RRE offers an excellent model with which to study the fine structure of how specificity evolves. Copyright © 2015 John Wiley & Sons, Ltd.
Copyright © 2015 John Wiley & Sons, Ltd.
The most suitable approach as an analytical alternative methodology for the detection of trace clinical analytes is based on affinity sensing, in which the immobilized biological element (receptor) can be an antibody, a receptor (natural or synthetic), a nucleic acid, or an aptamer.
Immunosensors, based on antibody–antigen interactions, and DNA‐based sensing, based on nucleic acid hybridization, are first reported. Finally an aptasensor, based on a new category of relevant receptors, nucleic acid sequences selected in vitro named aptamers, is shown. The reported examples deal with two different transduction principles, electrochemical and piezoelectric, applied to analyte detection of clinical interest. The development of immunosensors for progesterone detection by electrochemical sensing is first reported. Then DNA‐based sensing, for point mutation detection in the b‐globin gene, occurring in b‐thalassemia, is shown. Finally aptasensing for thrombin detection is discussed.
Both single‐use and multiuse sensing have been shown to be suitable for trace analysis in clinical chemistry. Both approaches possess sensitivity, reproducibility, and analysis times compatible with the final application.
RNA aptamers showing affinity and specificity for different strains of human influenza virus were assembled onto gold nanoparticles that subsequently formed a gold nanoshell (AuNS) around the viral envelope. These shells could be visualised by transmission electron microscopy (TEM). Changes in size and structure of the AuNS coated virus can be used to detect the viruses. We show that sedimentation with a low cost centrifuge and visual determination can detect 3 × 108 viral particles. This journal is
Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug "Macugen" is FDA approved and a series of aptamer-based drugs are in clinical pipelines. The present review discusses the aspects of design, unique properties, applications, and development of different aptamers to aid in cancer diagnosis, prevention, and/or treatment under defined conditions.
The concept of a biosensor is well established and the idea of integrating a molecular recognition layer with a base sensor, such as analyte binding or reaction at the former results in a measureable change (in current or voltage) in the latter has seen many ingenious embodiments. Despite this and the huge amount of research worldwide in biosensors, as outlined in Chap. 2, many challenges still remain in building reliable, long-lived biosensors, especially in the hostile environment of the human body. The enormous potential for in vivo sensing of pathophysiological molecules over time and space has led to many attempts to achieve this and the rewards, both in terms of clinical benefit and improved understanding, cannot be underestimated. As new tools for producing biosensors become available, they are rapidly recruited. In recent years, developments in two areas of science and engineering have provided new opportunities to look again at how biosensors are built and deployed. These developments were not driven by the needs of those building and using biosensors but by much broader scientific and technological trends, which nonetheless have found ready applicability in this area.
A simple α-helical N-model-peptide was designed to investigate the role of the arginine-rich motif of bacteriophage λ N-peptide in selective binding with boxB RNA. The five-arginine arrangement of native N-peptide was retained; all other residues were replaced with alanine. In vitro selection of RNA (30 random-nucleotide region) was carried out with N-model-peptide immobilized on a 27 MHz quartz-crystal microbalance (QCM). Selected RNAs were evaluated on the same QCM plate to obtain binding constants (Ka=107–108 M−1). Many selected RNAs contained GNR(N)A-type loops (similar to the boxB RNA motif recognized by the native N-peptide). Fragments and minimal RNAs containing the GNRA-type loop also bound to N-model-peptide (Ka=106–107 M−1). The RNA recognition specificity of the peptide was studied by changing the “closing” U–A base pair and one base in the tetraloop of the RNA aptamers, and by peptide mutations (18th residue of N-model-peptide). It was concluded that the five-arginine arrangement of the peptide performs selective recognition of the GNRA tetraloop and GNR(N)A pentaloop RNA structures, and that substitution of another functional amino acid residue at the 18th position in N-peptide adds the recognition ability for a loop-RNA sequence.
To export intron-containing viral mRNAs that encode the structural components of new viral particles from the nucleus to the cytoplasm, HIV-1 uses the cellular CRM1 export pathway that is exploited by the viral Rev protein. Rev multimerizes on the Rev response element (RRE) present in the intron-containing RNA species to bridge these to the cellular export factor CRM1. As a result, this Rev-RRE complex is exported to the cytoplasm. This review provides a systematic overview of different aspects of the crucial function of Rev multimerization, such as co-operative Rev-Rev and Rev-RNA interactions, the biological function of Rev multimerization, the relevance of Rev multimerization in the absence of RRE and its potential as a therapeutic target.
Celiac disease represents a significant public health problem in large parts of the world. A major hurdle in the effective management of the disease by celiac sufferers is the sensitivity of the current available methods for assessing gluten contents in food. In response, we report a highly sensitive approach for gluten analysis using aptamers as specific receptors. Gliadins, a fraction of gluten proteins, are the main constituent responsible for triggering the disease. But they are highly hydrophobic and large molecules, regarded as difficult targets for in-vitro evolution of aptamers without nucleobase modification. We describe the successful selection of aptamers for these water insoluble prolamins that was achieved choosing the immunodominant apolar peptide from -gliadin as a target for selection. All aptamers evolved are able to bind the target in its native environment within the natural protein. The best non-protein receptor is the basis for an electrochemical competitive enzyme-linked assay on magnetic particles, which allows the measurement of as low as 0.5 ppb of gliadin standard (0.5 ppm of gluten). Reference immunoassay for detecting the same target has a limit of detection of 3 ppm, six times less sensitive than this method. Importantly, it also displays high specificity, detecting the other three prolamins toxic for celiac patients and not showing cross-reactivity to non-toxic cereal proteins such as maize, soya and rice. These features make the proposed method a valuable tool for gluten detection in foods.
We report several classes of human interspersed repeats that resemble fossils of DNA transposons, elements that move by excision and reintegration in the genome, whereas previously characterized mammalian repeats all appear to have accumulated by retrotransposition, which involves an RNA intermediate. The human genome contains at least 14 families and > 100,000 degenerate copies of short (180-1200 bp) elements that have 14- to 25-bp terminal inverted repeats and are flanked by either 8 bp or TA target site duplications. We describe two ancient 2.5-kb elements with coding capacity, Tigger1 and -2, that closely resemble pogo, a DNA transposon in Drosophila, and probably were responsible for the distribution of some of the short elements. The deduced pogo and Tigger proteins are related to products of five DNA transposons found in fungi and nematodes, and more distantly, to the Tc1 and mariner transposases. They also are very similar to the major mammalian centromere protein CENP-B, suggesting that this may have a transposase origin. We further identified relatively low-copy-number mariner elements in both human and sheep DNA. These belong to two subfamilies previously identified in insect genomes, suggesting lateral transfer between diverse species.
An antiserum raised against a peptide was used to select a unique RNA species from a degenerate pool of RNAs designed to resemble an autoantibody recognition site in U1 RNA. The peptide and the selected RNA epitope could compete for antibody binding, suggesting that both RNA and peptide epitopes occupy the same or overlapping antigen-combining sites. Thus, the RNA epitope functioned as a specific inhibitor of the antibody-antigen interaction. We demonstrate that the RNA epitope can be used to tag unrelated RNA molecules and also to detect the presence of the antibody. We propose that sequence-specific recognition of RNA by antibodies may involve protein-RNA contacts similar to those occurring in other nucleic acid-binding proteins. In addition, these findings are compatible with the suggestion that nucleic acid-binding autoantibodies may arise through immunological cross-reactivity between proteins and nucleic acids.
The messenger RNAs of human immunodeficiency virus-1 (HIV-1) have an RNA hairpin structure, TAR, at their 5' ends that contains a six-nucleotide loop and a three-nucleotide bulge. The conformations of TAR RNA and of TAR with an arginine analog specifically bound at the binding site for the viral protein, Tat, were characterized by nuclear magnetic resonance (NMR) spectroscopy. Upon arginine binding, the bulge changes conformation, and essential nucleotides for binding, U23 and A27.U38, form a base-triple interaction that stabilizes arginine hydrogen bonding to G26 and phosphates. Specificity in the arginine-TAR interaction appears to be derived largely from the structure of the RNA.
Human immunodeficiency virus type 1 (HIV-1) replication requires the expression of two classes of viral mRNA. The early class of HIV-1 transcripts is fully spliced and encodes viral regulatory gene products. The functional expression of one of these nuclear regulatory proteins, termed Rev (formerly Art or Trs), induces the cytoplasmic expression of the incompletely spliced, late class of HIV-1 mRNAs that encode the viral structural proteins, including Gag and Env. Here, we provide evidence that this induction reflects the export from the cell nucleus to the cytoplasm of a pool of unspliced viral RNA constitutively expressed in the nucleus. The hypothesis that Rev acts on RNA transport, rather than splicing, is further supported by the observation that the cytoplasmic expression of a non-spliceable HIV-1 env gene sequence is also subject to Rev regulation. Here we show that this Rev response requires a specific target sequence which coincides with a complex RNA secondary structure present in the env gene. The response to Rev is fully maintained when this sequence is relocated to other exonic or intronic locations within env but is ablated by inversion. These results indicate that the HIV-1 rev gene product induces HIV-1 structural gene expression by activating the sequence-specific nuclear export of incompletely spliced HIV-1 RNA species.
Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies.
In vitro selection technology has been used to purify RNA aptamers from a random sequence pool that can bind to, and specifically inhibit, protein kinase C beta II. Two of the selected RNA aptamers bind to this isozyme of protein kinase C with nanomolar affinities and inhibit activation with unprecedented selectivity; the highly related, alternatively spliced beta I isozyme, which differs by 23 residues, is inhibited with 1 order of magnitude lower potency; the next most similar isozyme, alpha, shows no detectable inhibition. The production of isozyme-specific inhibitors of protein kinase C opens the possibilities for dissecting the roles of specific protein kinase Cs in the myriad of intracellular signalling pathways.
A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.
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Four types of antigenic sites found in viruses are discussed: cryptotopes, neotopes, metatopes and neutralization epitopes. The role played by conformation on the specificity of viral epitopes is illustrated in the case of tobacco mosaic virus and influenza virus. It appears that mechanisms reminiscent of induced fit contribute to the recognition of viral epitopes by antibodies.
Peptide antigens frequently induce antibodies which recognise the denatured form of a protein from which their sequences are derived. However, the ability to induce antibodies which crossreact with the native, fully folded form of the protein is less commonly observed. Although there is a growing number of examples in which this is the case, the ability to predict peptides having this property is extremely limited. Given the large surface areas involved in antibody/antigen interaction it is surprising that peptides could ever induce antibodies which would recognise the native protein well enough to have biological activity, such as the neutralization of infectivity. A mechanism is proposed to explain such observations which is compatible with many of the properties of antipeptide antibodies.
The three-dimensional structure of a specific antibody (Fab 17/9) to a peptide immunogen from influenza virus hemagglutinin [HA1(75-110)] and two independent crystal complexes of this antibody with bound peptide (TyrP100-LeuP108) have been determined by x-ray crystallographic techniques at 2.0 A, 2.9 A, and 3.1 A resolution, respectively. The nonapeptide antigen assumes a type I beta turn in the antibody combining site and interacts primarily with the Fab hypervariable loops L3, H2, and H3. Comparison of the bound and unbound Fab structures shows that a major rearrangement in the H3 loop accompanies antigen binding. This conformational change results in the creation of a binding pocket for the beta turn of the peptide, allowing TyrP105 to be accommodated. The conformation of the peptide bound to the antibody shows similarity to its cognate sequence in the HA1, suggesting a possible mechanism for the cross-reactivity of this Fab with monomeric hemagglutinin. The structures of the free and antigen bound antibodies demonstrate the flexibility of the antibody combining site and provide an example of induced fit as a mechanism for antibody-antigen recognition.
Human immunodeficiency virus type I (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev activity is mediated through specific binding to a cis-acting Rev responsive element (RRE) located within the env region of HIV-1. A monomer Rev binding site corresponding to 37 nucleotides of the RRE (IIB RNA) was studied by RNA footprinting, modification interference experiments and mutational analysis. Surprisingly, a 17 amino acid peptide, corresponding to the basic domain of Rev, binds specifically to this site at essentially identical nucleotides and probably induces additional base pairing. The Rev protein and related peptide interact primarily with two sets of nucleotides located at the junction of single and double stranded regions, and at an additional site located within a helix. This suggests that the domains of proteins responsible for specific RNA binding can be remarkably small and that the interaction between RNA and protein can probably induce structure in both constituents.
There are two methods of predicting secondary structure: phylogeny and energy minimization. Phylogeny relies on alignment and subsequent folding of several sequences into similar structures for functionally analogous RNA. Energy minimization relies on thermodynamic parameters and computer. There are two versions of the program: LRNA folds linear sequences, and CRNA folds circular sequences. LRNA and CRNA have the same user interface. Information about the structure helps the program fold the sequence into a better secondary structure. The locations of single-stranded sites, double-stranded sites, or known helixes are easily entered in LRNA. In batch mode, a sequence of commands in a batch file is run non-interactively, usually in nonprime time. The user can type in the batch file using an editor or the interactive program BATGEN. BATGEN first asks for the batch file name and the header file name. The header file contains information placed at the top of the batch file, such as a system command to change directories.
Replication of human immunodeficiency virus requires binding of the viral Tat protein to its RNA target sequence TAR; peptides derived from Tat bind to a TAR "contact site" spanning 5 bp and a trinucleotide pyrimidine bulge. We find that high affinity binding requires a U residue in the bulge loop and 2 specific adjacent base pairs. Other bulged RNAs bind in a lower affinity nonspecific manner; sequence-specific binding requires a bulge loop of more than 1 nucleotide. Reaction with diethyl pyrocarbonate indicates that one effect of the bulge is to make the otherwise deep and narrow RNA major groove accessible. A model consistent with these data involves local distortion of A-form geometry at the bulge, which bends the helix and permits protein binding and interactive access in the RNA major groove.
Complex retroviruses, including Human Immunodeficiency Virus Type 1 (HIV-1), are characterized by the ordered temporal expression of the various viral gene products in infected cells. This effect is mediated by a novel class of RNA-sequence-specific regulatory proteins typified by the Rev trans-activator of HIV-1. Evidence suggests that Rev regulates HIV-1 gene expression by intervening in the normal pathway of eukaryotic mRNA processing and transport.
The three-dimensional structures of an antibody to a peptide and its complex with the peptide antigen have been determined at 2.8 A resolution. The antigen is a synthetic 19-amino acid peptide homolog of the C helix of myohemerythrin (Mhr). The unliganded Fab' crystals are orthorhombic with two molecules per asymmetric unit, whereas the complex crystals are hexagonal with one molecule per asymmetric unit. The Fab' and the Fab'-peptide complex structures have been solved independently by molecular replacement methods and have crystallographic R factors of 0.197 and 0.215, respectively, with no water molecules included. The amino-terminal portion of the peptide sequence (NH2-Glu-Val-Val-Pro-His-Lys-Lys) is clearly interpretable in the electron density map of the Fab'-peptide complex and adopts a well-defined type II beta-turn in the concave antigen binding pocket. This same peptide amino acid sequence in native Mhr is alpha-helical. The peptide conformation when bound to the Fab' is inconsistent with binding of the Fab' to native Mhr, and suggests that binding of the Fab' to conformationally altered forms of the native Mhr or to apo-Mhr. Immunological mapping previously identified this sequence as the peptide epitope, and its fine specificity correlates well with the structural analysis. The binding pocket includes a large percentage of hydrophobic residues. The buried surfaces of the peptide and the antibody are complementary in shape and cover 460 A2 and 540 A2, respectively. These two structures now enable a comparison of a specific monoclonal Fab' both in its free and antigen complexed state. While no major changes in the antibody were observed when peptide was bound, there were some small but significant side chain and main chain rearrangements.
Sets of peptides representing all the possible hepta-, octa-, nona- and decapeptides of sperm whale myoglobin were synthesized. An ELISA method was used to detect the ability of antibodies, present in antisera raised against native sperm whale myoglobin, to bind to these peptides. Antisera made in two species were compared. It was found that the peptides recognized by the antibodies were a function of the species in which the antiserum was prepared and of the individual outbred member of that species. Peptides corresponding to surface epitopes of the native antigen were identified by reacting the antisera with native antigen prior to ELISA testing on peptides. More detailed analysis of one epitope revealed that, for some sera, a leucine residue which is facing inwards in the crystal structure is critical for the binding of antibody to the peptide. This suggests that binding between native antigen and antibody can require a restructuring of the native antigen.
A procedure is described for rapid concurrent synthesis on solid supports of hundreds of peptides, of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner an immunogenic epitope of the immunologically important coat protein of foot-and-mouth disease virus (type O1) is located with a resolution of seven amino acids, corresponding to amino acids 146-152 of that protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope was synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. It was found that the leucine residues at positions 148 and 151 were essential for reaction with antisera raised against intact virus. A lesser contribution was derived from the glutamine and alanine residues at positions 149 and 152, respectively. Aside from the practical significance for locating and examining epitopes at high resolution, these findings may lead to better understanding of the basis of antigen-antibody interaction and antibody specificity.
It has become clear that antibodies in most regions of a protein can be induced by immunizing with short synthetic peptides. The synthetic immunogenic technology has already had impact on diverse areas of biology and medicine. Because the region in the protein with which antipeptide antibodies react is known in advance to the experimenter, they can be said to be of predetermined specificity. Also, the predetermined specificity of these antibodies allows protein chemists and cell biologists to carry out precise structure–function experiments and to orient proteins in cellular compartments and subcellular organelles. Protein reactive antipeptide antibodies are being used to study the chemistry and structure of antigen–antibody union. The synthetic immunogen technology has already led us a long way toward answers in this area. Most importantly, the frequency with which protein- reactive antipeptide antibodies can be generated has led to a conceptual and experimental merger between the antibody and protein dynamics problems. It is a curious paradox that although almost all biological processes are mediated by proteins, very few proteins are useful as therapeutics. Proteins are labile, often destroyed by the acid pH of the stomach, unable to cross cell membranes or the blood–brain barrier. The process to design organic mimics of proteins is that of antibody template directed organic synthesis.
Interactions between the Rex protein of HTLV-1 and the genomic Rex-binding element (XBE) mediate the cytoplasmic transport of viral mRNAs. However, it is uncertain which RNA sequences and structures contribute to Rex recognition. A portion of the viral genome that spanned the XBE was partially randomized, and functional Rex-binding variants were selected. Alignment of selected Rex-binding sequences revealed positions that were functionally conserved between different molecules. A model is presented in which a subset of the selected residues are in direct contact with Rex. Positions that covaried with one another were also found. These covariations support a secondary-structural model in which a central paired stem is symmetrically flanked by two bulge loops. On the basis of this model, site-directed mutations of the XBE were constructed and each half molecule was found to bind independently to Rex. The functional residues and secondary structures in the XBE half molecules bear a remarkable resemblance to the transactivation response region element of HIV-1. Since the transactivation response region element is known to interact specifically with arginine residues in the Tat protein, these results suggest that the XBE binds to the arginine-rich RNA-binding domain of Rex in a similar manner. This model is supported by the selection data.
RNA molecules that can bind to the Rev protein of HIV-1 have been isolated from random sequence nucleic acid pools based on
a minimal Rev-binding element (RBE) found within the Rev Responsive Element (RRE). While the selected sequences are related to the wild-type element, they also contain substitutions that allow them to bind
Rev up to 10-fold better in vitro. A hypothesized homopurine pairing at G48:G71 is generally replaced by A48:A71; the occasional selection of C48:A71 suggests
that R71 may be in a syn conformation. These data support the structural model for the RBE originally proposed by Bartel et al. (1). Additional interactions with the Rev protein are promoted by the sequence CUC … UYGAG, found in one class of high-affinity
aptamers, but absent from the wild-type element. Within each class of aptamers different residues and substructures covary
with one another to generate optimal Rev-binding surfaces. The interdependencies of different nucleotide substitutions suggest
structural models for both the wild-type RBE and the selected high-affinity aptamers.
Specific, high-affinity RNA ligands to avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases were isolated from a combinatorial RNA library using the SELEX (systematic evolution of ligands by exponential enrichment) procedure. The selected RNA ligands bound their respective reverse transcriptases with approximately nanomolar dissociation constants. The ligands did not exhibit primary sequence conservation from selections against different target enzymes. Moreover, the selected ligands competed with the binding of template/primer complex and inhibited both the RNA-dependent DNA polymerase and the RNase H activities of the cognate reverse transcriptase. SELEX can yield both high-affinity and high-specificity oligonucleotide antagonists against specific members of a protein family.
RNA ligands to the tachykinin substance P have been selected from a large pool of random sequence RNA molecules. Substance P is an undecapeptide that plays a variety of roles as a neurotransmitter and neuromodulator in the central and peripheral nervous system of mammals. A systematic evolution of ligands by exponential enrichment (SELEX) procedure was used to isolate RNAs that bind substance P immobilized on a solid support. RNAs that also bind substance P in solution were identified, and the tightest binder was subjected to mutagenesis in a second SELEX procedure to evolve ligands with a higher affinity for the peptide. A comparative analysis of 36 ligands isolated from the second SELEX experiment revealed two main sequence classes with highly conserved secondary structures within each class. Dissociation constants for the interaction of these ligands with substance P in solution were determined by equilibrium dialysis. The amino acid residues involved in the interaction with the highest affinity ligand (190 nM Kd) were mapped by determining which of a set of overlapping fragments of substance P can compete with the intact peptide for binding. A binding competition experiment also demonstrated the ability of the same ligand to discriminate between substance P and the reverse orientation of the same amino acid sequence. The results from this study demonstrate that SELEX can yield high affinity RNA ligands to small nonconstrained peptides.
The crystal structure of the complex between the Fab fragment of C3, a neutralizing antibody for poliovirus, and a peptide corresponding to the viral epitope has been determined at 3.0 A resolution. Although this antibody was originally raised to heat inactivated (noninfectious) virus particles, it strongly neutralizes the Mahoney strain of type 1 poliovirus. Eleven peptide residues are well-defined in the electron-density map and form two type I beta-turns in series. At the carboxyl end, the peptide is bound snugly in the antibody-combining site and adopts a conformation that differs significantly from the structure of the corresponding residues in the virus. Structural comparisons between the peptide in the complex and the viral epitope suggests that on binding to infectious virions, this antibody may induce structural changes important for neutralization.
SELEX is a technology for the identification of high affinity oligonucleotide ligands. Large libraries of random sequence single-stranded oligonucleotides, whether RNA or DNA, can be thought of conformationally not as short strings but rather as sequence dependent folded structures with high degrees of molecular rigidity in solution. This conformational complexity means that such a library is a source of high affinity ligands for a surprising variety of molecular targets, including nucleic acid binding proteins such as polymerases and transcription factors, non-nucleic acid binding proteins such as cytokins and growth factors, as well as small organic molecules such as ATP and theophylline. The range of applications of this technology for new discovery extends from basic research reagents to the identification of novel diagnostic and therapeutic reagents. Examples of these applications are described along with a discussion of underlying principles and future developments expected to further the utility of SELEX.
A 17 amino acid peptide containing the arginine-rich region of the HIV Rev protein binds specifically to Rev response element (RRE) RNA. Even though it is highly charged, the peptide forms an alpha helix in solution, but only when its N- and C-termini are modified to provide favorable electrostatic interactions with the helix macrodipole. Binding affinity for IIB RNA (the primary binding site within the RRE) increases with alpha helix content, whereas nonspecific binding affinity is independent of helix content. Binding of mutant peptides demonstrates that one threonine, one asparagine, and four arginine side chains are important for sequence-specific recognition. Transactivation of the HIV LTR using Tat-Rev peptide hybrids and the RRE IIB site indicates that the peptide adopts an alpha-helical conformation in vivo. The results suggest that interactions with the RNA backbone may help to orient the alpha helix in the major groove of RNA.
Proteins can interact with short peptide sequences in a variety of ways that can be sequence dependent or independent. The bound peptides are frequently in an extended conformation but may also adopt beta-turns or alpha-helices as motifs for recognition. The peptides can be completely buried in cavities, bound in grooves or pockets, or form beta-strand type interactions at the protein surface. These various recognition motifs are illustrated by peptide interactions with antibodies, calmodulin, OppA periplasmic binding protein, PapD chaperone, MHC class I and class II molecules, and Src homology (SH) domains 2 and 3.
Arginine-rich domains are used by a variety of RNA-binding proteins to recognize specific RNA hairpins. It has been shown previously that a 17-aa arginine-rich peptide from the human immunodeficiency virus Rev protein binds specifically to its RNA site when the peptide is in an alpha-helical conformation. Here we show that related peptides from splicing factors, viral coat proteins, and bacteriophage antiterminators (the N proteins) also have propensities to form alpha-helices and that the N peptides require helical conformations to bind to their cognate RNAs. In contrast, introducing proline mutations into the arginine-rich domain of the human immunodeficiency virus Tat protein abolishes its potential to form an alpha-helix but does not affect RNA-binding affinity in vitro or in vivo. Based on results from several peptide-RNA model systems, we suggest that helical peptides may be used to recognize RNA structures having particularly wide major grooves, such as those found near loops or large bulges, and that nonhelical or extended peptides may be used to recognize less accessible grooves.
An arginine-rich peptide corresponding to amino acids 34-50 of the human immunodeficiency virus Rev protein has been shown to bind specifically to its RNA-binding site (RRE) when the peptide is in an alpha-helical conformation. Mutation of any one of six amino acids (Thr34, Arg35, Arg38, Arg39, Asn40, or Arg44) was shown to strongly decrease specific RNA-binding affinity in vitro, suggesting that these residues may contact specific bases or distinct structural features of the RNA. We now show that the four arginine side chains, and not just their charge, are important for specific binding in vivo, and present evidence that three additional arginines (Arg46, Arg48, and Arg50) may make electrostatic contacts to the RRE. RNA-binding specificity of the Rev peptide is temperature-dependent in vitro, correlating with alpha-helix unfolding. Circular dichroism experiments indicate that the peptide helical structure is stabilized when bound specifically to the RRE and that the RNA undergoes a conformational change upon binding. Because the structures of the peptide and RNA in this model system appear to be mutually stabilized upon binding, it is suggested that the entire complex may be viewed as a single folding unit.
Tourist was originally described as a 128-bp insertion mutation in the maize wx-B2 allele. Subsequent analysis revealed that Tourist elements are in the introns or flanking sequences of 11 maize genes and a single barley gene. In this study we report that Tourist elements are frequently associated with the wild-type genes of two other grasses, rice and sorghum. Six of 35 rice and 5 of 8 sorghum complete gene sequences reported to date contain Tourist elements. Furthermore, 11 additional maize genes have been found to contain Tourist elements, bringing the current total of elements associated with maize genes to 23. Sequence comparison of Tourist elements has led to the identification of four subfamilies, designated A-D. Evidence is presented for the recent mobility of elements in three of these subfamilies and in three of the four grass species. These data suggest that Tourist elements are highly repetitive in the genomes of some and perhaps all members of the grasses.
The Rev responsive element (RRE) is an RNA secondary structural element within the env gene of HIV and is the binding site for the viral Rev protein. Formation of the Rev-RRE complex is involved in regulation of splicing and transport of mRNA from the nucleus. To understand the structural basis for the specific recognition of RRE by Rev, we have studied a model system for this interaction using NMR. We have obtained a specific 1:1 complex between an RNA derived from stem IIB of RRE, which contains the highest affinity Rev binding site, and a modified Rev34-50 peptide, which binds the RRE as an alpha-helix [Tan, R., et al. (1993) Cell 73, 1031-1040]. Binding of the peptide was accompanied by a conformational change in the RNA, which resulted in the formation of additional base pairs not present in the free RNA. Two of these induced base pairs are purine-purine pairs within the internal loop of RRE, which had been previously proposed on the basis of biochemical experiments [Bartel, D.P., et al. (1991) Cell 67, 529-536]. The formation of non-Watson-Crick base pairs, interactions in the major groove, and protein-induced conformational changes may prove to be common characteristics of RNA recognition of proteins.
Genomic and cDNA clones of a gene expressed after a blood meal in the mosquito, Aedes aegypti, were identified as having significant similarity to the vitelline membrane protein genes of Drosophila melanogaster. The predicted protein had unusually high contents of alanine, histidine, and proline and contained a region of hydrophobic amino acids that was highly conserved in the predicted protein of the D. melanogaster vitelline membrane protein genes. The 15a gene was expressed from 5 to 40 hr after a blood meal. It was expressed only in the follicle cells of the ovary, particularly in the cells surrounding the oocyte. The 15a gene was expressed in ovaries of the blood-fed, decapitated female in response to an injection of 20-hydroxyecdysone, and in ovaries from non-blood-fed females incubated with the hormone, even in the presence of cycloheximide. A second gene, with weaker homology to 15a, is presumably another member of a family of related genes, as is the case with D. melanogaster vitelline membrane protein genes. This second gene contained a coding sequence similar to a decapeptide recently isolated from mosquito ovaries as an "oostatic factor" (Borovsky et al., FASEB J. 4, 3015-3020, 1990).
A single point mutation in the insect gamma-aminobutyric acid receptor (GABAR)-encoding gene (Rdl) confers high levels of resistance to cyclodienes in Drosophila and other insects. We were interested in studying the promoter of this gene for two reasons. Firstly, to define the elements underlying Rdl expression. Secondly, to identify the minimum set of regulatory elements necessary for construction of a functional Rdl minigene. Such an insecticide-resistance-associated minigene should form a strong selectable marker for use in the genetic transformation of non-drosophilid pest insects, such as mosquitoes. Here, we report the identification of the region containing the rdl promoter, via transient expression of a luc reporter gene following micro-injection into embryos of the mosquito Aedes aegypti. Promoter activity is contained within a 2.53-kb fragment immediately upstream from the rdl start codon. Primer extension shows three closely linked sites for transcript initiation within this region and sequence analysis reveals anumber of putative consensus regulatory sequences shared by other genes expressed in the nervous system. The implications for construction of a functional minigene and the identification of cis-acting control elements underlying ion-channel gene regulation are discussed.
There are approximately 1.4 million organisms on this planet that have been described morphologically but there is no comparable coverage of biodiversity at the molecular level. Little more than 1% of the known species have been subject to any molecular scrutiny and eukaryotic genome projects have focused on a group of closely related model organisms. The past year, however, has seen an approximately 80% increase in the number of species represented in sequence databases and the completion of the sequencing of three prokaryotic genomes. Large-scale sequencing projects seem set to begin coverage of a wider range of the eukaryotic diversity, including green plants, microsporidians and diplomonads.
Vitellogenic carboxypeptidase is a 53 kDa yolk protein produced by the fat body of the female mosquito, Aedes aegypti, in response to a blood meal. Its expression is sex-, stage- and tissue-specific and is identical to that of the major yolk protein, vitellogenin. The gene is intronless and two alleles have been cloned and sequenced, including more than 1.5 kb on both sides of the coding region. A capsite consensus recently identified as an arthropod initiator is present at the start site of transcription. Upstream of this capsite is a 16 bp imperfect palindrome repeated four times showing strong homology to defined hormone-response elements. In addition, a region that closely resembles the fat body enhancer and double sex binding site from the Drosophila yolk protein genes and several potential fat body-specific regulatory protein binding sites were found.
In vitro selection, or SELEX, has been used both to characterize the interaction of natural nucleic acids with proteins and to generate novel nucleic acid-binding species, or aptamers. Although numerous reports have demonstrated the power of the technique, they have not expanded on the methodologies that can be used for selection. This review focuses on the considerations and problems involved in selecting protein-binding aptamers from a random-sequence RNA pool. As an illustration, we describe two approaches to selecting aptamers to a particular target, the HTLV-I Rex protein. In the first, complete randomization is used to find an artificial, high-affinity RNA binding site. In the second, the contributions of individual nucleotides and/or base pairs to the natural Rex-binding element are determined by mutating the wild-type sequence and selecting active binding variants.
It has been hypothesized that the fact that both ribosomal RNA and the group I intron can bind to aminoglycoside antibiotics implies that these RNAs are evolutionarily related. This hypothesis requires the assumption that there are relatively few ways for RNA molecules to form aminoglycoside-binding sites.
We have used in vitro selection to determine the diversity of aminoglycoside-binding sites that can be formed by RNA molecules. We have generated RNA 'lectins' that can bind aminoglycosides tightly and specifically. Sequence analysis indicates that there are many different ways to form tight and specific aminoglycoside binding sites. These artificially selected binding sites are functionally similar to those that have arisen from natural selection.
Our results suggest that the presence of aminoglycoside-binding sites on RNA molecules may not be a useful trait for determining evolutionary relatedness. Instead, the fact that RNA molecules can bind these 'low molecular-weight effectors' may indicate that natural products such as aminoglycosides have evolved to exploit sequence- and structure-specific recognition of nucleic acids, in much the same way that lexitropsins have been designed by chemists to recognise specific nucleic acid sequences.
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