Large DNA Libraries Research Articles

Bio-Constrained Codes with Neural Network for Density-Based DNA Data Storage

DNA has evolved as a cutting-edge medium for digital information storage due to its extremely high density and durable preservation to accommodate the data explosion. However, the strings of DNA are prone to errors during the hybridization process. In addition, DNA synthesis and sequences come with a cost that depends on the number of nucleotides present. An efficient model to store a large amount of data in a small number of nucleotides is essential, and it must control the hybridization errors among the base pairs. In this paper, a novel computational model is presented to design large DNA libraries of oligonucleotides. It is established by integrating a neural network (NN) with combinatorial biological constraints, including constant GC-content and satisfying Hamming distance and reverse-complement constraints. We develop a simple and efficient implementation of NNs to produce the optimal DNA codes, which opens the door to applying neural networks for DNA-based data storage. Further, the combinatorial bio-constraints are introduced to improve the lower bounds and to avoid the occurrence of errors in the DNA codes. Our goal is to compute large DNA codes in shorter sequences, which should avoid non-specific hybridization errors by satisfying the bio-constrained coding. The proposed model yields a significant improvement in the DNA library by explicitly constructing larger codes than the prior published codes.

Ultrahigh-Throughput Detection of Enzymatic Alcohol Dehydrogenase Activity in Microfluidic Droplets with a Direct Fluorogenic Assay.

The exploration of large DNA libraries of metagenomic or synthetic origin is greatly facilitated by ultrahigh‐throughput assays that use monodisperse water‐in‐oil emulsion droplets as sequestered reaction compartments. Millions of samples can be generated and analysed in microfluidic devices at kHz speeds, requiring only micrograms of reagents. The scope of this powerful platform for the discovery of new sequence space is, however, hampered by the limited availability of assay substrates, restricting the functions and reaction types that can be investigated. Here, we broaden the scope of detectable biochemical transformations in droplet microfluidics by introducing the first fluorogenic assay for alcohol dehydrogenases (ADHs) in this format. We have synthesized substrates that release a pyranine fluorophore (8‐hydroxy‐1,3,6‐pyrenetrisulfonic acid, HPTS) when enzymatic turnover occurs. Pyranine is well retained in droplets for >6 weeks (i. e. 14‐times longer than fluorescein), avoiding product leakage and ensuring excellent assay sensitivity. Product concentrations as low as 100 nM were successfully detected, corresponding to less than one turnover per enzyme molecule on average. The potential of our substrate design was demonstrated by efficient recovery of a bona fide ADH with an >800‐fold enrichment. The repertoire of droplet screening is enlarged by this sensitive and direct fluorogenic assay to identify dehydrogenases for biocatalytic applications.

DNAzymes as Activity-Based Sensors for Metal Ions: Recent Applications, Demonstrated Advantages, Current Challenges, and Future Directions.

Metal ions can be beneficial or toxic depending on their identity, oxidation state, and concentration. Therefore, the ability to detect and quantify different types of metal ions using portable sensors or in situ imaging agents is important for better environmental monitoring, in vitro medical diagnostics, and imaging of biological systems. While numerous metal ions in different oxidation states are present in the environment and biological systems, only a limited number of them can be detected effectively using current methods. In this Account, we summarize research results from our group that overcome this limitation by the development of a novel class of activity-based sensors based on metal-dependent DNAzymes, which are DNA molecules with enzymatic activity. First, we have developed an in vitro selection method to obtain DNAzymes from a large DNA library of up to 1015 sequences that can carry out cleavage of an oligonucleotide substrate only in the presence of a specific metal ion with high selectivity. Negative selection steps can further be used to improve the selectivity against potentially competing targets by removing sequences that recognize the competing metal ions. Second, we have developed a patented catalytic beacon method to transform the metal-dependent DNAzyme cleavage reaction into a turn-on fluorescent signal by attaching a fluorophore and quenchers to the DNAzyme complex. Because of the difference in the melting temperatures of DNA hybridization before and after metal-ion-dependent cleavage of the DNAzyme substrate, the fluorophore on the DNA cleavage product can be released from its quenchers to create a turn-on fluorescent signal. Because DNAzymes are easy to conjugate with other signaling moieties, such as gold nanoparticles, lanthanide-doped upconversion nanoparticles, electrochemical agents, and gadolinium complexes, these DNAzymes can also readily be converted into colorimetric sensors, upconversion luminescence sensors, electrochemical sensors, or magnetic resonance contrast agents. In addition to describing recent progress in developing and applying these metal ion sensors for environmental monitoring, point-of-care diagnostics, cellular imaging, and in vivo imaging in zebrafish, we summarize major advantages of this class of activity-based sensors. In addition to advantages common to most activity-based sensors, such as enzymatic turnovers that allow for signal amplification and the use of initial rates instead of absolute signals for quantification to avoid interferences from sample matrices, the DNAzyme-based sensors allow for in vitro selection to expand the method to almost any metal ion under a variety of conditions, negative selection to improve the selectivity against competing targets, and reselection of DNAzymes and combination of active and inactive variants to fine-tune the dynamic range of detection. The use of melting temperature differences to separate target binding from signaling moieties in the catalytic beacon method allows the use of different fluorophores and nanomaterials to extend the versatility and modularity of this sensing platform. Furthermore, sensing and imaging artifacts can be minimized by using an inactive mutant DNAzyme as a negative control, while spatiotemporal control of sensing/imaging can be achieved using optical, photothermal, and endogenous orthogonal caging methods. Finally, current challenges, opportunities, and future perspectives for DNAzymes as activity-based sensors are also discussed.

Recent developments in affinity-based selection of aptamers for binding disease-related protein targets

The early diagnosis of cancerous or infectious diseases is of interest in medical biology and biochemistry. For such purposes, specific proteins such as cancer biomarkers and viral/bacterial proteins have been molecularly targeted by conventional antibodies for rapid and on-site detection. Recently, DNA aptamers have emerged as superior alternatives to the antibodies based on their high binding affinities and advantages in production. To select aptamer sequences from large DNA libraries, Systematic Evolution of Ligands by EXponential enrichment, or SELEX, has been used for almost 30 years. To date, a number of SELEX techniques have been developed to select specific aptamer sequences against cancer biomarkers or viral/bacterial proteins, resulting in an increasing number of discovery of aptamers exhibiting high binding affinities for the target proteins. In this review, we discuss recent (within 3 years) developments in affinity-based selection of DNA aptamers that are specific to disease-related proteins, such as proteins from tumors and viral/bacterial pathogens. The medical and physiological significance of these proteins motivated researchers to make chemical and physical variations to SELEX, which are introduced and discussed.

Competition-Enhanced Ligand Selection to Identify DNA Aptamers.

Competition-enhanced ligand screening (CompELS) was employed to rapidly screen through large DNA libraries to identify single-stranded, oligonucleotide-based ligands called aptamers that bind to a nonbiological target. This previously unreported aptamer screening approach involves the repeated introduction of unenriched random sequence populations during the biopanning process, but avoids iterative elution and polymerase chain reaction (PCR) amplification steps inherent to traditional SELEX (systematic evolution of ligands by exponential enrichment) screening. In this study, 25 aptamers were identified against a gold surface via CompELS and evaluated to identify patterns in primary structures and predicted secondary structures. Following a final one-round competition experiment with the 25 identified aptamers, one particular aptamer sequence (1N) emerged as the most competitive adsorbate species for the gold substrate. Binding analysis indicated at least an order of magnitude difference in the binding affinity of 1N ( Kd = 5.6 × 10-10 M) compared to five other high affinity aptamer candidates ( Kd = 10-8-10-9 M) from identical secondary structure families. Collectively, these studies introduce a rapid, reliable screening and ranking platform along with a classification scheme well-suited for identifying and characterizing aptamers for nonbiological as well as biological targets.

In Vitro Selection in Serum: RNA-Cleaving DNAzymes for Measuring Ca2+ and Mg2+

RNA-cleaving DNAzymes have been attempted as in vivo analytical probes and gene silencing reagents over the past two decades. Despite progress already achieved, concerns still exist regarding the activity of DNAzymes in biological fluids. An example is the low activity of the 10–23 DNAzyme in intracellular Mg2+ concentrations. To obtain DNAzymes that work optimally in biological samples, we herein report the first DNAzyme in vitro selection in undiluted human blood serum. The selection starts with a large DNA library containing 50 random nucleotides, and sequences that can be cleaved in serum were isolated and amplified. After deep sequencing analysis, 80% of the final library are a variant of the 8–17 DNAzyme (named 17EV1). The main difference between 17E and 17EV1 is a single mutation at the N12 position of the catalytic core. 17EV1 is ∼6-fold faster in serum than 17E, since 17EV1 is preferentially activated by Ca2+ and serum is rich in Ca2+ over Mg2+. On the other hand, 17E has a similar activity with ...

A colony multiplex quantitative PCR-Based 3S3DBC method and variations of it for screening DNA libraries.

A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC) method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements.

Understanding DNA-binding specificity by bacteria hybrid selection

Understanding how sequence-specific protein-DNA interactions direct cellular function is of great interest to the research community. High-throughput methods have been developed to determine DNA-binding specificities; one such technique, the bacterial one-hybrid (B1H) system, confers advantages including ease of use, sensitivity and throughput. In this review, we describe the evolution of the B1H system as a tool capable of screening large DNA libraries to investigate protein-DNA interactions of interest. We discuss how DNA-binding specificities produced by the B1H system have been used to predict regulatory targets. Additionally, we examine how this approach has been applied to characterize two common DNA-binding domain families-homeodomains and Cys2His2 zinc fingers-both in organism-wide studies and with synthetic approaches. In the case of the former, the B1H system has produced large catalogs of protein specificity and nuanced information about previously recovered DNA targets, thereby improving our understanding of these proteins' functions in vivo and increasing our capacity to predict similar interactions in other species. In the latter, synthetic screens of the same DNA-binding domains have further refined our models of specificity, through analyzing comprehensive libraries to uncover all proteins able to bind a complete set of targets, and, for instance, exploring how context-in the form of domain position within the parent protein-may affect specificity. Finally, we recognize the limitations of the B1H system and discuss its potential for use in the production of designer proteins and in studies of protein-protein interactions.

Computational methods for the construction, editing, and error correction of DNA molecules and their libraries.

The field of synthetic biology is fueled by steady advances in our ability to produce designer genetic material on demand. This relatively new technological capability stems from advancements in DNA construction biochemistry as well as supporting computational technologies such as tools for specifying large DNA libraries, as well as planning and optimizing their actual physical construction. In particular, the design, planning, and construction of user specified, combinatorial DNA libraries are of increasing interest. Here we present some of the computational tools we have built over the past decade to support the multidisciplinary task of constructing DNA molecules and their libraries. These technologies encompass computational methods for [1] planning and optimizing the construction of DNA molecules and libraries, [2] the utilization of existing natural or synthetic fragments, [3] identification of shared fragments, [4] planning primers and overlaps, [5] minimizing the number of assembly steps required, and (6) correcting erroneous constructs. Other computational technologies that are important in the overall process of DNA construction, such as [1] computational tools for efficient specification and intuitive visualization of large DNA libraries (which aid in debugging library design pre-construction) and [2] automated liquid handling robotic programming [Linshiz et al., Mol Syst Biol 4:191, 2008; Shabi et al., Syst Synth Biol 4:227-236, 2010], which aid in the construction process itself, have been omitted due to length limitations.

Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation

Genetic selection provides the most powerful method to assay large libraries of biomolecules for function. However, harnessing the power of genetic selection for the detection of specific, nonendogenous small-molecule targets in vivo remains a significant challenge. The ability to genetically select for small molecules would provide a reaction-independent mechanism to clone biosynthesis genes from large DNA libraries and greatly facilitate the exploration of large libraries of mutant enzymes for improved synthetic capabilities including altered substrate specificities and enhanced regio- or stereoselectivities. While remarkable progress has been made in developing genetic methods to detect small molecules in vivo, many of these methods rely on engineering small-molecule-protein interactions which remains a difficult problem, and the potential for some of these systems to assay large libraries is limited by the low transformation efficiency and long doubling time of yeast relative to bacteria. Herein, we demonstrate that synthetic riboswitches that activate protein translation in response to a specific small molecule can be used to perform sensitive genetic screens and selections for the presence of small molecules in Escherichia coli. We further demonstrate that the exquisite molecular discrimination properties of aptamers selected in vitro translate directly into an in vivo genetic selection system. Finally, we demonstrate that a cell harboring a synthetic riboswitch with a particular ligand specificity can be selectively amplified from a million-fold larger pool of cells containing mutant riboswitches that respond to a closely related ligand, suggesting that it is possible to use genetic selection in E. coli to discover synthetic riboswitches with new ligand specificities from libraries of mutant riboswitches.

A microarray approach for identifying mutated genes

This study was performed to evaluate if microarray technology can identify genes using their transcriptionally defective mutant alleles. Three barley ( Hordeum vulgare L.) mutant strains, xantha-h 57, xantha-f 27 and xantha-g 28, with mutations in the genes encoding the three subunits of the chlorophyll biosynthetic enzyme magnesium chelatase, were used in a reconstruction experiment. The mutation xantha-h 57 prevents transcription of Xantha-h mRNA. Microarrays were prepared by robotic spotting of PCR products from 968 clones at 1760 positions. Most of the materials were from cloned expressed sequence tags (ESTs). The barley Xantha-h gene was printed at six positions in duplicate. Crude bacterial lysates as well as purified plasmids were used as template in the PCR reactions. The bacterial lysates did not affect the printing quality of the DNA. This simplification is important, as it increases the throughput capacity and decreases costs. cDNA from the three mutants and the wild-type strain were differentially labeled with fluorescing nucleotides. Labeled cDNA from one mutant was mixed in equal amounts with labeled cDNA of another mutant or wild type and competitively hybridized to the microarrayed clones. The combination of labeled cDNA from xantha-h 57 with that of xantha-f 27 or xantha-g 28 specifically highlighted the positions representing the Xantha-h gene on the microarrays. Therefore, we regard this experiment as a demonstration that microarrays provide a very promising method for screening large DNA libraries in order to clone genes known only through their mutant phenotype. This opens up a new way of using the microarray technology for cloning genes from eukaryotes with complex genomes for which genome sequencing is unlikely to proceed. Our results also put the many available plant mutant collections in focus as treasuries for gene hunters.