Publications by authors named "Phillip Ordoukhanian"

29 Publications

  • Page 1 of 1

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

Transl Lung Cancer Res 2021 Dec;10(12):4336-4352

State Key Laboratory of Molecular Oncology, Department of Medical Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

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.

Methods: The novel real-time digital PCR instrument was compared to an endpoint digital PCR system to determine the sensitivity and quantification accuracy of both instruments. Samples were all thermal cycled on the real-time digital PCR instrument but were analyzed on both endpoint and real-time digital PCR instruments to compare the performance without introducing other variables. Contrived samples for epidermal growth factor receptor () exon 19 deletion, T790M, and L858R point mutations as well as human epidermal growth factor receptor 2 () amplification were tested. Different mutant allele frequencies and wildtype to mutant gene copy number ratios were tested for and , respectively.

Results: By removing false positive datapoints using real-time amplification curves, real-time digital PCR improved sensitivity by lowering the baseline for wildtype samples. For 19del assay, samples with 2 or more fluorescein amidite (FAM) labeled positive wells are determined positive by real-time digital PCR, while a minimum of 5 FAM positive datapoints is needed by endpoint digital PCR. Improved limit of detection for 19del mutation was also observed. Real-time digital PCR also had better quantification accuracy and sensitivity, resulting in the mutant allele frequencies being closer to the expected values for all mutations, especially at very low allele frequencies. However, at high allele frequencies or for gene amplification assays, real-time digital PCR is comparable with endpoint digital PCR.

Conclusions: This novel technology with improved sensitivity is important and needed because it addresses current issues with liquid biopsy tests. Due to limited amounts of circulating tumor DNA (ctDNA) obtained for liquid biopsy tests, few copies of mutant alleles are expected. With the lower baseline of real-time digital PCR, false negative test results from tissue biopsy would be more effectively reduced, leading to more patients receiving the targeted therapy they need for better survival.
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http://dx.doi.org/10.21037/tlcr-21-728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8743530PMC
December 2021

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

Biotechniques 2021 01 23;70(1):7-20. Epub 2020 Nov 23.

Gnomegen, 6440 Lusk Blvd, D207, San Diego, CA 92121, USA.

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. Real-time dPCR showed increased linear dynamic range compared with end point dPCR based on quantitation from amplification curves. Real-time dPCR can improve the performance of TaqMan assays beyond real-time qPCR and end point dPCR with better sensitivity and specificity, absolute quantification and a wider linear range of detection.
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http://dx.doi.org/10.2144/btn-2020-0133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888512PMC
January 2021

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

Gastroenterology 2020 03 4;158(4):1058-1071.e6. Epub 2019 Dec 4.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, California. Electronic address:

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.gov identifier NCT00500747) and compared them with serum or plasma samples from C57BL/6J mice (n = 28) and rhesus macaques (n = 4) immunized with the same HCV E1E2 antigen. Blood samples were collected at different time points and analyzed for antibody binding, neutralizing activity, and epitope specificity. Monoclonal antibodies from the immunized NHPs were isolated from single plasmablasts and memory B cells, and their immunogenetic properties were characterized.

Results: Antibody responses of the volunteers, NHPs, and mice to the non-neutralizing epitopes on the E1 N-terminus and E2 hypervariable region 1 did not differ significantly. Antibodies from volunteers and NHPs that neutralized heterologous strains of HCV primarily interacted with epitopes in the antigen region 3. However, the neutralizing antibodies were not produced in sufficient levels for broad neutralization of diverse HCV isolates. Broadly neutralizing antibodies similar to the human V1-69 class antibody specific for antigen region 3 were produced in the immunized NHPs.

Conclusions: In an analysis of vaccinated volunteers, NHPs, and mice, we found that recombinant E1E2 vaccine antigen induces high-antibody titers that are insufficient to neutralize diverse HCV isolates. Antibodies from volunteers and NHPs bind to the same neutralizing epitopes for virus neutralization. NHPs can therefore be used as a preclinical model to develop HCV vaccines. These findings also provide useful baseline values for development of vaccines designed to induce production of neutralizing antibodies.
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http://dx.doi.org/10.1053/j.gastro.2019.11.282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371413PMC
March 2020

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

Methods Mol Biol 2018 ;1712:253-261

Next Generation Sequencing and Microarray Core Facility, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA, 92037, USA.

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 utilization of solid-phase bead chemistries facilitates simple workflow and efficient library yields.
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http://dx.doi.org/10.1007/978-1-4939-7514-3_16DOI Listing
July 2018

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

PLoS One 2015 7;10(12):e0144409. Epub 2015 Dec 7.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America.

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. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as "central" interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, "peripheral" interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144409PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671683PMC
June 2016

Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.

Methods Mol Biol 2016 ;1352:67-83

DNA Array Core, Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA.

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. Each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface.
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http://dx.doi.org/10.1007/978-1-4939-3037-1_6DOI Listing
August 2016

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

J Mol Biol 2015 Aug 24;427(16):2610-6. Epub 2015 Jun 24.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

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. This generates ssDNA molecules containing an unnatural triazole-linked DNA backbone that is sufficiently biocompatible for PCR amplification to generate a cDNA library for RNAseq. Here, we analyze viral RNAs and mRNA to demonstrate that ClickSeq produces unbiased NGS libraries with low error rates comparable to standard methods. Importantly, ClickSeq is robust against common artifacts of NGS such as chimera formation and artifactual recombination with fewer than 3 aberrant events detected per million reads.
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http://dx.doi.org/10.1016/j.jmb.2015.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523409PMC
August 2015

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

J Invest Dermatol 2015 Feb 9;135(2):481-9. Epub 2014 Sep 9.

Division of Dermatology, Department of Medicine, Institute for Genomic Medicine, Stem Cell Program, University of California, San Diego, La Jolla, California, USA.

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. Spinous and granular differentiation were restored by pharmacologic inhibition of MAPK/ERK kinase or BRAF. However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies. These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.
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http://dx.doi.org/10.1038/jid.2014.388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4289449PMC
February 2015

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

Nucleic Acids Res 2014 13;42(16):10235-44. Epub 2014 Aug 13.

Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA

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. While we found that dTPT3-dNaM is generally the most promising UBP, we identified several others that are replicated nearly as well and significantly better than d5SICS-dNaM, and are thus viable candidates for the expansion of the genetic alphabet of a living organism. Moreover, the results suggest that continued optimization should be possible, and that the putatively essential hydrogen-bond acceptor at the position ortho to the glycosidic linkage may not be required. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new structure-activity relationship data and importantly identify multiple new candidates for in vivo evaluation and further optimization.
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http://dx.doi.org/10.1093/nar/gku715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176363PMC
January 2015

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

Biotechniques 2014 1;56(2):61-4, 66, 68, passim. Epub 2014 Feb 1.

NGS and Microarray Core Facility, The Scripps Research Institute, La Jolla, CA.

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. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed.
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http://dx.doi.org/10.2144/000114133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351865PMC
September 2014

Technical variations in low-input RNA-seq methodologies.

Sci Rep 2014 Jan 14;4:3678. Epub 2014 Jan 14.

1] Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, La Jolla, California, USA [2] Department of Bioengineering, University of California at San Diego, La Jolla, California, USA [3] Departments of Cellular and Molecular Medicine and Chemistry and Biochemistry, University of California at San Diego, La Jolla, California, USA.

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. Smart-seq, DP-seq and CEL-seq, and demonstrated significant technical variations in these libraries. Reduction in mRNA levels led to inefficient amplification of the majority of low to moderately expressed transcripts. Furthermore, noise in primer hybridization and/or enzyme incorporation was magnified during the amplification step resulting in significant distortions in fold changes of the transcripts. Consequently, the majority of the differentially expressed transcripts identified were either high-expressed and/or exhibited high fold changes. High technical variations ultimately masked subtle biological differences mandating the development of improved amplification-based strategies for quantitative transcriptomics from limiting amounts of mRNA.
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http://dx.doi.org/10.1038/srep03678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890974PMC
January 2014

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

J Immunol Methods 2014 Jan 19;402(1-2):35-42. Epub 2013 Nov 19.

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, United States. Electronic address:

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. Microscopic glass slide pre-coated with N-Hydroxysuccinimide (NHS)-ester (Slide H) was the preferred surface for conjugation of aminooxy-tagged peptides. This combination provides a simple chemical means to orient the peptides to the conjugation surface via an orthogonal covalent linkage at the N- or C-terminus of each peptide. The addition of polyvinyl alcohol to printing buffer gave uniform spot morphology and improved sensitivity and specificity of binding signals. Libraries of overlapping peptides covering the HCV E1 and E2 glycoprotein polypeptides (15-mer, 10 amino acids overlap) of 6 major HCV genotypes and the entire polypeptide sequence of the prototypic strain H77 were synthesized and printed in quadruplets in the assays. The utility of the peptide arrays was confirmed using HCV monoclonal antibodies (mAbs) specific to known continuous epitopes and immune sera of rabbits immunized with HCV antigens. The methods developed here can be easily adapted to studying antibody responses to antigens relevant in vaccine and autoimmune research.
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http://dx.doi.org/10.1016/j.jim.2013.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4009380PMC
January 2014

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

J Am Chem Soc 2014 Jan 23;136(3):826-9. Epub 2013 Oct 23.

Department of Chemistry and ‡Center for Protein and Nucleic Acid Research, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

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.
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http://dx.doi.org/10.1021/ja408814gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979842PMC
January 2014

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

Chemistry 2013 Oct 11;19(42):14205-14209. Epub 2013 Sep 11.

Department of Chemistry and Dr. P. Ordoukhanian Center for Protein and Nucleic Acid Research The Scripps Research Institute 10550 North Torrey Pines Road La Jolla, CA 92037.

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. It was also found that with the dMMO2 and dDMO analogues, the dMMO2 position para to the glycosidic linkage is best suited for linker attachment and that although aliphatic and ether-based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogues, a variety of them were used to site-selectively attach a biotin tag to the amplified DNA. Finally, we use d5SICS(CO) -dNaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps "evolution" of nanomaterials.
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http://dx.doi.org/10.1002/chem.201302496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983968PMC
October 2013

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

Methods Mol Biol 2013 ;1034:385-403

Microarray and Next Generation Sequencing Core Facility, The Scripps Research Institute, La Jolla, CA, USA.

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.
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http://dx.doi.org/10.1007/978-1-62703-493-7_25DOI Listing
February 2014

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

J Am Chem Soc 2013 Apr 2;135(14):5408-19. Epub 2013 Apr 2.

Department of Chemistry and Center for Protein and Nucleic Acid Research, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

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. Subject to size constraints, we find that para substituents can optimize replication via both steric and electronic effects and that meta methoxy groups are unfavorable, while fluoro substituents can be beneficial or deleterious depending on the para substituent. In addition, we find that improvements in the efficiency of unnatural triphosphate insertion translate most directly into higher fidelity replication. Importantly, we identify multiple, unique base pair derivatives that when incorporated into DNA are well replicated. The most promising, d5SICS-dFEMO, is replicated under some conditions with greater efficiency and fidelity than d5SICS-dNaM. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new SAR data, and importantly identify multiple new candidates for eventual in vivo evaluation.
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http://dx.doi.org/10.1021/ja312148qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3690937PMC
April 2013

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

J Mol Biol 2012 Dec 13;424(5):257-69. Epub 2012 Oct 13.

Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

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. We detect hundreds of thousands of recombination events, with single-nucleotide resolution, which result in diversity in the encapsidated genome rivaling that due to mismatch mutation. We detect previously identified defective RNAs as well as many other abundant and novel defective RNAs. Our approach is exceptionally sensitive and unbiased and requires no prior knowledge beyond the virus genome sequence. RNA recombination is a powerful driving force behind the evolution and adaptation of RNA viruses. The strategy implemented here is widely applicable and provides a highly detailed description of the complex mutational landscape of the transmissible viral genome.
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http://dx.doi.org/10.1016/j.jmb.2012.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502730PMC
December 2012

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

Proc Natl Acad Sci U S A 2012 Jul 6;109(30):12005-10. Epub 2012 Jul 6.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.

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. Under standard conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates containing the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that, for PCR and PCR-based applications, d5SICS-dNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet.
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http://dx.doi.org/10.1073/pnas.1205176109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409741PMC
July 2012

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

Nat Chem Biol 2012 Jul 3;8(7):612-4. Epub 2012 Jun 3.

Department of Chemistry, Universität Konstanz, Konstanz, Germany.

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.
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http://dx.doi.org/10.1038/nchembio.966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3690913PMC
July 2012

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

Nucleic Acids Res 2012 Feb 29;40(3):e24. Epub 2011 Nov 29.

Laboratory of Pharmaceutical Biotechnology, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.

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. To solve this problem, we developed a mate-paired protocol for use with Illumina sequencing technology that uses Cre-Lox recombination instead of blunt end circularization. In this method, a LoxP sequence is incorporated at the junction site. This sequence allows screening reads for junctions without using a reference genome. Junction reads can be trimmed or split at the junction. Moreover, the location of the LoxP sequence in the reads distinguishes mate-paired reads from spurious paired-end reads. We tested this new method by preparing and sequencing a mate-paired library with an insert size of 3 kb from Saccharomyces cerevisiae. We present an analysis of the library quality statistics and a new bio-informatics tool called DeLoxer that can be used to analyze an IlluminaCre-Lox mate-paired data set. We also demonstrate how the resulting data significantly improves a de novo assembly of the S. cerevisiae genome.
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http://dx.doi.org/10.1093/nar/gkr1000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273786PMC
February 2012

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

PLoS One 2011 28;6(10):e26969. Epub 2011 Oct 28.

Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.

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. The Illumina TruSeq small RNA strategy introduces the barcode during the PCR step using differentially barcoded primers, while the TruSeq DNA strategy introduces the barcode before the PCR step by ligation of differentially barcoded adaptors. Results show virtually no bias between the differentially barcoded miRNA and DNA samples, both for the PALM and the TruSeq sample preparation methods. We also multiplexed miRNA reference samples using a pre-PCR barcode ligation. This barcoding strategy results in significant bias.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026969PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203936PMC
March 2012

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

J Am Chem Soc 2011 Dec 18;133(49):19878-88. Epub 2011 Nov 18.

Department of Chemistry and Center for Protein and Nucleic Acid Research, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.

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. We also synthesized and evaluated the α-phosphorothioate variant of d5SICSTP, which provides a route to backbone thiolation and an additional strategy for the postamplification site-specific labeling of DNA. The deoxynucleotides were characterized via steady-state kinetics and PCR, while the ribonucleosides were characterized by the transcription of both a short, model RNA as well as full length tRNA. The data reveal that while there are interesting nucleotide and polymerase-specific sensitivities to linker attachment, both (d)MMO2 and (d)5SICS may be used to produce DNA or RNA site-specifically modified with multiple, different functional groups with sufficient efficiency and fidelity for practical applications.
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http://dx.doi.org/10.1021/ja207907dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988912PMC
December 2011

Evolution and protein packaging of small-molecule RNA aptamers.

ACS Nano 2011 Oct 7;5(10):7722-9. Epub 2011 Sep 7.

Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, United States.

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. The system therefore comprises a general approach to the production and sequestration of functional RNA molecules, characterized by a convenient label-free analytical technique.
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http://dx.doi.org/10.1021/nn2006927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209476PMC
October 2011

Application of microdroplet PCR for large-scale targeted bisulfite sequencing.

Genome Res 2011 Oct 14;21(10):1738-45. Epub 2011 Jul 14.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.

Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, few current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream of and downstream from their transcription start sites. This method yielded 96% coverage of the targeted CpGs and demonstrated high correlation between CpG island (CGI) DNA methylation and transcriptional regulation. The method was scaled to interrogate the methylation status of 77,674 CpGs in the promoter regions of 2100 genes in primary CD4 T cells. The 2100 gene library yielded 97% coverage of all targeted CpGs and 99% of the target amplicons.
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http://dx.doi.org/10.1101/gr.116863.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3202290PMC
October 2011

Method for improved Illumina sequencing library preparation using NuGEN Ovation RNA-Seq System.

Biotechniques 2011 Mar;50(3):177-80

Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, CA 92040, USA.

In this study, we tested the NuGEN Ovation RNA-Seq System for library preparation followed by next-generation sequencing on an Illumina GAIIx. The cDNA product of the NuGEN kit may have significant amounts of ssDNA with hairpin structures that are generated during the amplification process. These structures interfere with efficient downstream end repair, A-tailing, and adapter ligation, all standard steps in post-amplification sequencing library construction. We were able to increase the efficiency of sequencing library yields 4- to 6-fold or greater by treatment of NuGEN-amplified cDNA product with the single-strand endonuclease S1. These results suggest that this treatment effectively cleaves hairpin structures generated during amplification that are resistant to the standard enzyme cocktails used for the end-repair step.
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http://dx.doi.org/10.2144/000113613DOI Listing
March 2011

PCR with an expanded genetic alphabet.

J Am Chem Soc 2009 Oct;131(41):14620-1

Department of Chemistry and Center for Protein and Nucleic Acid Research, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Expansion of the genetic alphabet with a third base pair would lay the foundation for a semisynthetic organism with an expanded genetic code and also have immediate in vitro applications. Previously, the unnatural base pairs formed between d5SICS and either dNaM or dMMO2 were shown to be well-replicated by DNA polymerases under steady-state conditions and also transcribed by T7 RNA polymerase efficiently in either direction. We now demonstrate that DNA containing either the d5SICS-dNaM or d5SICS-dMMO2 unnatural base pair may be amplified by PCR with fidelities and efficiencies that approach those of fully natural DNA. These results further demonstrate that the determinants of a functional unnatural base pair may be designed into predominantly hydrophobic nucleobases with no structural similarity to the natural purines or pyrimidines. Importantly, the results reveal that the unnatural base pairs may function within an expanded genetic alphabet and make possible many in vitro applications.
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http://dx.doi.org/10.1021/ja906186fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978235PMC
October 2009

Optimization of an unnatural base pair toward natural-like replication.

J Am Chem Soc 2009 Mar;131(9):3246-52

Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Predominantly hydrophobic unnatural nucleotides that selectively pair within duplex DNA as well as during polymerase-mediated replication have recently received much attention as the cornerstone of efforts to expand the genetic alphabet. We recently reported the results of a screen and subsequent lead hit optimization that led to identification of the unnatural base pair formed between the nucleotides dMMO2 and d5SICS. This unnatural base pair is replicated by the Klenow fragment of Escherichia coli DNA polymerase I with better efficiency and fidelity than other candidates reported in the literature. However, its replication remains significantly less efficient than a natural base pair, and further optimization is necessary for its practical use. To better understand and optimize the slowest step of replication of the unnatural base pair, the insertion of dMMO2 opposite d5SICS, we synthesized two dMMO2 derivatives, d5FM and dNaM, which differ from the parent nucleobase in terms of shape, hydrophobicity, and polarizability. We find that both derivatives are inserted opposite d5SICS more efficiently than dMMO2 and that overall the corresponding unnatural base pairs are generally replicated with higher efficiency and fidelity than the pair between dMMO2 and d5SICS. In fact, in the case of the dNaM:d5SICS heteropair, the efficiency of each individual step of replication approaches that of a natural base pair, and the minimum overall fidelity ranges from 10(3) to 10(4). In addition, the data allow us to propose a generalized model of unnatural base pair replication, which should aid in further optimization of the unnatural base pair and possibly in the design of additional unnatural base pairs that are replicated with truly natural-like efficiency and fidelity.
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http://dx.doi.org/10.1021/ja807853mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901498PMC
March 2009

Major biocontrol of plant tumors targets tRNA synthetase.

Science 2005 Sep;309(5740):1533

Skaggs Institute for Chemical Biology, Scripps Research Institute, La Jolla, CA 92037, USA.

Crops can be devastated by pathogenic strains of Agrobacterium tumefaciens that cause crown gall tumors. This devastation can be prevented by the nonpathogenic biocontrol agent A. radiobacter K84, which prevents disease by production of the "Trojan horse" toxin agrocin 84, which is specifically imported into tumorgenic A. tumefaciens strains to cause cell death. We demonstrate that this biocontrol agent targets A. tumefaciens leucyl-tRNA synthetase (LeuRS), an essential enzyme for cell viability, while the agent itself survives by having a second, self-protective copy of the synthetase. In principle, this strategy from nature could be applied to other crop diseases by direct intervention.
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http://dx.doi.org/10.1126/science.1116841DOI Listing
September 2005

RNA-cleaving DNA enzymes with altered regio- or enantioselectivity.

J Am Chem Soc 2002 Oct;124(42):12499-506

Departments of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5'-phosphodiester following a D-ribonucleotide or a 3',5'-phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5'-phosphodiester exhibits a k(cat) of approximately 0.01 min(-1) and catalytic efficiency, k(cat)/K(m), of approximately 10(8) M(-1) min(-1). The enzyme that cleaves an L-ribonucleotide is about 10-fold slower and has a catalytic efficiency of approximately 4 x 10(5) M(-1) min(-1). Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 degrees C. In a comparison of each enzyme's activity with either its corresponding substrate that contains an unnatural ribonucleotide or a substrate that instead contains a standard ribonucleotide, the 2',5'-phosphodiester-cleaving DNA enzyme exhibited a regioselectivity of 6000-fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 40-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.
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http://dx.doi.org/10.1021/ja027467pDOI Listing
October 2002
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