Off-target Hybridization Research Articles

Selective miRNA quantitation with high-temperature thermal gel electrophoresis

MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression and correlate to the prognosis of numerous diseases. To support research efforts elucidating the roles of miRNAs in pathogenesis, rapid and inexpensive analytical methods are required to quantify miRNAs from biological samples. The challenge of developing new analyses with these time and cost constraints is compounded by the short sequence lengths and high degrees of homology between miRNAs that hinder detection selectivity. This report describes the development of a high-temperature thermal gel electrophoresis (TGE) method to rapidly quantify miRNAs with single-nucleotide resolution using low-cost microfluidic devices. Fluorescent probes were designed for three miRNAs that differed in sequence by one or two nucleotides. A microfluidic analysis was optimized to enrich miRNA-probe hybrids into a high-concentration band and then automatically initiate a separation to resolve each species. Analyses conducted at 30 °C exhibited significant off-target hybridization, as the different-yet-structurally-similar miRNAs bound to each probe, which biased measurements. To overcome this problem, the stability of thermal gels at elevated temperatures was exploited to conduct analyses. At 50 °C, off-target hybrids melted to prevent their detection without impeding the enrichment or separation of on-target hybrids. Selectivity studies validated that high-temperature TGE prevented off-target hybrids from interfering with the quantitative responses of the target miRNAs. This work demonstrates that TGE affords rapid, highly selective analyses of structurally similar miRNAs in low-complexity microfluidic devices, which is expected to facilitate diverse biomedical research.

Whole-Genome Sequencing of Pathogens in Saliva : A Target-Enrichment Approach for SARS-CoV-2.

Outbreak analysis and transmission surveillance of viruses can be performed via whole-genome sequencing after viral isolation. Such techniques have recently been applied to characterize and monitor SARS-CoV-2 , the etiological agent of the COVID-19 pandemic. However, the isolation and culture of SARS-CoV-2 is time consuming and requires biosafety level 3 containment, which is not ideal for many resource-constrained settings. An alternate method, bait capture allows target enrichment and sequencing of the entire SARS-CoV-2 genome eliminating the need for viral culture. This method uses a set of hybridization probes known as "baits" that span the genome and provide sensitive, accurate, and minimal off-target hybridization. Baits can be designed to detect any known virus or bacteria in a wide variety of specimen types, including oral secretions. The bait capture method presented herein allows the whole genome of SARS-CoV-2 in saliva to be sequenced without the need to culture and provides an outline of bait design and bioinformatic analysis to guide a bioinformatician.

Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae.

RNA fluorescence in situ hybridization (FISH) and antibody staining/immunofluorescence (IF) are widely used to detect distributions of mRNAs and proteins. Here we describe a combined FISH and IF protocol to simultaneously detect multiple mRNAs and proteins in whole-mount zebrafish embryos and larvae. In our approach, FISH is performed before IF to prevent mRNA degradation during the IF procedure. Instead of proteinase K digestion, Triton X-100 treatment and skin removal are used to permeate tissues and preserve antigen epitopes, making this protocol applicable to both whole-mount embryos and larvae. Off-target hybridization and FISH background are reduced by using PCR-amplified DNA templates and stringent buffers. This protocol simultaneously detects multiple mRNAs and proteins with high sensitivity, and enables detection at single-cell resolution. The protocol can be completed within 6 days, overcoming the shortage of reliable antibodies available for zebrafish and exploiting the advantages of zebrafish for studying organ development and regeneration.

Sequence characteristics define trade-offs between on-target and genome-wide off-target hybridization of oligoprobes.

Off-target oligoprobe’s interaction with partially complementary nucleotide sequences represents a problem for many bio-techniques. The goal of the study was to identify oligoprobe sequence characteristics that control the ratio between on-target and off-target hybridization. To understand the complex interplay between specific and genome-wide off-target (cross-hybridization) signals, we analyzed a database derived from genomic comparison hybridization experiments performed with an Affymetrix tiling array. The database included two types of probes with signals derived from (i) a combination of specific signal and cross-hybridization and (ii) genomic cross-hybridization only. All probes from the database were grouped into bins according to their sequence characteristics, where both hybridization signals were averaged separately. For selection of specific probes, we analyzed the following sequence characteristics: vulnerability to self-folding, nucleotide composition bias, numbers of G nucleotides and GGG-blocks, and occurrence of probe’s k-mers in the human genome. Increases in bin ranges for these characteristics are simultaneously accompanied by a decrease in hybridization specificity—the ratio between specific and cross-hybridization signals. However, both averaged hybridization signals exhibit growing trends along with an increase of probes’ binding energy, where the hybridization specific signal increases significantly faster in comparison to the cross-hybridization. The same trend is evident for the S function, which serves as a combined evaluation of probe binding energy and occurrence of probe’s k-mers in the genome. Application of S allows extracting a larger number of specific probes, as compared to using only binding energy. Thus, we showed that high values of specific and cross-hybridization signals are not mutually exclusive for probes with high values of binding energy and S. In this study, the application of a new set of sequence characteristics allows detection of probes that are highly specific to their targets for array design and other bio-techniques that require selection of specific probes.

Loop-mediated isothermal amplification using self-avoiding molecular recognition systems and antarctic thermal sensitive uracil-DNA-glycosylase for detection of nucleic acid with prevention of carryover contamination

Loop-mediated isothermal amplification (LAMP) is the most popular technique to amplify nucleic acid sequence without the use of temperature cycling. However, LAMP is often confounded by false-positive results, arising from interactions between (hetero-dimer) or within (self-dimerization) primers, off-target hybrids and carryover contaminants. Here, we devised a new LAMP technique that is self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme-assisted, termed AUDG-SAMRS-LAMP. Incorporating SAMRS components into 3′-ends of LAMP primers can improve assay's specificity, which completely prevents the non-specific amplification yielding from off-target hybrids and undesired interactions between or within primers. Adding AUDG into reaction mixtures can effectively eliminate the false-positive results arising from carryover contamination, thus the genuine positive reactions are generated from the amplification of target templates. Furthermore, AUDG-SAMRS-LAMP results are confirmed using a new analysis strategy, which is developed for detecting LAMP amplicons by lateral flow biosensor (LFB). Only a single labeled primer is required in the analysis system, thus the false positive results arising from hybridization (the labeled primer and probe, or between two labeled primers) are avoided. Hence, the SAMRS components, AUDG and LFB convert traditional LAMP from a technique suited for the research laboratory into one that has practical value in the field of diagnosis. Human Tuberculosis (TB) is caused by infection with members of Mycobacterium tuberculosis complex (MTC), which are detected by the AUDG-SAMRS-LAMP technique to demonstrate the availability of target analysis. The proof-of-concept method can be reconfigured to detect various nucleic acids by redesigning the specific primers.

A label-free technique for accurate detection of nucleic acid–based self-avoiding molecular recognition systems supplemented multiple cross-displacement amplification and nanoparticles based biosensor

Here, we devised a novel isothermal technique on the basis of standard multiple cross-displacement amplification (MCDA), which is assisted with self-avoiding molecular recognition system (SAMRS) components and antarctic thermal-sensitive uracil-DNA-glycosylase enzyme (AUDG), termed AUDG–SAMRS–MCDA. To enable product detection on the dipsticks, we firstly developed an analysis strategy, which did not require the labelled primers or probes, and thus, the analysis system avoids the false-positive results arising from undesired hybridization (between two labelled primers, or the labelled probe and primer). The SAMRS components are incorporated into MCDA primers for improve the assay’s specificity, which can prevent the false-positive results yielding from off-target hybrids, undesired interactions between (hetero-dimer) or within (self-dimerization) primers. Two additional components (AUDG enzyme and dUTP) were added into the reaction mixtures, which were used for removing the false-positive results generating from carryover contamination, and thus, the genuine positives results were produced from the amplification of target templates. For the demonstration, the label-free AUDG–SAMRS–MCDA technique was successfully applied to detect Pseudomonas aeruginosa from pure culture and blood samples. As a proof-of-concept technique, the label-free AUDG–SAMRS–MCDA method can be reconfigured to detect different target sequences by redesigning the specific primers.

Nanoparticles-based lateral flow biosensor coupled with multiple cross displacement amplification Plus for simultaneous detection of nucleic acid and prevention of carryover contamination

Abstract Isothermal amplification techniques, which avoid thermal cycling, are often confounded by non-specific amplification arising from off-target hybrids, interactions between (hetero-dimer) or within (self-dimerization) primers and carryover contaminants. Here, we developed a new method, termed multiple cross displacement amplification Plus (MCDA Plus), which uses self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme to improve the specificity of MCDA and prevent carryover contamination, respectively. In the presence of SAMRS components, the false positive results arising from off-target hybrids and undesired interactions between or within primers are completely eliminated, which increase the reproducibility of the assay. In the presence of AUDG, the false positive results generating from carryover contaminants are removed, thus the genuine positive results are obtained from the amplification of target sequences. Moreover, we also devised a new analysis strategy, which was intended for the detection of amplified products using lateral flow biosensor (LFB). The analysis system only required a single labeled primer, thus eliminated the false positive result yielding from hybridization (the labeled primer and probe, or between two labeled primers). Therefore, the SAMRS components, AUDG and LFB convert standard MCDA from an assay suited only for the research laboratory into one that has applicable value at points-of-care testing, in the field environments and in poor-resource settings. For demonstration purpose, Mycobacterium tuberculosis complex (MTC), which is the causes of human Tuberculosis (TB), are detected by the novel MCDA Plus technique to demonstrate the capability of target analysis. The performance of MCDA Plus assay in identifying MTC from pure culture and clinical samples is successfully evaluated. The proof-of-concept technique can be reconfigured to detect various nucleic acid sequences by redesigning the specific MCDA Plus primers.

Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance.

Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide “letters”, the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson–Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so.Results: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson–Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2’-deoxyisocytidine and 2’-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted.Conclusion: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology.

Defining the factors that contribute to on-target specificity of antisense oligonucleotides.

To better understand the factors that influence the activity and specificity of antisense oligonucleotides (ASOs), we designed a minigene encoding superoxide dismutase 1 (SOD-1) and cloned the minigene into vectors for T7 transcription of pre-mRNA and splicing in a nuclear extract or for stable integration in cells. We designed a series of ASOs that covered the entire mRNA and determined the binding affinities and activities of the ASOs in a cell-free system and in cells. The mRNA bound known RNA-binding proteins on predicted binding sites in the mRNA. The higher order structure of the mRNA had a significantly greater effect than the RNA-binding proteins on ASO binding affinities as the ASO activities in cells and in the cell-free systems were consistent. We identified several ASOs that exhibited off-target hybridization to the SOD-1 minigene mRNA in the cell-free system. Off-target hybridization occurred only at highly accessible unstructured sites in the mRNA and these interactions were inhibited by both the higher order structure of the mRNA and by RNA-binding proteins. The same off-target hybridization interactions were identified in cells that overexpress E. coli RNase H1. No off-target activity was observed for cells expressing only endogenous human RNase H1. Neither were these off-target heteroduplexes substrates for recombinant human RNase H1 under multiple-turnover kinetics suggesting that the endogenous enzyme functions under similar kinetic parameters in cells and in the cell-free system. These results provide a blueprint for design of more potent and more specific ASOs.

Use of PCR Template-Derived Probes Prevents Off-Target Whole Mount In Situ Hybridization in Transgenic Zebrafish

Transgenic zebrafish have been utilized for in vivo analysis of cell behaviors using advanced imaging techniques, for analyzing spatiotemporal gene regulation, and for targeted mis-expression of transgenes. The Tg(fli1a:EGFP)y1 vascular reporter has been particularly useful for examining the development of blood and lymphatic vessels, but it has been suggested that whole-mount in situ hybridization may result high background staining in this line, potentially limiting its usefulness. Here, we show that off-target hybridization of plasmid vector-derived probes to tissues expressing transgenes occurs in a number of different commonly used transgenic lines as a result of multiple cloning site sequences present in the cloning vectors, suggesting this may be a more general problem. However, we also show that this problem is easily avoided by performing in situ hybridization using probes synthesized from PCR templates lacking vector sequences.

An efficient algorithm for systematic analysis of nucleotide strings suitable for siRNA design

BackgroundThe "off-target" silencing effect hinders the development of siRNA-based therapeutic and research applications. Existing solutions for finding possible locations of siRNA seats within a large database of genes are either too slow, miss a portion of the targets, or are simply not designed to handle a very large number of queries. We propose a new approach that reduces the computational time as compared to existing techniques.FindingsThe proposed method employs tree-based storage in a form of a modified truncated suffix tree to sort all possible short string substrings within given set of strings (i.e. transcriptome). Using the new algorithm, we pre-computed a list of the best siRNA locations within each human gene ("siRNA seats"). siRNAs designed to reside within siRNA seats are less likely to hybridize off-target. These siRNA seats could be used as an input for the traditional "set-of-rules" type of siRNA designing software. The list of siRNA seats is available through a publicly available database located at http://web.cos.gmu.edu/~gmanyam/siRNA_db/search.phpConclusionsIn attempt to perform top-down prediction of the human siRNA with minimized off-target hybridization, we developed an efficient algorithm that employs suffix tree based storage of the substrings. Applications of this approach are not limited to optimal siRNA design, but can also be useful for other tasks involving selection of the characteristic strings specific to individual genes. These strings could then be used as siRNA seats, as specific probes for gene expression studies by oligonucleotide-based microarrays, for the design of molecular beacon probes for Real-Time PCR and, generally, any type of PCR primers.

In situ analysis of cross-hybridisation on microarrays and the inference of expression correlation.

BackgroundMicroarray co-expression signatures are an important tool for studying gene function and relations between genes. In addition to genuine biological co-expression, correlated signals can result from technical deficiencies like hybridization of reporters with off-target transcripts. An approach that is able to distinguish these factors permits the detection of more biologically relevant co-expression signatures.ResultsWe demonstrate a positive relation between off-target reporter alignment strength and expression correlation in data from oligonucleotide genechips. Furthermore, we describe a method that allows the identification, from their expression data, of individual probe sets affected by off-target hybridization.ConclusionThe effects of off-target hybridization on expression correlation coefficients can be substantial, and can be alleviated by more accurate mapping between microarray reporters and the target transcriptome. We recommend attention to the mapping for any microarray analysis of gene expression patterns.