Research Labs

Erbay Yigit

Associate Director
Ph.D., University of Massachusetts Medical School, 2007

Area of focus:

RNA modifications and RNA-modifying enzymes

Current Research:

There are more than one hundred known chemical modifications of RNA. Modifications are sites specifically positioned in RNA molecules and are found in all three domains of life (archaea, prokaryotes, eukaryotes). tRNA and rRNA contain numerous different RNA modifications, while six are currently known to occur in mRNA (2'-O-methyl, pseudouridine, 5-methylcytosine, 5- hydoxymethycytosine, N1-methyladenosine, N6-methyladenosine). The precise roles of mRNA modifications are not known. In cells, post-transcriptional RNA modifications are introduced in two ways. The first pathway uses a stand-alone enzyme that modifies specific positions on the target RNA, while a second pathway uses a ribonucleoprotein complex containing a short guide-RNA called small nucleolar RNA (snoRNA) to achieve this. My group is interested in the identification and characterization of both types of RNA-modifying enzymes and determining their substrate specificity and biological role in the cell.

Laurence Ettwiller

Senior Scientist
Ph.D., University of Cambridge, 2005

Area of focus:

NGS and computational biology

Current Research:

The major topic of the Ettwiller group is to bridge the large enzyme discovery and optimization efforts conducted at NEB with the latest massive parallel sequencing technologies.

With the central aim to develop innovative large scale applications, our research involves the elaboration of both experimental methodologies and data analysis strategies inherent to such applications. The outcomes of these efforts are twofold: first, unique new abilities to address biological questions; and second, the perspective of improving existing or novel technologies.

Sir Richard Roberts

Chief Scientific Officer
Ph.D., University of Sheffield, 1968

Area of focus:

Bioinformatics of restriction and modification

Current Research:

My laboratory has a long history of research on restriction enzymes and their associated DNA methyltransferases (MTases). We are developing methods to find new enzymes with novel properties using a combination of bioinformatics and biochemical experimentation to analyze the DNA sequence information from the many microbial genome sequences that are now available. Current work is focused on the use of PacBio sequencing to identify novel restriction systems and to determine recognition sequences for MTases (m4C and m6A). We are also developing new methodology for m5C recognition sequence determination. We run REBASE (www.neb.com/rebase), which is a manually-curated database of information about restriction enzymes and their associated proteins, such as MTases, control proteins, nicking enzymes, etc. We welcome collaborations from outside users with PacBio data as the methylation analysis can be biologically important and cannot easily be obtained in any other way.

Learn more about Rich Roberts in a lecture he gave at California State University.

Sriharsa Pradhan

Distinguished Scientist
Ph.D., University of Glasgow, 1995

Area of focus:

Epigenetic gene regulation and inheritance

Current Research:

In eukaryotes, the epigenome plays an essential role in regulation of gene expression. DNA hypermethylation in chromatin usually leads to gene silencing and is essential for a variety of epigenetic processes in the majority of eukaryotes, including animals, fungi and plants. Similarly, post-translational modifications (methylation, acetylation, phosphorylation, ubiquitination and ADP ribosylation) of histones determine the interaction of regulatory and structural proteins with the nucleosome thereby, altering chromatin structure. There are numerous examples of aberrant DNA methylation in pathological conditions, including cancer. The acquisition of a cancer specific DNA methylation pattern plays an important role in oncogenic transformation and thus can be used as an early diagnostic marker.

My laboratory is focused on understanding the regulation of DNA and protein modifications, particularly the enzymes that act on chromatin. We also investigate the role of various transcription factors and tumor suppressor proteins as epigenetic modulators during cellular growth, development and programmed cell death. Finally, we integrate epigenomic information including chromatin accessibility and architectural studies with biochemistry, structural biology and live cell imaging to decipher the intricate epigenetic mechanisms involved in cellular function.

Lana Saleh

Associate Director
Ph.D., Pennsylvania State University, 2005

Area of focus:

Ten-eleven translocase (TET) proteins

Current Research:

In eukaryotes, methylation on both DNA and RNA is reversible and exists in the form 2'-deoxy-5-methylcytidine (5mC) on DNA and N6-methyladenosine (m6A) on RNA. The primary interest of my group is the biochemical characterization of oxygenases involved in the demethylation processes of these bases, such as the ten eleven translocation (TET) enzymes in the case of 5mC and FTO, and AlkBH5 in the case of m6A. These oxygenases are members of the Fe(II)/alpha-ketoglutarate-dependent dioxygenases, which employ a radical-based mechanism for the oxidation of the methyl moiety on their corresponding substrates. My group is interested in understanding the chemical mechanism that these enzymes employ to generate these oxidized bases, as well as deciphering their modes of substrate recognition and product release. We are also interested in how the various members of the nucleic acid oxygenase sub-family evolved to perform various oxidative functionalities and understand the factors that ultimately dictate their specificity.

1. Oxidative demethylation of 5mC on DNA and RNA

The TET enzymes have been implicated in the oxidation of 5mC into three oxidized forms, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). These bases have been thought to be intermediate products on the pathway to active demethylation in mammals but recent literature has presented evidence of the possibility of these modified bases functioning as epigenetic markers involved in cellular regulation and developmental processes. We are in the process of performing in vitro biochemical characterization of the mammalian TETs (mTETs) and their evolutionary distant relative, NgTET1, from the unicellular protist Naegleria gruberi, and relating the in vitro modus operandi to the in vivo biological reports. We are also interested in studying various other members of the TET/JBP family of which mTETs and NgTET1 are members. Specifically, we are keen to identify novel oxygenases that exhibit oxidative functionality to 5mC on mRNA and long non-coding RNA. Last but not least, we are utilizing structural and bioinformatics tools to identify TET homologs with altered oxidized-product distribution and substrate specificity.

2. Oxidative demethylation of m6A on RNA and DNA

m6A on RNA is known to play a role in various RNA metabolism processes such as splicing, nuclear export, translation ability, and stability. My group focuses on the functional and biochemical characterization of various m6A demethylases, which catalyze the conversion of m6A to adenosine (A) on RNA, such as FTO and AlkBH5, and DNA, such as a predicted AlkB homolog from Caenorhabditis elegans. In its demethylation reaction, FTO has been shown to catalyze the formation of two oxidized intermediates, hydroxy-6-methyladenosine (hm6A) and formyl-6-adenosine (f6A) on the pathway to the formation of A. On the other hand, neither intermediate has been detected in the reaction of mammalian AlkBH5. Therefore, one of the aspects we are interested in studying in the lab is the possible divergences in the chemical demethylation mechanisms of these two enzymes or whether the lack of detection of the same intermediates in the reaction of AlkBH5 is merely related to low in vitro activity of this enzyme. We are also focused on identifying the nature of optimum substrates of the aforementioned enzymes using an array of biochemical tools such as liquid chromatography (LC)-triple quad mass (QQQ) spectrometry-based kinetic assays, UV-visible spectroscopy, stopped-flow spectroscopy, and next generation sequencing methods.

3. Biotechnological tools for methylome sequencing on DNA and RNA.

My group is exploring the biotechnological application of the TET enzymes and the m6A demethylases in single-base resolution methylome sequencing of DNA and RNA, respectively, with the ultimate goal of developing commercial sequencing kits, which will serve as a molecular diagnostic tool for the epigenome and transcriptome-wide mapping of 5mC and m6A to understand the role of these bases and their modifications in various biological processes and disease states.

James Eaglesham

Staff Scientist
Ph.D., Harvard Medical School, USA, 2020

Area of focus:

Cell defense against viruses

Current Research:

Mehmet Berkmen

Senior Scientist
Ph.D., University of Vienna, University of Houston, 2000

Area of focus:

Strain engineering and disulfide bonded protein folding

Current Research:

We are interested in understanding the molecular mechanisms that govern oxidative folding of proteins. Many important proteins require disulfide bonds for their folding and stability, including antibodies, interferons, proteases and signaling proteins. The formation of the correct disulfide bonds in a protein requires the coordinated activity of enzymes belonging to the thioredoxin family. In eukaryotes, disulfide bond formation is compartmentalized to the endoplasmic reticulum where 30% of all proteins fold; of those, half are predicted to form disulfide bonds. In Gram negative prokaryotes, disulfide bond formation occurs in the periplasm. A deep understanding of the processes involved in disulfide bond formation is critical to the production of complex disulfide-bonded proteins.

Our research focuses on how disulfide bond formation is catalyzed. In addition, we are interested in how mis-oxidized proteins are isomerized back to their native correctly folded state. We investigate these principles in vivo using the model organism E. coli. Specifically, our research is focused on understanding the mechanisms of forming disulfide bonds within the two distinct compartments of E. coli, the reducing cytoplasm and the oxidizing periplasm. We hope to, (i) improve our understanding of the mechanism of oxidative-folding, (ii) identify novel enzymes which can assist in the oxidative-folding of proteins and (iii) engineer mutant E. coli strains that can express multi-disulfide bonded proteins at high yields.

Jeremy M. Foster

Senior Scientist
Ph.D., University of Liverpool, 1989

Area of focus:

Genomics and glycobiology of filarial nematodes

Current Research:

Parasitic filarial nematodes threaten the wellbeing of 1 in 5 people on the planet and trap heavily infected communities in poverty.  The few available therapies do not kill adult worms but prevent transmission by targeting the larval forms transmitted by insect vectors. These drugs are losing their effectiveness and furthermore there is no vaccine available. Wolbachia endosymbionts of filarial nematodes are essential for parasite development and adult worm survival providing a new avenue towards filarial disease control. Research in the laboratory aims to uncover aspects of filarial and Wolbachia biology that might highlight molecules or processes for further development as drug targets, vaccine candidates or diagnostic reagents. 

Filarial nematodes survive in immunocompetent mammalian hosts for many years. The worm surface is rich in glycoconjugates implicated in mechanisms of immunomodulation and immune evasion. We are applying new reagents and technologies to characterize specific classes of surface glycoconjugates and identify unusual sugar modifications that might serve as vaccine components or diagnostic tools. We also apply serum glycoprofiling to highlight potential biomarkers of disease progression.

We use NextGen sequencing to examine diversity within and between different geographic isolates and laboratory strains of filarial nematodes and their Wolbachia endosymbionts.  Genes under varying selection pressures and required for life cycle progression between the human and insect hosts are subjected to detailed functional characterization.

Peter Weigele

Senior Scientist
Ph.D., University of Utah, 2003

Area of focus:

The biochemistry of complex nucleic acid modifications

Current Research:

My group seeks to discover novel complex nucleic acid modifications and figure out how they are made. We have multiple goals: identify non-canonical nucleosides of biological origin, determine their exact chemical structures, find the genes responsible for their formation, reconstitute the biosynthesis of modifications in vitro, and where possible, harness these activities as tools to manipulate nucleic acids in new and useful ways.

In collaboration with the laboratory of Ivan Corrêa, we have recently identified two novel thymidine base "hypermodifications": 5-aminoethoxymethyl-2'-deoxyuridine (5-NeOmdU) and 5-aminoethyl-2'-deoxyuridine (5-NedU) in the chromosomal DNA of certain bacteriophages. My group has subsequently determined the genes responsible for their biosynthesis, characterized the major pathway intermediates en route to their formation, and uncovered the metabolic origins of the modifying chemical groups. These discoveries now enable us to reconstitute the formation of 5-NeOmdU and 5-NedU from purified enzymes and substrates in vitro. We are now exploring ways in which we can use these base-modifying enzymatic activities to introduce useful chemical functionalities into DNA.

In addition to the technological applications of base hypermodification, we will continue to investigate the biology of complex DNA modification. Homologs of genes encoding DNA hypermodifying functions are also found encoded in mobile DNA, diverse bacteria, and even fungi, suggesting that base-hypermodification is "repurposed" for a variety of epigenetically-directed DNA-based functions. Future work will take advantage of our proven biochemical approaches to identify the functionalities of predicted DNA hypermodifying gene products found in cellular organisms. Biochemical identification and characterization of their activities will reveal new biological functions of complex base modifications and expand our understanding of the epigenetic coding capacity of nucleic acid polymers.

Tom Evans

Executive Director, Research
Ph.D., University of Minnesota, 1996

Area of focus:

Enzymes and technologies involved in DNA repair and replication

Current Research:

Previously my group developed an in vitro DNA repair mix that was commercialized under the name “PreCR” and, later, a next generation sequencing (NGS) focused mix termed “NEBNext FFPE DNA Repair Mix”.  Both mixes contain 7 enzymes and were designed to be as general as possible in their repair capabilities.  These mixes improve NGS library preparation quality from damaged DNA and allow longer reads in long read technologies.  Our interest in DNA damage and repair led us to ask if damage during DNA manipulation is impacting the quality of sequencing data.  In collaboration with Laurence Ettwiller and Pingfang Liu we demonstrated that in vitro DNA damage does indeed lead to sequencing miscalls at a level that obscures the identification of low frequency somatic variants, even data in commonly used databases (Chen, L, et. al., Science (2017), 355(6326):752).  We are continuing these studies looking at whether regions of the genome are particularly sensitive to certain types of in vitro DNA damage.

In addition, we are developing methodologies that use current sequencing instruments to generate large datasets to more accurately identify in vivo DNA damage and initiation/termination sites in the genome.

Elisabeth Raleigh

Emeritus Scientist
Ph.D., Massachusetts Institute of Technology, 1981

Area of focus:

Adding new genes to bacterial chromosomes and controlling their spread.

Current Research:

My research interests concern mechanisms that shape horizontal gene transfer. The main focus is the molecular mechanisms that determine the distribution and expression of RM systems.

Of particular interest are modification-dependent restriction systems (MDRS), which cleave when DNA IS modified rather than when it is not. This action of MDRS affects the “clonability” of DNA from many sources whether by scientists or by natural transfer; it can also affect what spectrum of RM systems can co-exist in the cell. Study of the recognition specificity and mode of action of these enzymes was pioneered here.

We have turned to query what genetic mechanisms contribute to RM system acquisition, to understand how the diversification of the RM repertoire occurs in populations. This specific problem exemplifies a more general one, the genesis of “genome island variation”, which allows a lineage to explore many modes of interaction with its environment. There are anecdotal answers in individual cases--transposable elements, site-specific recombination systems, and favorable local DNA sequence are inferred to play roles in different instances for example. We are focusing on the diversification process at a single location, the highly variable “immigration control region” of the E. coli chromosome. We have found new genes and proteins that contribute to nonhomologous gene acquisition in E. coli and are extending the inquiry to other enteric bacteria.

Nathan Tanner

Associate Director
Ph.D., Harvard University, 2010

Area of focus:

Isothermal amplification, molecular diagnostics, single-molecule analysis of replication enzymes

Current Research:

Amplification of nucleic acids is a fundamental process in biotechnology, used in applications from molecular cloning to sequencing and molecular diagnostics. This wide variety of amplification techniques and applications requires a flexible toolbox of enzymes and methods to enable such a diverse user base. We are focused on studying and improving this collection of reagents and techniques, with efforts to develop improved amplification polymerases and enzymes as well as novel amplification methods for applications in both laboratory and point-of-need settings. Using a variety of methods, particularly isothermal amplification techniques (LAMP, SDA, NASBA, etc.), we work to ensure NEB offers unique and optimized reagents and can support a variety of nucleic acid amplification and molecular diagnostics applications.

Jennifer Ong

Scientific Director
Ph.D., University of Cambridge, 2005

Area of focus:

DNA polymerases, RNA polymerases, reverse transcriptases, replication fidelity, next-generation sequence, protein engineering

Current Research:

The replication of genetic material is fundamentally important to biology and the maintenance of genetic information. DNA polymerases, the enzymes that replicate DNA, also enable many technologies in modern molecular biology, such as DNA amplification, nucleic acid-based diagnostics and next-generation sequencing. Errors made by DNA polymerases can have significant consequences for such applications. Our lab studies replication errors by single-molecule sequencing to assess the frequency, identity and context of mistakes made by DNA polymerases and reverse transcriptases. We are also interested in studying the effect of DNA lesions and modified bases on polymerase fidelity.

In addition to studying replication fidelity, we are also interested in protein engineering to create new enzyme variants to enable new technology development. While nature has been a rich source of new enzymes and activities, many new technologies require enzymes to act under conditions that were not selected by nature. We utilize a combination of computational protein design, high-throughput screening, machine-learning and evolution strategies to alter enzymatic function and properties. Our aim is to approach protein engineering from a research and discovery perspective, by hypothesizing, testing and developing new tools for evolving new enzymes.

Bill Jack

Senior Scientist
Ph.D., Duke University, 1983

Area of focus:

DNA polymerase function

Current Research:

My lab focuses on DNA polymerases, more particularly on the ability to incorporate modified nucleotides, including chain terminators. This focus is expanding to include polymerase activity on modified DNA templates, for example, those of modified phage genomes.

Greg Lohman

Senior Scientist
Ph.D., Massachusetts Institute of Technology, 2007

Area of focus:

DNA & RNA ligase mechanisms, kinetics and applications

Current Research:

The primary goal of research in the Lohman lab is the deep understanding of key enzymes in DNA repair. We study enzymes that have important roles in both biology and biotechnology, aiming to profile substrate specificity, elucidate the mechanism and characterize the detailed kinetics of enzyme reaction. We study nucleic acid enzymes with the goal both of publication of basic research expanding knowledge of these systems, and applying the information in the development and improvement of molecular biology applications.

Polynucleotide ligases, a core focus of my work to date, are critical players in the maintenance of genome integrity, replication and repair. These enzymes further play key roles in methodologies including traditional cloning methods, modern synthetic biology gene assembly methods such as Golden Gate and Gibson Assembly, the preparation of libraries for next generation sequencing, and molecular diagnostics methods such as the ligase detection reaction and the ligase chain reaction.

Despite this central importance in both biology and biotechnology, there is still a great deal to learn about the mechanism of DNA ligases. Many questions remain, including the kinetics of enzyme dynamics, the details of DNA binding and searching, determinants of substrate specificity and ligation fidelity, and the mechanism of end-joining ligation. We aim to develop high throughput, information-dense assays for studying the details of DNA ligation, utilizing rapid chemical quench, stopped-flow, high throughput capillary electrophoresis, and next generation sequencing methodologies.

Our recent study of DNA ligase fidelity and bias in end-joining reactions revealed trends in DNA ligation that have been applied to the improvement of DNA assembly methods. We have demonstrated that one-pot, 25+ fragment Golden Gate Assembly reactions are easily achieved when using data-driven junction sequence selection, enabled by our comprehensive study of ligase fidelity.

The Lohman lab has also begun the detailed study of DNA exonucleases. Current projects aim to study the substrate specificity of commercially available exonucleases in great detail, including investigation of the mechanistic origins of inhibition by a range of modified substrates, hybrid helix specificity, and the mechanistic details of DNA binding and exonuclease processivity. As with DNA ligases, this knowledge will enable improvements to existing exonuclease applications and the development of new technologies.

Kelly Zatopek

Staff Scientist
Ph.D., Brown University, 2014

Area of focus:

Nucleic acid maintenance pathways of archaeal extremophiles

Current Research:

Ivan Correa

Associate Director
Ph.D., State University of Campinas, 2003

Area of focus:

Nucleic acid modifications, bioorthogonal probes, synthetic chemistry, mass spectrometry.

Current Research:

A major focus of my laboratory is the design and synthesis of chemical probes for studying biomolecules in vitro and in living cells. We strive to bring concepts from synthetic chemistry into biological systems to track, manipulate, separate, and interrogate nucleic acids, proteins, glycans, and other cellular species. We are particularly interested in the development of chemical and bioanalytical tools to study DNA, RNA, and their modifications with biological significance. Projects involve the application of multi-stage mass spectrometry, chemical synthesis, and biochemical assays toward the screening, identification, and quantification of epigenetic and epitranscriptomic modifications. Another area of focus is the development of novel chemical and biological strategies to assemble RNA 5’-cap structures for targeted enrichment and sequencing of cellular transcripts. Our lab is engaged in multiple research collaborations aimed at the investigation of nucleic acid modifications from various organisms as well as the characterization of the respective modification enzymes. 

We are also interested in the design of innovative chemical probes for the SNAP-tag and CLIP-tag protein labeling systems. Our efforts toward the synthesis of fluorescent dyes, blockers, biotin labels, and functionalized building blocks with unique properties have enabled researchers to image biological structures with greater resolution and lesser background. Current projects are focused on advancing methods for immobilization of SNAP-tag fusion proteins and expanding the breadth of multifunctional probes for detection of protein-protein and protein-nucleic acid interactions.

S. Hong Chan

Staff Scientist
Ph.D., The Chinese University of Hong Kong, 2000

Area of focus:

mRNA capping and 5’ modifying enzymes and technologies

Current Research:

Ira Schildkraut

Emeritus Scientist
Ph.D., University of Miami, 1974

Area of focus:

Enzymatic manipulation of RNA

Current Research:

My current research interests are concerned with identifying enzymes/activities that can be used to manipulate RNA. We have characterized activities that react specifically with various eukaryotic cap structures to remove caps from eukaryotic RNA. These particular activities are of interest as they leave a 5′ diphosphate. We have also shown that Vaccinia Capping Enzyme (VCE) can add a 3′ biotinylated GMP to the 5′ triphosphate or diphosphate of RNA. Together these newly described characteristics have enabled us to develop a method for the enrichment of primary transcripts of prokaryotic RNA as well as eukaryotic capped RNA. This method overcomes the challenge of the overabundant ribosomal RNA inherent in all microbial RNA preparations regardless of genus. Biotinylated transcripts are subsequently selected on streptavidin magnetic beads while the unwanted processed and ribosomal transcripts are eliminated. The method yields NextGen sequencing libraries for determination of the transcriptional start sites to one base pair resolution, with prokaryotes or eukaryotes. The methods are currently being adapted to reveal eukaryotic splice variants using long read sequencing technologies.

Hoong Lim

Staff Scientist
Ph.D., Yale University, 2014

Area of focus:

Molecular origins of microbial genetic innovations

Current Research:

Robert Trachman

Staff Scientist
Ph.D., Johns Hopkins University, USA, 2014

Area of focus:

DNA, RNA, viruses, immunity

Current Research:

Dora Posfai

Associate Director of Core Facilities & Group Leader Sequencing Core

Area of focus:

Capillary electrophoresis and NGS

Current Research:

Léa Chuzel

Group Leader Metagenomics Screening Research

Area of focus:

Metagenomics screening

Current Research:

Zhiyi Sun

Group Leader Bioinformatics Research

Area of focus:

Bioinformatics solutions for research

Current Research: