Random Sequence Libraries Research Articles

Generalized Strategy for Engineering Mammalian Cell-Compatible RNA-Based Biosensors from Random Sequence Libraries.

Fluorescent RNA-based biosensors are useful tools for real-time detection of molecules in living cells. These biosensors typically consist of a chromophore-binding aptamer and a target-binding aptamer, whereby the chromophore-binding aptamer is destabilized until a target is captured, which causes a conformational change to permit chromophore binding and an increase in fluorescence. The target-binding region is typically fabricated using known riboswitch motifs, which are already known to have target specificity and undergo structural changes upon binding. However, known riboswitches only exist for a limited number of molecules, significantly constraining biosensor design. To overcome this challenge, we designed a framework for producing mammalian cell-compatible biosensors using aptamers selected from a large random library by Capture-SELEX. As a proof-of-concept, we generated and characterized a fluorescent RNA biosensor against L-dopa, the precursor of several neurotransmitters. Overall, we suggest that this approach will have utility for generating RNA biosensors that can reliably detect custom targets in mammalian cells.

Rescue of Escherichia coli auxotrophy by de novo small proteins.

Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here, we show that by upregulating hisB expression, de novo small proteins (≤50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5' end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.

Aptamer-based cell-free detection system to detect target protein

Biomarkers of disease, especially protein, show great potential for diagnosis and prognosis. For detecting a certain protein, a binding assay implementing antibodies is commonly performed. However, antibodies are not thermally stable and may cause false-positive when the sample composition is complicated. In recent years, a functional nucleic acid named aptamer has been used in many biochemical analysis cases, which is commonly selected from random sequence libraries by using the systematic evolution of ligands by exponential enrichment (SELEX) techniques. Compared to antibodies, the aptamer is more thermal stable, easier to be modified, conjugated, and amplified. Herein, an Aptamer-Based Cell-free Detection (ABCD) system was proposed to detect target protein, using epithelial cell adhesion molecule (EpCAM) as an example. We combined the robustness of aptamer in binding specificity with the signal amplification ability of CRISPR-Cas12a′s trans-cleavage activity in the ABCD system. We also demonstrated that the ABCD system could work well to detect target protein in a relatively low limit of detection (50–100 nM), which lay a foundation for the development of portable detection devices. This work highlights the superiority of the ABCD system in detecting target protein with low abundance and offers new enlightenment for future design and development.

Secondary Structure Libraries for Artificial Evolution Experiments.

Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.

Developing RNA aptamers for potential treatment of neurological diseases.

AMPA receptor antagonists are drug candidates for potential treatment of a number of CNS diseases that involve excessive receptor activation. To date, small-molecule compounds are the dominating drug candidates in the field. However, lower potency, cross activity and poor water solubility are generally associated with these compounds. Here we show the potential of RNA-based antagonists or RNA aptamers as drug candidates and some strategies to discover these aptamers from a random sequence library (∼1014 sequences). As an alternative to small molecule compounds, our aptamers exhibit higher potency and selectivity toward AMPA receptors. Because aptamers are RNA molecules, they are naturally water soluble. We also discuss the major challenges of translating RNA aptamers as lead molecules into drugs/treatment options.

Selection of self-priming molecular replicators.

Self-priming amplification of oligonucleotides is possible based on foldback of 3′ ends, self-priming, and concatemerization, especially in the presence of phosphorothioate linkages. Such a simple replicative mechanism may have led to the accumulation of specific replicators at or near the origin of life. To determine how early replicators may have competed with one another, we have carried out selections with phosphorothiolated hairpins appended to a short random sequence library (N10). Upon the addition of deoxynucleoside triphosphates and a polymerase, concatemers quickly formed, and those random sequences that templated the insertion of purines, especially during initiation, quickly predominated. Over several serial transfers, particular sequences accumulated, and in isolation these were shown to outcompete less efficient replicators.

Enzymatic Preparation of Modified DNA: Study of the Kinetics by Real-Time PCR

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).

Engineering Riboswitches in Vivo Using Dual Genetic Selection and Fluorescence-Activated Cell Sorting.

Riboswitches, noncoding RNAs that bind a small molecule effector to control gene expression at the level of transcription or translation, are uniquely suited to meet challenges in synthetic biology. To expand the limited set of existing riboswitches, we developed a riboswitch discovery platform that couples dual genetic selection and fluorescence-activated cell sorting to identify novel riboswitches from a 108 random-sequence library in which the aptamer domain of the ThiM#2 riboswitch was replaced with an N40 sequence. In a proof-of-principle validation, we identified novel riboswitches for the small molecule theophylline. Our best riboswitch (Hit 3-5) displays 2.3-fold activation of downstream gene expression in the presence of theophylline. Random mutagenesis of Hit 3-5, coupled with selections and screens, afforded improved riboswitches displaying nearly 3-fold activation. To the best of our knowledge, this is the first report of in vivo directed evolution of an aptamer domain to generate a functional riboswitch.

The FGF2 aptamer inhibits the growth of FGF2-FGFR pathway driven lung cancer cells

Cancers, including lung cancer, are a leading cause of death worldwide. To overcome this deadly disease, multiple modality inhibitors have been developed. These include cytotoxic agents, molecular targeted small molecules, such as tyrosine kinase inhibitors, and neutralizing antibodies. An aptamer is a short single-stranded nucleic acid molecule that is selected in vitro from a large random sequence library based on its high and specific affinity to a target molecule. Aptamers can be applied to therapeutics of various types of diseases, including cancer, due to their strong and specific neutralizing activities. However, the efficacy of aptamer-based therapy for cancer cells is not well characterized. In this study, we aimed to show that the FGF2 aptamer is effective for the treatment of FGF2 dependent lung cancer cells. We previously developed PC9GR lung cancer cells, whose proliferation is dependent on EGFR and FGF2-FGFR pathways in a cell autonomous manner. Using PC9GR cells, we demonstrate that the addition of the FGF2 aptamer induces more significant inhibition of PC9GR cell proliferation than does the addition of EGFR inhibitor alone. Furthermore, the addition of the FGF2 aptamer more significantly inhibits the downstream signals and induces apoptosis to a higher extent than does the addition of EGFR inhibitor alone. Our results show that the FGF2 aptamer inhibits the growth of FGF2-FGFR pathway-dependent lung cancer cells. The findings provide preclinical evidence that aptamers can be useful for cancer treatment.

Design of a glutamine substrate tag enabling protein labelling mediated by Bacillus subtilis transglutaminase.

Transglutaminases (TGases) are enzymes that catalyse protein cross-linking through a transamidation reaction between the side chain of a glutamine residue on one protein and the side chain of a lysine residue on another. Generally, TGases show low substrate specificity with respect to their amine substrate, such that a wide variety of primary amines can participate in the modification of specific glutamine residue. Although a number of different TGases have been used to mediate these bioconjugation reactions, the TGase from Bacillus subtilis (bTG) may be particularly suited to this application. It is smaller than most TGases, can be expressed in a soluble active form, and lacks the calcium dependence of its mammalian counterparts. However, little is known regarding this enzyme and its glutamine substrate specificity, limiting the scope of its application. In this work, we designed a FRET-based ligation assay to monitor the bTG-mediated conjugation of the fluorescent proteins Clover and mRuby2. This assay allowed us to screen a library of random heptapeptide glutamine sequences for their reactivity with recombinant bTG in bacterial cells, using fluorescence assisted cell sorting. From this library, several reactive sequences were identified and kinetically characterized, with the most reactive sequence (YAHQAHY) having a kcat/KM value of 19 ± 3 μM-1 min-1. This sequence was then genetically appended onto a test protein as a reactive ‘Q-tag’ and fluorescently labelled with dansyl-cadaverine, in the first demonstration of protein labelling mediated by bTG.

Native Electrospray Ionization Mass Spectrometry Reveals Multiple Facets of Aptamer-Ligand Interactions: From Mechanism to Binding Constants.

Aptamers are oligonucleotide receptors obtained through an iterative selection process from random-sequence libraries. Though many aptamers for a broad range of targets with high affinity and selectivity have been generated, a lack of high-resolution structural data and the limitations of currently available biophysical tools greatly impede understanding of the mechanisms of aptamer-ligand interactions. Here we demonstrate that an approach based on native electrospray ionization mass spectrometry (ESI-MS) can be successfully applied to characterize aptamer-ligand complexes in all details. We studied an adenosine-binding aptamer (ABA), a l-argininamide-binding aptamer (LABA), and a cocaine-binding aptamer (CBA) and their noncovalent interactions with ligands by native ESI-MS and complemented these measurements by ion mobility spectrometry (IMS), isothermal titration calorimetry (ITC), and circular dichroism (CD) spectroscopy. The ligand selectivity of the aptamers and the respective complex stoichiometry could be determined by the native ESI-MS approach. The ESI-MS data can also help refining the binding model for aptamer-ligand complexes and deliver accurate aptamer-ligand binding affinities for specific and nonspecific binding events. For specific ligands, we found Kd1 = 69.7 μM and Kd2 = 5.3 μM for ABA (two binding sites); Kd1 = 22.04 μM for LABA; and Kd1 = 8.5 μM for CBA.

High‐throughput discovery of functional disordered regions: investigation of transactivation domains

Over 40% of proteins in any eukaryotic genome encode intrinsically disordered regions (IDRs) that do not adopt defined tertiary structures. Certain IDRs perform critical functions, but discovering them is non‐trivial as the biological context determines their function. We present IDR‐Screen, a framework to discover functional IDRs in a high‐throughput manner by simultaneously assaying large numbers of DNA sequences that code for short disordered sequences. Functionality‐conferring patterns in their protein sequence are inferred through statistical learning. Using yeast HSF1 transcription factor‐based assay, we discovered IDRs that function as transactivation domains (TADs) by screening a random sequence library and a designed library consisting of variants of 13 diverse TADs. Using machine learning, we find that segments devoid of positively charged residues but with redundant short sequence patterns of negatively charged and aromatic residues are a generic feature for TAD functionality. We anticipate that investigating defined sequence libraries using IDR‐Screen for specific functions can facilitate discovering novel and functional regions of the disordered proteome as well as understand the impact of natural and disease variants in disordered segments.

Engineering riboswitch‐based whole cell biosensors in Escherichia coli

In vitro diagnostics (IVD) have become increasingly popular tool for tackling global health concerns, but conventional IVD tests, such as microscopy and nucleic‐acid amplification methods, are often expensive and complex to use, limiting their use in low‐resource settings. Whole‐cell biosensors in bacterial systems offer an attractive solution as an easy‐to‐use, portable device that non‐specialists could use for clinical purposes outside clinical locations. However, a strategy is needed to engineer ligand specific sensors for new targets. An appealing candidate for a sensing module is the riboswitch. Genetic RNA switches, termed riboswitches, are genetic regulatory elements found in the 5′‐UTR (untranslated region) of some prokaryotic mRNA and contain two functional components: an aptamer binding domain and an expression platform that modulates downstream gene expression. We developed an engineering platform coupling genetic selections and Fluorescence‐Activated Cell Sorting (FACS) screens to identify novel riboswitches from a 108 random‐sequence library in which we replaced the aptamer sequence of the ThiM#2 riboswitch with a degenerate 40‐nucleotide sequence. In a proof‐of‐principle validation of the platform, we identified novel riboswitches for the small molecule theophylline, which are distinct from the existing synthetic theophylline switches. Our best riboswitch hit displays a 2.3‐fold activation in response to theophylline and does not respond to the structurally unrelated molecule thiamine. We performed directed evolution on this riboswitch hit by mutagenizing its sequence and performing additional rounds of selections and screens, resulting in a variant with a 3.6‐fold activation. Current efforts are focused on assessing the efficacy of the selection platform when isolating riboswitches for dopamine. Furthermore, a variant of the engineering platform is being used side‐by‐side to compare if a dopamine riboswitch can be selected from a library composed of an in vitro selected aptamer coupled with a 15‐nucleotide degenerate sequence. We anticipate that these efforts will provide general guidelines toward the development of novel riboswitches for a variety of natural and non‐natural ligands.Support or Funding InformationNSF CBET 1258307Arnold and Mabel Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterizing mRNA Sequence Motifs in the 3'-UTR Using GFP Reporter Constructs.

GFP reporter constructs are widely used as an expression system for studying the function of regulatory sequence motifs (cis elements) within the 3'-UTRs (3' untranslated regions) of mRNAs. Here we provide details on the characterization of individual sequence motifs, which typically regulate mRNA decay and translation. In addition, we describe methods to identify trans factors required for the function of such elements. To facilitate efficient identification of novel functional 3'-UTR motifs, we describe a screening approach based on dual-color fluorescence reporter constructs. Such screening approaches can be used to test large collections of defined sequence or libraries of random sequences.

Foldability of a Natural De Novo Evolved Protein

The de novo evolution of protein-coding genes from noncoding DNA is emerging as a source of molecular innovation in biology. Studies of random sequence libraries, however, suggest that young de novo proteins will not fold into compact, specific structures typical of native globular proteins. Here we show that Bsc4, a functional, natural de novo protein encoded by a gene that evolved recently from noncoding DNA in the yeast S.cerevisiae, folds to a partially specific three-dimensional structure. Bsc4 forms soluble, compact oligomers with high β sheet content and a hydrophobic core, and undergoes cooperative, reversible denaturation. Bsc4 lacks a specific quaternary state, however, existing instead as a continuous distribution of oligomer sizes, andbinds dyes indicative of amyloid oligomers or molten globules. The combination of native-like and non-native-like properties suggests a rudimentary fold that could potentially act as a functional intermediate in the emergence of new folded proteins de novo.

A Method for Large-Scale Screening of Random Sequence Libraries to Determine the Function of Unstructured Regions from Essential Proteins.

In this chapter we present a method allowing the screening of random sequences to discover essential aspects of unstructured protein regions in yeast. The approach can be applied to any protein with unstructured peptide sequences for which functions are difficult to decipher, for example the N-terminal tails of histones. The protocol first describes the building and preparation of a large library of random peptides in fusion with a protein of interest. Recent technical advances in oligonucleotide synthesis allow the construction of long random sequences up to 35 residues long. The protocol details the screening of the library in yeast for sequences that can functionally replace an unstructured domain in an essential protein in vivo. Our method typically identifies sequences that, while being totally different from the wild type, retain essential features allowing yeast to live. This collection of proteins with functional synthetic sequences can subsequently be used in phenotypic tests or genetic screens in order to discover genetic interaction.

The efficient cell-SELEX strategy, Icell-SELEX, using isogenic cell lines for selection and counter-selection to generate RNA aptamers to cell surface proteins.

Aptamers are short single-stranded nucleic acid molecules that are selected invitro from a large random sequence library based on their high and specific affinity to a target molecule by a process known as SELEX. Cell-SELEX that employs whole living cells overexpressing the defined cell surface proteins (for selection) and appropriate mock cells (for counter-selection) has been widely used as a valid and feasible method for generating aptamers against specific cell surface proteins. However, the endogenous expression of target proteins in mock cells or the heterogeneity of surface proteins between selection and counter-selection cells often impeded the isolation of proper aptamers against target proteins. To solve this problem, we developed "Isogenic cell-SELEX" (Icell SELEX in short) method, in which isogenic cell lines were manipulated for counter-selection by microRNA-mediated silencing and for selection by overexpression of target proteins. As a model experiment, we targeted integrin alpha V (ITGAV), which is a major transmembrane receptor expressed in almost all the cells, and established ITGAV-overexpressed and -downregulated HEK293cells for selection and counter-selection, respectively. By taking advantage of a hundred-fold difference in the expression level of ITGAV between these two isogenic cell lines, we easily isolated several anti-ITGAV aptamers, whose binding to the cell-surface ITGAV was confirmed by flow cytometry with the dissociation constant of 300-400nM range. We assume that Icell-SELEX could be applicable to a wide range of cell-surface proteins including various transmembrane proteins of biological and pharmacological significance.

A General Method to Discover Epitopes from Sera.

Antigen-antibody complexes are central players in an effective immune response. However, finding those interactions relevant to a particular disease state can be arduous. Nonetheless many paths to discovery have been explored since deciphering these interactions can greatly facilitate the development of new diagnostics, therapeutics, and vaccines. In silico B cell epitope mapping approaches have been widely pursued, though success has not been consistent. Antibody mixtures in immune sera have been used as handles for biologically relevant antigens, but these and other experimental approaches have proven resource intensive and time consuming. In addition, these methods are often tailored to individual diseases or a specific proteome, rather than providing a universal platform. Most of these methods are not able to identify the specific antibody’s epitopes from unknown antigens, such as un-annotated neo antigens in cancer. Alternatively, a peptide library comprised of sequences unrestricted by naturally-found protein space provides for a universal search for mimotopes of an antibody’s epitope. Here we present the utility of such a non-natural random sequence library of 10,000 peptides physically addressed on a microarray for mimotope discovery without sequence information of the specific antigen. The peptide arrays were probed with serum from an antigen-immunized rabbit, or alternatively probed with serum pre-absorbed with the same immunizing antigen. With this positive and negative screening scheme, we identified the library-peptides as the mimotopes of the antigen. The unique library peptides were successfully used to isolate antigen-specific antibodies from complete immune serum. Sequence analysis of these peptides revealed the epitopes in the immunized antigen. We present this method as an inexpensive, efficient method for identifying mimotopes of any antibody’s targets. These mimotopes should be useful in defining both components of the antigen-antibody complex.

An RNA Aptamer Binding With Tumor Endothelial Marker I

The commonest strategy of dealing with cancer depends on availability of tools to detect them at early stage and selectively eliminate the cancer cells. Certain molecules differentially expressed on cancer cell surface offer a basis for developing diagnostic as well as therapeutic tools. Tumor endothelial marker I (TEM I) is one such marker expressed on surface of solid tumors. Beginning with a random sequence library we have selected for RNA molecules specifically binding with a TEMI peptide following a SELEX protocol. Surface plasmon resonance assays showed Kd for some of the TEMI‐binding aptamers to be in nanomolar range. We have also put this molecule in context with cytotoxin‐coding sequence along with an internal ribosome entry site borrowed from ras I. We hope to use this chimeric construct for targeted delivery of cytotoxin to tumor cells. This would result in targeted killing of the tumor cells and can also be used as supplement to surgical removal to ensure any transformed cells escaping surgical removal. We are also developing chimeric molecules with fluorophores for detection of TEM I on cell surface.

Development of a mimotope vaccine targeting the Staphylococcus aureus quorum sensing pathway.

A major hurdle in vaccine development is the difficulty in identifying relevant target epitopes and then presenting them to the immune system in a context that mimics their native conformation. We have engineered novel virus-like-particle (VLP) technology that is able to display complex libraries of random peptide sequences on a surface-exposed loop in the coat protein without disruption of protein folding or VLP assembly. This technology allows us to use the same VLP particle for both affinity selection and immunization, integrating the power of epitope discovery and epitope mimicry of traditional phage display with the high immunogenicity of VLPs. Previously, we showed that using affinity selection with our VLP platform identifies linear epitopes of monoclonal antibodies and subsequent immunization generates the proper antibody response. To test if our technology could identify immunologic mimotopes, we used affinity selection on a monoclonal antibody (AP4-24H11) that recognizes the Staphylococcus aureus autoinducing peptide 4 (AIP4). AIP4 is a secreted eight amino acid, cyclized peptide produced from the S. aureus accessory gene regulator (agrIV) quorum-sensing operon. The agr system coordinates density dependent changes in gene expression, leading to the upregulation of a host of virulence factors, and passive transfer of AP4-24H11 protects against S. aureus agrIV-dependent pathogenicity. In this report, we identified a set of peptides displayed on VLPs that bound with high specificity to AP4-24H11. Importantly, similar to passive transfer with AP4-24H11, immunization with a subset of these VLPs protected against pathogenicity in a mouse model of S. aureus dermonecrosis. These data are proof of principle that by performing affinity selection on neutralizing antibodies, our VLP technology can identify peptide mimics of non-linear epitopes and that these mimotope based VLP vaccines provide protection against pathogens in relevant animal models.