Human-specific duplicate CHRFAM7A gene is associated with more severe osteoarthritis and amplifies pain behaviours.

Objectives: CHRFAM7A is a uniquely human fusion gene that functions as a dominant negative regulator of alpha 7 acetylcholine nicotinic receptor (α7nAChR) in vitro. This study determined the impact of CHRFAM7A on α7nAChR agonist responses, osteoarthritis (OA) severity and pain behaviours and investigated mechanisms.

Methods: Transgenic CHRFAM7A (TgCHRFAM7A) mice were used to determine the impact of CHRFAM7A on knee OA histology, pain severity in OA and other pain models, response to nAchR agonist and IL-1β.

Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.

Understanding antibody specificity and defining response profiles to antigens continue to be essential to both vaccine research and therapeutic antibody development. Peptide scanning assays enable mapping of continuous epitopes in order to delineate antibody-antigen interactions beyond traditional immunoassay formats. We have developed a relatively low-cost method to generate peptide microarray slides for antibody binding studies that allow for interrogation of up to 1536 overlapping peptides derived from the target antigens on a single microslide.

Real-time digital polymerase chain reaction (PCR) as a novel technology improves limit of detection for rare allele assays.

Background: Tumor heterogeneity may lead to false negative test results for tissue biopsy-based companion diagnostic tests. Real-time polymerase chain reaction (PCR) and digital PCR assays are used to detect rare alleles in cell-free circulating DNA for liquid biopsies; however, those tests lack strong sensitivity at low allele frequencies. We show here a novel real-time digital PCR instrument that utilizes cycle-based amplification curves to further improve the sensitivity and quantification accuracy of digital PCR.

Increased sensitivity using real-time dPCR for detection of SARS-CoV-2.

A real-time dPCR system was developed to improve the sensitivity, specificity and quantification accuracy of end point dPCR. We compared three technologies - real-time qPCR, end point dPCR and real-time dPCR - in the context of SARS-CoV-2. Some improvement in limit of detection was obtained with end point dPCR compared with real-time qPCR, and the limit of detection was further improved with the newly developed real-time dPCR technology through removal of false-positive signals.

Antibody Responses to Immunization With HCV Envelope Glycoproteins as a Baseline for B-Cell-Based Vaccine Development.

Background & Aims: We investigated antibody responses to hepatitis C virus (HCV) antigens E1 and E2 and the relevance of animal models for vaccine development. We compared antibody responses to vaccination with recombinant E1E2 complex in healthy volunteers, non-human primates (NHPs), and mice.

Methods: We analyzed 519 serum samples from participants in a phase 1 vaccine trial (ClinicalTrials.

Primer Extension, Capture, and On-Bead cDNA Ligation: An Efficient RNAseq Library Prep Method for Determining Reverse Transcription Termination Sites.

In this chapter, we describe a method for making Illumina-compatible sequencing libraries from RNA. This protocol can be used for standard RNAseq analysis for detecting differentially expressed genes. In addition, this protocol is ideally suited for adapting to RIPseq, 5'-RACE, RNA structural probing, nascent RNA sequencing, and other protocols where polymerase termination sites need to be profiled.

The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.

Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2.

Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.

With the increasing need for understanding antibody specificity in antibody and vaccine research, pepscan assays provide a rapid method for mapping and profiling antibody responses to continuous epitopes. We have developed a relatively low-cost method to generate peptide microarray slides for studying antibody binding. Using a setup of an IntavisAG MultiPep RS peptide synthesizer, a Digilab MicroGrid II 600 microarray printer robot, and an InnoScan 1100 AL scanner, the method allows the interrogation of up to 1536 overlapping, alanine-scanning, and mutant peptides derived from the target antigens.

ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3'-Azido cDNAs.

We present a simple method called "ClickSeq" for NGS (next-generation sequencing) library synthesis that uses click chemistry rather than enzymatic reactions for the ligation of Illumina sequencing adaptors. In ClickSeq, randomly primed reverse transcription reactions are supplemented with azido-2',3'-dideoxynucleotides that randomly terminate DNA synthesis and release 3'-azido-blocked cDNA fragments in a process akin to dideoxy-Sanger sequencing. Purified fragments are "click ligated" via copper-catalyzed alkyne-azide cycloaddition to DNA oligos modified with a 5'-alkyne group.

Fate and plasticity of the epidermis in response to congenital activation of BRAF.

Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin. Here we demonstrate that activation of BRAF in the embryonic mouse ectoderm triggers both craniofacial and skin defects, including hyperproliferation, loss of spinous and granular keratinocyte differentiation, and cleft palate. RNA sequencing of the epidermis confirmed these findings but unexpectedly revealed evidence of continued epidermal maturation, expression of >80% of epidermal differentiation complex genes, and formation of a hydrophobic barrier.

Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabet.

We have developed a family of unnatural base pairs (UBPs), which rely on hydrophobic and packing interactions for pairing and which are well replicated and transcribed. While the pair formed between d5SICS and dNaM (d5SICS-dNaM) has received the most attention, and has been used to expand the genetic alphabet of a living organism, recent efforts have identified dTPT3-dNaM, which is replicated with even higher fidelity. These efforts also resulted in more UBPs than could be independently analyzed, and thus we now report a PCR-based screen to identify the most promising.

Library construction for next-generation sequencing: overviews and challenges.

High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources.

Technical variations in low-input RNA-seq methodologies.

Recent advances in RNA-seq methodologies from limiting amounts of mRNA have facilitated the characterization of rare cell-types in various biological systems. So far, however, technical variations in these methods have not been adequately characterized, vis-à-vis sensitivity, starting with reduced levels of mRNA. Here, we generated sequencing libraries from limiting amounts of mRNA using three amplification-based methods, viz.

Optimization of peptide arrays for studying antibodies to hepatitis C virus continuous epitopes.

Accurate and in-depth mapping of antibody responses is of great value in vaccine and antibody research. Using hepatitis C virus (HCV) as a model, we developed an affordable and high-throughput microarray-based assay for mapping antibody specificities to continuous antibody epitopes of HCV at high resolution. Important parameters in the chemistry for conjugating peptides/antigens to the array surface, the array layout, fluorophore choice and the methods for data analysis were investigated.

Natural-like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applications.

We synthesized a panel of unnatural base pairs whose pairing depends on hydrophobic and packing forces and identify dTPT3-dNaM, which is PCR amplified with a natural base pair-like efficiency and fidelity. In addition, the dTPT3 scaffold is uniquely tolerant of attaching a propargyl amine linker, resulting in the dTPT3(PA)-dNaM pair, which is amplified only slightly less well. The identification of dTPT3 represents significant progress toward developing an unnatural base pair for the in vivo expansion of an organism's genetic alphabet and for a variety of in vitro biotechnology applications where it is used to site-specifically label amplified DNA, and it also demonstrates for the first time that hydrophobic and packing forces are sufficient to mediate natural-like replication.

Site-specifically arraying small molecules or proteins on DNA using an expanded genetic alphabet.

A class of replicable unnatural DNA base pairs formed between d5SICS and either dMMO2, dDMO, or dNaM were developed. To explore the use of these pairs to produce site-specifically labeled DNA, the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase-mediated replication, and subsequent site-specific modification of the amplified DNA by Click chemistry is reported. With the d5SICS scaffold a propynyl ether linker is accommodated better than its aliphatic analogue, but not as well as the protected propargyl amine linker explored previously.

RNA purification and expression analysis using microarrays and RNA deep sequencing.

Transcriptome analysis or global gene expression profiling is a powerful tool for discovery as well as -understanding biological mechanisms in health and disease. We present in this chapter a description of methods used to isolate mRNA from cells and tissues that has been optimized for preservation of RNA quality using clinical materials and implemented successfully in several large, multicenter studies by the authors. In addition, two methods, gene expression microarrays and RNAseq, are described for mRNA profiling of cells and tissues from clinical or laboratory sources.

Expanding the scope of replicable unnatural DNA: stepwise optimization of a predominantly hydrophobic base pair.

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventually in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs exemplified by d5SICS-dMMO2 and d5SICS-dNaM. When incorporated into DNA, the latter is replicated and transcribed with greater efficiency and fidelity than the former; however, previous optimization efforts identified the para and methoxy-distal meta positions of dMMO2 as particularly promising for further optimization. Here, we report the stepwise optimization of dMMO2 via the synthesis and evaluation of 18 novel para-derivatized analogs of dMMO2, followed by further derivatization and evaluation of the most promising analogs with meta substituents.

Nucleotide-resolution profiling of RNA recombination in the encapsidated genome of a eukaryotic RNA virus by next-generation sequencing.

Next-generation sequencing has been used in numerous investigations to characterize and quantify the genetic diversity of a virus sample through the mapping of polymorphisms and measurement of mutation frequencies. Next-generation sequencing has also been employed to identify recombination events occurring within the genomes of higher organisms, for example, detecting alternative RNA splicing events and oncogenic chromosomal rearrangements. Here, we combine these two approaches to profile RNA recombination within the encapsidated genome of a eukaryotic RNA virus, flock house virus.

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet.

The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chemical and synthetic biology. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs in a wide variety of sequence contexts.

KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry.

Many candidate unnatural DNA base pairs have been developed, but some of the best-replicated pairs adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replication for one such pair, dNaM-d5SICS, and show that efficient replication results from the polymerase itself, inducing the required natural-like structure.

Illumina mate-paired DNA sequencing-library preparation using Cre-Lox recombination.

Standard Illumina mate-paired libraries are constructed from 3- to 5-kb DNA fragments by a blunt-end circularization. Sequencing reads that pass through the junction of the two joined ends of a 3-5-kb DNA fragment are not easy to identify and pose problems during mapping and de novo assembly. Longer read lengths increase the possibility that a read will cross the junction.

Quantitative bias in Illumina TruSeq and a novel post amplification barcoding strategy for multiplexed DNA and small RNA deep sequencing.

Here we demonstrate a method for unbiased multiplexed deep sequencing of RNA and DNA libraries using a novel, efficient and adaptable barcoding strategy called Post Amplification Ligation-Mediated (PALM). PALM barcoding is performed as the very last step of library preparation, eliminating a potential barcode-induced bias and allowing the flexibility to synthesize as many barcodes as needed. We sequenced PALM barcoded micro RNA (miRNA) and DNA reference samples and evaluated the quantitative barcode-induced bias in comparison to the same reference samples prepared using the Illumina TruSeq barcoding strategy.

Site-specific labeling of DNA and RNA using an efficiently replicated and transcribed class of unnatural base pairs.

Site-specific labeling of enzymatically synthesized DNA or RNA has many potential uses in basic and applied research, ranging from facilitating biophysical studies to the in vitro evolution of functional nucleic acids and the construction of various nanomaterials and biosensors. As part of our efforts to expand the genetic alphabet, we have developed a class of unnatural base pairs, exemplified by d5SICS-dMMO2 and d5SICS-dNaM, which are efficiently replicated and transcribed, and which may be ideal for the site-specific labeling of DNA and RNA. Here, we report the synthesis and analysis of the ribo- and deoxyribo-variants, (d)5SICS and (d)MMO2, modified with free or protected propargylamine linkers that allow for the site-specific modification of DNA or RNA during or after enzymatic synthesis.

Evolution and protein packaging of small-molecule RNA aptamers.

A high-affinity RNA aptamer (K(d) = 50 nM) was efficiently identified by SELEX against a heteroaryldihydropyrimidine structure, chosen as a representative drug-like molecule with no cross reactivity with mammalian or bacterial cells. This aptamer, its weaker-binding variants, and a known aptamer against theophylline were each embedded in a longer RNA sequence that was encapsidated inside a virus-like particle by a convenient expression technique. These nucleoprotein particles were shown by backscattering interferometry to bind to the small-molecule ligands with affinities similar to those of the free (nonencapsidated) aptamers.