Founded in 1975 as an independent research institution, now part of Vilnius University, the Institute of Biotechnology strives to maintain the high standards of excellence in scientific endeavour, research training and technological advance with its main focus ina broadly defined field of molecular biotechnology including nucleic acid and protein technologies, bioinformatics, molecular diagnostics, drug design, next generation epigenomic and gene editing technologies. The Institute provides an interface between advanced education, basic research and technological development for the economic and social benefit of Lithuania.

Department of Biological DNA Modification


Chief Scientist and Head

Dr. Habil., FRSC

ORCID; Google Scholar; ResearcherID

phone: +370 5 2234350
fax: +370 5 2234367
e-mail: saulius.klimasauskas (at)

NEWS: ERC Advanced Grant support for the EpiTrack project!

Employees                                    PhD students

Giedrius Vilkaitis, Ph.D.                    Milda Rudytė, M.Sc.
Edita Kriukienė,  Ph.D.                     Povilas Gibas, M.Sc.
Rasa Rakauskaitė, Ph.D.                   Milda Mickutė, M.Sc.
Viktoras Masevičius, Ph.D.                Stasė Gasiulė. M.Sc.
Juozas Gordevičius, Ph.D.                 Janina Ličytė, M.Sc.
Miglė Tomkuvienė, Ph.D.
Vaidotas Stankevičius, Ph.D.
Zdislav Staševskij, M.Sc.

Giedrė Urbanavičiūtė, M.Sc.
Audronė Rukšėnaitė, M.Sc.
Kotryna Kvederavičiūtė, M.Sc.
Aleksandr Osipenko, M.Sc.

Research Overview

AdoMet-dependent methyltransferases (MTases), which represent more than 3% of the proteins in the cell, catalyze the transfer of the methyl group from S-adenosyl-L-methionine (AdoMet) to N-, C-, O- or S-nucleophiles in DNA, RNA, proteins or small biomolecules.

In DNA, enzymatic methylation of nucleobases serves to expand the information content of the genome in organisms ranging from bacteria to mammals. Postreplicative methylation is accomplished by DNA methyltransferases yielding 5-methylcytosine, N4-methylcytosine or N6-methyladenine. Genomic DNA methylation is a key epigenetic regulatory mechanism in high eukaryotes. Aberrant DNA methylation correlates with a number of pediatric syndromes and cancer, or predisposes individuals to various other human diseases. However, research into the epigenetic misregulation and its diagnostics is hampered by the limitations of available analytical techniques. We aim to develop new approaches to genome-wide profiling of DNA methylation for epigenome studies and improved diagnostics.

Besides their diverse biological roles, DNA MTases are attractive models to study the structural aspects of DNA-protein interaction. Bacterial enzymes recognize an impressive variety (over 200) of short sequences in DNA. Although the methylation of biopolymers generally occurs in a highly specific manner, the naturally transferred methyl group has limited utility for practical applications. Our goal is to redesign the methyltransferase reactions for targeted covalent deposition of desired functional or reporter groups onto biopolymer molecules such as DNA and RNA.

Targeted covalent labeling of biopolymers

Our goal is to convert MTases into alkyltransferases for sequence-specific covalent modification of DNA and other biopolymers. Our strategy is based on designing novel synthetic analogues of the natural cofactor AdoMet. We have synthesized a series of model AdoMet analogs with sulfonium-bound extended side chains replacing the methyl group (Klimašauskas and Weinhold, 2007). This novel enabling technology named mTAG (methyltransferase-directed Transfer of Activated Groups) is a convenient and robust technique that is suitable for routine laboratory use. In particular, we demonstrated that propargylic side chains can be efficiently transferred by DNA MTases with high sequence- and base-specificity (Lukinavičius et al., 2007, 2012 and 2013; Masevičius et al., 2016; Tomkuvienė et al., 2016; Tomkuvienė et al., 2019) offering many potential applications for genomic (Neely et al., 2010) and epigenomic (see below) studies. Moreover, the newly developed cofactors are suitable for targeted transfer of functional groups or other chemical entities to RNA (Tomkuvienė et al., 2012; Plotnikova et al., 2014; Osipenko et al., 2017; Mickutė et al., 2018) using appropriate MTases as catalysts.

Novel approaches to epigenome profiling

Genomic DNA methylation is a key epigenetic regulatory mechanism in high eukaryotes, however, research into the epigenetic regulation is hampered by limitations of current analytical techniques (Daniūnaitė et al., 2019). We therefore aim to develop new experimental approaches to genome-wide profiling of DNA methylation for epigenome studies and improved diagnostics. Our approach is based on selective mTAG labeling and enrichment of unmethylated CpG sites (Gerasimaitė et al., 2009; Kriukienė et al. 2013; Labrie et al., 2016) in the genome followed by analysis of the enriched fractions on tiling microarrays (in collaboration with Prof. Art Petronis, CAMH, Toronto, Canada). Recently, we have advanced DNA methylome profiling in by developing a high-resolution economical technique named Tethered Oligonucleotide-Primed sequencing, TOP-seq, which exploits non-homologous priming of the DNA polymerase at covalently tagged CpG sites to directly produce adjoining regions for their sequencing and precise genomic mapping (Staševskij et al., 2017).

In the absence of the S-adenosylmethionine cofactor, bacterial cytosine-5 MTases can catalyze catalyze reversible covalent addition of exogenous aliphatic aldehydes to their target residues in DNA, thus yielding corresponding 5-hydroxyalkylcytosines (Liutkevičiūtė al., 2009). Moreover, our most recent studies demonstrate the ability of the MTases to direct condensation of aliphatic thiols and selenols with 5-hydroxymethylcytosine in DNA to yield 5-alkylchalcogenomethyl derivatives (Liutkevičiūtė et al., 2011) or decarboxylation of 5-carboxylcytosines (Liutkevičiūtė et al., 2014) in DNA. These atypical reactions demonstrate a surprizing catalytic versatility of these enzymes and pave new ways for the sequence-specific derivatization and analysis of 5-hydroxymethylcytosine in DNA (Kriukienė et al., 2012).

Methylation of small non-coding RNA

MicroRNAs and siRNAs are small non-coding double-stranded RNA molecules that control gene activity in a homology-dependent manner - a process named RNA interference. Since their discovery in 1993, numerous microRNAs have been identified and recognized as important regulators of gene expression in both plants and animals. Many microRNAs have well-defined developmental and tissue-specific expression pattern, but a great number of microRNAs and their roles are still unknown.

The biogenesis of miRNAs and siRNAs in plants differs from that in animals as it involves an additional methylation step catalyzed by the HEN1 methyltransferase. HEN1 from Arabidopsis catalyzes the transfer methyl groups from AdoMet onto the 2'OH group of the 3'-terminal nucleotide of small RNAs, like miRNA/miRNA* and siRNA/siRNA*. The methylation is imperative in the biogenesis of microRNA in Arabidopsis since microRNAs in hen1 mutants are reduced in abundance or are totally absent. A number of biochemical approaches have been developed in our laboratory for examining the unique methyltransferase HEN1 (Yang et al., 2007; Vilkaitis et al., 2010; Plotnikova et al., 2013; Baranauskė et al., 2015).


Recent publications

M. Tomkuvienė, M. Mickutė, G. Vilkaitis, and S. Klimašauskas
Repurposing enzymatic transferase reactions for targeted labeling and analysis of DNA and RNA.
Curr. Opin. Biotechnol., 2019, 55: 114-123.

K. Daniūnaitė, S. Jarmalaitė, and E. Kriukienė

Epigenomic tehcnologies for diciphering circulating tumor DNA.
Curr. Opin. Biotechnol., 2019, 55: 23-29.

M. Mickutė, M. Nainytė, L. Vasiliauskaitė. A. Plotnikova, V. Masevičius, S. Klimašauskas and G. Vilkaitis
Animal Hen1 2′-O-methyltransferases as tools for 3′-terminal functionalization and labelling of single-stranded RNAs.
Nucleic Acids Res., 2018, 46: e104.

M. Alexeeva, P. Guragain, A.N. Tesfahun, M. Tomkuvienė, A. Arshad, R. Gerasimaitė, A. Rukšėnaitė, G. Urbanavičiūtė, M.Bjørås, J.K. Laerdahl, A. Klungland, S. Klimašauskas and S. Bjelland
Excision of the double methylated base N4,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins.
Phil. Trans. R. Soc. B, 2018, 373(1748): 20170337.

J. Gordevičius, A. Kriščiūnas, D.E. Groot, S.M. Yip, M. Susic, A. Kwan, R. Kustra, A.M. Joshua, K.N. Chi, A. Petronis, and G. Oh.
Cell-free DNA modification dynamics in abiraterone acetate-treated prostate cancer patients.
Clin. Cancer Res., 2018, 24(14): 3317-3324.

M. Tomkuvienė, J. Ličytė, I. Olendraitė, Z. Liutkevičiūtė, B. Clouet-d'Orval and S. Klimašauskas
Archaeal fibrillarin-Nop5 heterodimer 2'-O-methylates RNA independently of the C/D guide RNP particle.
RNA, 2017, 23(9): 1329-1337.

A. Osipenko, A. Plotnikova, M. Nainytė, V. Masevičius, S. Klimašauskas, and G. Vilkaitis
Oligonucleotide-addressed covalent 3'-terminal derivatization of small RNA strands for enrichment and visualization.
Angew. Chem. Int. Ed., 2017, 56(23): 6507-6510.

Z. Staševskij, P. Gibas, J. Gordevičius, E. Kriukienė, and S. Klimašauskas

Tethered Oligonucleotide-Primed sequencing, TOP-seq: a high resolution economical approach for DNA epigenome profiling.
Mol. Cell, 2017, 65(3): 554-564.

M. Tomkuvienė, E. Kriukienė, and S. Klimašauskas

DNA labeling using DNA methyltransferases.
Adv. Exp. Med. Biol., 2016, 945: 511-535.

V. Labrie, O. J. Buske, E. Oh, R. Jeremian, C. Ptak, G. Gasiūnas, A. Maleckas, R. Petereit, A. Žvirbliene, K. Adamonis, E. Kriukienė, K. Koncevičius, J. Gordevičius, A. Nair, A. Zhang, S. Ebrahimi, G. Oh, V. Šikšnys, L. Kupčinskas, M. Brudno, and A. Petronis

Lactase nonpersistence is directed by DNA-variation-dependent epigenetic aging.
Nature Struct. Mol. Biol. 2016, 23(6): 566-573.

V. Myrianthopoulos, P. F. Cartron, Z. Liutkevičiūtė, S. Klimašauskas, D. Matulis, C. Bronner, N. Martinet, and E. Mikros
Tandem virtual screening targeting the SRA domain of UHRF1 identifies a novel chemical tool modulating DNA methylation.
Eur. J. Med. Chem., 2016, 114: 390-396.

V. Masevičius, M. Nainytė, and S. Klimašauskas
Synthesis of S-adenosyl-L-methionine analogs with extended transferable groups for methyltransferase-directed labeling of DNA and RNA.
Curr. Protoc. Nucleic Acid Chem., 2016, 64: 1.36.1-1.36.13.

R. Rakauskaitė, G. Urbanavičiūtė, A. Rukšėnaitė, Z. Liutkevičiūtė, R. Juškėnas, V. Masevičius, and S. Klimašauskas
Biosynthetic selenoproteins with genetically-encoded photocaged selenocysteines.
Chem. Commun., 2015, 51(39): 8245-8248.

S. Baranauskė, M. Mickutė, A. Plotnikova, A. Finke, Č. Venclovas, S. Klimašauskas, and G. Vilkaitis
Functional mapping of the plant small RNAmethyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins.
Nucleic Acids Res., 2015, 43(5): 2802-2812.

A. Plotnikova, A. Osipenko, V. Masevičius, G. Vilkaitis, and S. Klimašauskas
Selective covalent labeling of miRNA and siRNA duplexes using HEN1 methyltransferase.
J. Am. Chem. Soc., 2014, 136(39):

Z. Liutkevičiūtė, E. Kriukienė, J. Ličytė, M. Rudytė, G. Urbanavičiūtė, and S. Klimašauskas
Direct decarboxylation of 5-carboxylcytosine by DNA C5-methyltransferases.
J. Am. Chem. Soc
., 2014, 136(16): 5884?5887.

E. Kriukienė, V. Labrie,
T. Khare, G. Urbanavičiūtė, A. Lapinaitė, K. Koncevičius, D. Li, T. Wang, S. Pai, C. Ptak, J. Gordevičius, S.C. Wang, A. Petronis, and S. Klimašauskas
DNA unmethylome profiling by covalent capture of CpG sites.
Nature Commun., 2013, 4: 2190.

A. Plotnikova, S. Baranauskė, A. Osipenko, S. Klimašauskas, and
G. Vilkaitis

Mechanistic insights into small RNA recognition and modification by the HEN1 methyltransferase.
Biochem J., 2013, 453: 281-290.

G. Lukinavičius, M. Tomkuvienė, V. Masevičius, and S. Klimašauskas
Enhanced chemical stability of AdoMet analogues for improved methyltransferase-directed labeling of DNA.
ACS Chem. Biol., 2013, 8: 1134-1139.

T. Khare, S. Pai, K. Koncevičius, M. Pal, E. Kriukienė, Z. Liutkevičiūtė, M. Irimia, P. Jia, C. Ptak, M. Xia, R. Tice, M. Tochigi, S. Morera, A. Nazarians, D. Belsham, A.H.C. Wong, B.J. Blencowe, S.C. Wang, P. Kapranov, R. Kustra, V. Labrie, S. Klimašauskas, and A. Petronis
5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary.
Nature Struct. Mol. Biol., 2012, 19: (10) 1037-1043.

E. Kriukienė, Z. Liutkevičiūtė, and S. Klimašauskas
5-Hydroxymethylcytosine - the elusive epigenetic mark in mammalian DNA.
Chem. Soc. Rev., 2012, 41: (21) 6916-6930.

G. Lukinavičius, A. Lapinaitė, G. Urbanavičiūtė, R. Gerasimaitė, and S. Klimašauskas
Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA.
Nucleic Acids Res., 2012, 40, (22) 11594-11602.

M. Tomkuvienė, B. Clouet-d'Orval, I. Černiauskas, E. Weinhold, and S. Klimašauskas

Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases.
Nucleic Acids Res., 2012, 40, (14) 6765-6773.

R. Sakaguchi, A. Giessing, Q. Dai, G. Lahoud, Z. Liutkevičiūtė, S. Klimašauskas, J. Piccirilli, F. Kirpekar, and Y.-M. Hou
Recognition of guanosine by dissimilar tRNA methyltransferases.
RNA, 2012, 18: 1687-1701.

Z. Liutkevičiūtė, E. Kriukienė, I. Grigaitytė, V. Masevičius and S. Klimašauskas
Methyltransferase-directed derivatization of 5-hydroxymethylcytosine in DNA.
Angew. Chem. Int. Ed., 2011, 50: 2090-2093; Very Important Paper.

R. Gerasimaitė, E. Merkienė and S. Klimašauskas
Direct observation of cytosine flipping and covalent catalysis in a DNA methyltransferase.
Nucleic Acids Res., 2011, 39: 3771-3780; Featured Article.

R.K. Neely, P. Dedecker, J. Hotta, G. Urbanavičiūtė, S. Klimauskas, and J. Hofkens
DNA fluorocode: A single molecule, optical map of DNA with nanometre resolution.
Chem. Sci., 2010, 1: 453-460; Edge Article, commentary in Highlights Chem. Sci.

G. Vilkaitis, A. Plotnikova, and S. Klimašauskas
Kinetic and functional analysis of the small RNA methyltransferase HEN1:
The catalytic domain is essential for preferential modification of duplex RNA.
RNA, 2010, 16: 1935-1942.

N. Miropolskaya, I. Artsimovitch, S. Klimašauskas, V. Nikiforov, and A. Kulbachinskiy 
Allosteric control of catalysis by the F loop of RNA polymerase. 
Proc. Natl. Acad. Sci. USA, 2009, 106: 18942-18947. F1000 evaluation

R. Gerasimaitė, G. Vilkaitis, and S. Klimašauskas
A directed evolution design of a GCG-specific DNA hemimethylase.
Nucleic Acids Res.
2009, 37: 7332-7341.

Z. Liutkevičiūtė, G. Lukinavičius, V. Masevičius, D. Daujotytė, and S. Klimašauskas
Cytosine-5 methyltransferases add aldehydes to DNA.
Nature Chem. Biol., 2009, 5: 400-402. F1000 evaluation

D. Daujotytė, Z. Liutkevičiūtė, G. Tamulaitis and S. Klimašauskas
Chemical mapping of cytosines enzymatically flipped out of the DNA helix.
Nucleic Acids Res.
, 2008, 36: e57.

G. Lukinavičius, V. Lapienė, Z. Staševskij, C. Dalhoff, E. Weinhold, and S. Klimašauskas
Targeted labeling of DNA by methyltransferase-directed Transfer of Activated Groups (mTAG).
J. Am. Chem. Soc., 2007, 129: 2758-2759.

S. Klimašauskas and E. Weinhold
A new tool for biotechnology: AdoMet-dependent methyltransferases.
Trends Biotechnol., 2007, 25: 99-104.