Postdoctoral Researcher. Supervisor: Professor Peter E Nielsen, University of Copenhagen, Center for Biomolecular Recognition (CBR), Institut for Cellulær og Molekylær Medicin (ICMM), Faculty of Health Sciences (Det Sundhedsvidenskabelige Fakultet), The Panum Institute, Copenhagen, Denmark. 2001 - 2009.

Outline: Nucleic acids (PNA, DNA) analyzed by Optical Spectroscopy (UV-Vis, fluorescence), Surface Plasmon Resonance (BIAcore), FACS, ITC, AFM.

Highlights of the main aspects explored:

To accomplish non-enzymatic PNA ligation (free in solution as well as attached on magnetic beads), as the basic foundation for protocell design involving compartmentalized replication and recycle of genetic information in order for understanding the origin of life (using FRET, mass spectrometry and HPLC analyses). (EU “PACE” project, 2004-2008.)

To explore spectroscopic, thermodynamic and kinetic aspects of PNA•DNA hybridization using thermal melting technique (UV-Vis spectrophotometer) and Optical Biosensor technique that utilizes surface plasmon resonance (BIAcore instrument), along with solution conformations of PNA•DNA using Circular Dichroism (spectropolarimetric technique).

To pinpoint the molecular mechanisms of hydrogen bonding versus stacking forces by incorporation of modified nucleobases in PNA•DNA Duplexes.

To investigate PNA vs. DNA in non-aqueous medium, DMF, dioxane, formamide, ethylene glycol and glycerol (thermodynamics and ITC).

To examine the interaction of potential cell-penetrating PNA-peptide conjugates with phospholipid bilayer (constructed in Langmuir-Blodgett trough) analyzed by AFM and CD (done in Prof. Thomas Bjørnholm’s lab.).

• To attempt a real-time transfer and entrapment of fluorescent labeled PNAs into POPC vesicles, analyzed by the Fluorescence-activated Cell Sorting (FACS).

Collaboration with James Graham Brown Cancer Center (USA): Targeting c-myc proto-oncogene G-quadruplex by PNA

With utter solo effort, I initiated this collaborative multi-dimensional project in 2014, which is dedicated to explore the fascinating world of guanine.

c-myc proto-oncogene is overexpressed in a majority of human malignancies. Transcriptional inhibition of specific cancer-related genes has functioned as a therapeutic miracle for over thirty years. It has not been possible to demonstrate gene-specific transcriptional inhibition under biologically relevant conditions, because DNA repair mechanisms do not allow tertiary structures to exist intra-cellularly, such as, triplex. That’s where guanine quadruplex structure (high prevalence of clustered G units throughout human genome) came into the scene in the last decade. G-quadruplex serves as therapeutic target in vivo and supra-molecular objects in nanoscience. Prof. Donald M. Miller’s group was the first to show the growth inhibitory activity of quadruplex-forming DNA aptamers utilizing such approach in gene therapy clinical trials (Journal of Biological Chemistry, (2001), 276, 43221).

In an attempt of achieving

◦persistent gene-specific down-regulation of c-myc expression,

◦death of malignant cells,

◦identifying mutations leading to c-myc dysregulation,

◦maximizing growth inhibitory activity,

◦inhibition of cell line proliferation,

◦stronger block to polymerase extension,

we (the Medical Oncology group and gene medicine (PNA) group) have teamed up together, contemplating and designing, in search for the key aspect of a novel generation of selectivity-optimized probe for genomic G-quadruplex-targeting, which could represent the basis of a promising new design strategy.

My task involved the structural aspects of quadruplex-targeting.

We are utilizing strategies, such as:

◦strand invasion of double stranded c-myc target

◦higher order secondary structure that sustains the NHE III1 element in “off” form (transcriptionally inactive state)

◦backbone, sugar, nucleobase modifications for elevated affinity

◦suitable PNA conjugates for easier cell delivery

The elucidation of the NHE III1 family member sequences in regulating cell growth will have important therapeutic implications. Successful completion of this project will open up a new generation of design and will result in an increased understanding of the role of quadruplex-forming DNA in the integration of control mechanisms for cellular proliferation.

2013-2015: Interaction of PNA with special structural mimics of DNA “Ferrocene Nucleic Acids” (international collaboration, finishing soon, going to be published soon).

Collaboration with University of Birmingham (UK): Interactions of Ferrocene Nucleic Acid FCNA

In an attempt of

◦inventing a synthetic mimic of the natural nucleic acids,

◦inventing a more novel approach than M-DNA (an effective semi-conductor in the world of molecular Electronics, Journal of Inorganic Biochemistry, 2003, 94, 94-99),

◦particularly, inventing a superior alternative to the backbone-modified versions, such as PNA and LNA, for instance,

a Bioorganometallic structural mimic Ferrocene Nucleic Acid FcNA has recently been discovered (Chem. Comm.Camb., 2012, 48, 12165-12167), which is the very first example in this new family of artificial nucleic acids.

With utter solo effort, I initiated this collaborative multi-dimensional project in 2013, which is dedicated to explore and define the role of FcNA, targeting natural nucleic acids as well as PNA, by FcNA probes, which contain repeating nucleobase-tagged Ferrocene units as integral component of the backbone, in order for getting an insight into the binding mechanisms, and scrutinizing them, as well as an eventual utilization of metal-incorporated helical structures as functional building blocks for nanostructures in near future (not part of the current project).

Postdoctoral Researcher. Supervisor: Professor Peter E Nielsen, University of Copenhagen. Head, Department of Cellular and Molecular Medicine II (ICMM II) (The Panum Institute), & Department of Drug Design and Pharmacology. Faculty of Health and Medical Sciences, Copenhagen, Denmark. 2001 - 2010, 2013 - present time.

Outline: Nucleic acids and mimics (PNA, DNA, FcNA) analyzed by Optical Spectroscopy (UV-Vis, fluorescence, CD), SPR (BIAcore), FACS, ITC, AFM.

Highlights of the main aspects explored:

◦To accomplish non-enzymatic PNA ligation (free in solution as well as attached on magnetic beads), as the basic foundation for protocell design involving compartmentalized replication and recycle of genetic information in order for understanding the origin of life (using FRET, mass spectrometry and HPLC analyses). (EU “PACE” project, 2004-2008.)

◦To explore spectroscopic, thermodynamic and kinetic aspects of PNA•DNA hybridization using thermal melting technique (UV-Vis spectrophotometer) and Optical Biosensor techniquethat utilizes surface plasmon resonance (BIAcore instrument, in collaboration with Professor Wolfgang Knoll, at the Max Planck Institute for Polymer Research, Mainz, Germany), along with solution conformations of PNA•DNA using Circular Dichroism (spectropolarimetric technique).

◦To pinpoint the molecular mechanisms of hydrogen bonding versus stacking forces by incorporation of modified nucleobases in PNA•DNA Duplexes.

◦To investigate PNA vs. DNA in non-aqueous medium, DMF, dioxane, formamide, ethylene glycol and glycerol (thermodynamics and ITC).

◦To examine the interaction of potential cell-penetrating PNA-peptide conjugates with phospholipid bilayer (constructed in Langmuir-Blodgett trough) analyzed by AFM and CD (done in Prof. Thomas Bjørnholm’s lab.).

◦To attempt a real-time transfer and entrapment of fluorescent labeled PNAs into POPC vesicles, analyzed by the Fluorescence-activated Cell Sorting (FACS).

December2001-June2008: Postdoctoral Fellow (supervisor: Prof Peter E Nielsen, Københavns Universitet, Center for Biomolekylær Genkendelse, Institut for Cellulær og Molekylær Medicin (ICMM), Det Sundhedsvidenskabelige Fakultet, Panum Instituttet, København, Denmark).
Including a 4 years EU-sponsored project, Programmable Artificial Cell Evolution (PACE), 2004-2008. www.protocell.org.

Outline: Nucleic acids (PNA, DNA) analyzed by Optical Spectroscopy (UV-Vis, fluorescence), Surface Plasmon Resonance (BIAcore), FACS, ITC, AFM.

Highlights:

Investigate spectroscopic, thermodynamic and kinetic aspects of PNA•DNA hybridization using thermal melting technique (UV-Vis spectrophotometer) and Optical Biosensor technique that utilizes surface plasmon resonance (BIAcore instrument), and solution conformations of PNA•DNA using Circular Dichroism (spectropolarimetric technique).

Investigate and explore the molecular mechanisms of hydrogen bonding versus stacking forces by incorporation of modified nucleobases in PNA∙DNA Duplexes.

Investigate structural and thermodynamic properties of PNA in non-aqueous solvents.

Investigate non-enzymatic PNA ligation (free in solution as well as on magnetic beads) with an ultimate goal to design protocells (techniques used for analysis were mainly spectrofluorimetry, along with MALDI-TOF mass spectrometry and HPLC analysis).

Investigate the interaction of PNA-peptide conjugates with phospholipid bilayer, analyzed by AFM and CD.

• Investigate the real-time transfer and entrapment of fluorescent labeled PNAs into liposomes, analyzed by FACS.

December2001-June2008: Postdoctoral Fellow (supervisor: Prof Peter E Nielsen, Københavns Universitet, Center for Biomolekylær Genkendelse, Institut for Cellulær og Molekylær Medicin (ICMM), Det Sundhedsvidenskabelige Fakultet, Panum Instituttet, København, Denmark).
Including a 4 years EU-sponsored project, Programmable Artificial Cell Evolution (PACE), 2004-2008. www.protocell.org, (Detailed description on pages 8-11)
Outline: Nucleic acids (PNA, DNA) analyzed by Optical Spectroscopy (UV-Vis, fluorescence), Surface Plasmon Resonance (BIAcore), FACS, ITC, AFM.
Highlights:

Investigate spectroscopic, thermodynamic and kinetic aspects of PNA•DNA hybridization using thermal melting technique (UV-Vis spectrophotometer) and Optical Biosensor technique that utilizes surface plasmon resonance (BIAcore instrument), and solution conformations of PNA•DNA using Circular Dichroism (spectropolarimetric technique).

Investigate and explore the molecular mechanisms of hydrogen bonding versus stacking forces by incorporation of modified nucleobases in PNA∙DNA Duplexes.

Investigate structural and thermodynamic properties of PNA in non-aqueous solvents.

Investigate non-enzymatic PNA ligation (free in solution as well as on magnetic beads) with an ultimate goal to design protocells (techniques used for analysis were mainly spectrofluorimetry, along with MALDI-TOF mass spectrometry and HPLC analysis).

Investigate the interaction of PNA-peptide conjugates with phospholipid bilayer, analyzed by AFM and CD.

• Investigate the real-time transfer and entrapment of fluorescent labeled PNAs into liposomes, analyzed by FACS.

December2001-June2008: Postdoctoral Fellow (supervisor: Prof Peter E Nielsen, Københavns Universitet, Center for Biomolekylær Genkendelse, Institut for Cellulær og Molekylær Medicin (ICMM), Det Sundhedsvidenskabelige Fakultet, Panum Instituttet, København, Denmark).
Including a 4 years EU-sponsored project, Programmable Artificial Cell Evolution (PACE), 2004-2008. www.protocell.org.

Outline: Nucleic acids (PNA, DNA) analyzed by Optical Spectroscopy (UV-Vis, fluorescence), Surface Plasmon Resonance (BIAcore), FACS, ITC, AFM.

Highlights:

Investigate spectroscopic, thermodynamic and kinetic aspects of PNA•DNA hybridization using thermal melting technique (UV-Vis spectrophotometer) and Optical Biosensor technique that utilizes surface plasmon resonance (BIAcore instrument), and solution conformations of PNA•DNA using Circular Dichroism (spectropolarimetric technique).

Investigate and explore the molecular mechanisms of hydrogen bonding versus stacking forces by incorporation of modified nucleobases in PNA∙DNA Duplexes.

Investigate structural and thermodynamic properties of PNA in non-aqueous solvents.

Investigate non-enzymatic PNA ligation (free in solution as well as on magnetic beads) with an ultimate goal to design protocells (techniques used for analysis were mainly spectrofluorimetry, along with MALDI-TOF mass spectrometry and HPLC analysis).

Investigate the interaction of PNA-peptide conjugates with phospholipid bilayer, analyzed by AFM and CD.

• Investigate the real-time transfer and entrapment of fluorescent labeled PNAs into liposomes, analyzed by FACS.

December2001-June2008: Postdoctoral Fellow (supervisor: Prof Peter E Nielsen, Københavns Universitet, Center for Biomolekylær Genkendelse, Institut for Cellulær og Molekylær Medicin (ICMM), Det Sundhedsvidenskabelige Fakultet, Panum Instituttet, København, Denmark).
Including a 4 years EU-sponsored project, Programmable Artificial Cell Evolution (PACE), 2004-2008. www.protocell.org, (Detailed description on pages 8-11)
Outline: Nucleic acids (PNA, DNA) analyzed by Optical Spectroscopy (UV-Vis, fluorescence), Surface Plasmon Resonance (BIAcore), FACS, ITC, AFM.
Highlights:

Investigate spectroscopic, thermodynamic and kinetic aspects of PNA•DNA hybridization using thermal melting technique (UV-Vis spectrophotometer) and Optical Biosensor technique that utilizes surface plasmon resonance (BIAcore instrument), and solution conformations of PNA•DNA using Circular Dichroism (spectropolarimetric technique).

Investigate and explore the molecular mechanisms of hydrogen bonding versus stacking forces by incorporation of modified nucleobases in PNA∙DNA Duplexes.

Investigate structural and thermodynamic properties of PNA in non-aqueous solvents.

Investigate non-enzymatic PNA ligation (free in solution as well as on magnetic beads) with an ultimate goal to design protocells (techniques used for analysis were mainly spectrofluorimetry, along with MALDI-TOF mass spectrometry and HPLC analysis).

Investigate the interaction of PNA-peptide conjugates with phospholipid bilayer, analyzed by AFM and CD.

• Investigate the real-time transfer and entrapment of fluorescent labeled PNAs into liposomes, analyzed by FACS.

January2001-December2001: Postdoctoral Fellow (supervisor: Prof Gary B Schuster, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332 0400, USA).
Outline: Nucleic acid conjugate analyzed by Optical and Magnetic Spectroscopy.
Highlights:
Investigating the structure and conformation of metal-DNA conjugate called M-DNA, characterizing and analyzing by Fluorescence, Circular Dichroism and NMR spectra.

January2001-December2001: Postdoctoral Fellow (supervisor: Prof Gary B Schuster, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332 0400, USA).
Outline: Nucleic acid conjugate analyzed by Optical and Magnetic Spectroscopy.

Highlights:
Investigating the structure and conformation of metal-DNA conjugate called M-DNA, characterizing and analyzing by Fluorescence, Circular Dichroism and NMR spectra.

January2001-December2001: Postdoctoral Fellow (supervisor: Prof Gary B Schuster, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332 0400, USA).
Outline: Nucleic acid conjugate analyzed by Optical and Magnetic Spectroscopy.
Highlights:
Investigating the structure and conformation of metal-DNA conjugate called M-DNA, characterizing and analyzing by Fluorescence, Circular Dichroism and NMR spectra.

Postdoctoral Fellow. Supervisor: Professor Gary B Schuster, (Dean, College of Sciences, at the time), School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332 0400, USA.

January 2001 - December 2001.

Outline: Nucleic acid conjugate analyzed by Optical and Magnetic Spectroscopy.

Highlights:

Investigation and reassessment of the structure and conformation of divalent metal-DNA conjugate called M-DNA under extreme conditions of pH and ion-concentration, characterizing and diagnosing by Ethidium Bromide intercalation and analyzing by UV-Vis absorption, Fluorescence, Circular Dichroism and 1-2 dimensional 1H NMR spectroscopy.

January2001-December2001: Postdoctoral Fellow (supervisor: Prof Gary B Schuster, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332 0400, USA).
Outline: Nucleic acid conjugate analyzed by Optical and Magnetic Spectroscopy.

Highlights:
Investigating the structure and conformation of metal-DNA conjugate called M-DNA, characterizing and analyzing by Fluorescence, Circular Dichroism and NMR spectra.

Postdoctoral Fellow. Supervisor: Prof Gary B Schuster, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332 0400, USA. January 2001 - December 2001.

Outline: Nucleic acid conjugate analyzed by Optical and Magnetic Spectroscopy.

Highlights:
Investigation and reassessment of the structure and conformation of divalent metal-DNA conjugate called M-DNA under extreme conditions of pH and ion-concentration, characterizing and diagnosing by Ethidium Bromide intercalation and analyzing by UV-Vis absorption, Fluorescence, Circular Dichroism and 1-2 dimensional 1H NMR spectroscopy.

Postdoctoral Fellow. Supervisor: Prof Astrid Gräslund, Stockholm University, Dept of Biochemistry and Biophysics (Institutionen för biokemi och biofysik), The Arrhenius Laboratories (Arrheniuslaboratoriet), SE-106 91 Stockholm, Sweden.
1998 - 2000.

Outline: DNA analyzed by Optical and Magnetic Spectroscopy.

Highlights of the main aspects studied:

To investigate the structure and dynamics of sequence-specific triplex-forming efficiency/propensity of oligonucleotides.

To explore the unique conformational features of A-tracts.

To pinpoint the structural constraints regulating triple helix formation by oligonucleotides containing A-tracts. The inability to support a triplex was interpreted as a probe of altered major groove geometry.

To examine the triplex formation with and without flanking G•C base pairs in order to probe the role of length of the A-tract and the flanking sequences.

• To analyze the base-pair opening dynamics in double helical DNAs catalyzed by imino proton exchange catalysts analyzed by NMR technique, UV-Vis Spectrophotometry and Circular Dichroism.

Postdoctoral Fellow, 1998 - 2000. Supervisor: Professor Astrid Gräslund, Secretary, Nobel Committee for Chemistry (since 1996). That time Head, Dept of Biochemistry and Biophysics, (Institutionen för biokemi och biofysik), The Arrhenius Laboratories (Arrheniuslaboratoriet), Stockholm University, SE-106 91 Stockholm, Sweden.

Outline: DNA analyzed by Optical and Magnetic Spectroscopy.

Highlights of the main aspects studied:

◦To investigate the structure and dynamics of sequence-specific triplex-forming efficiency/propensity of oligonucleotides.

◦To explore the unique conformational features of A-tracts.

◦To pinpoint the structural constraints regulating triple helix formation by oligonucleotides containing A-tracts. The inability to support a triplex was interpreted as a probe of altered major groove geometry.

◦To examine the triplex formation with and without flanking G•C base pairs in order to probe the role of length of the A-tract and the flanking sequences.

◦To analyze the base-pair opening dynamics in double helical DNAs catalyzed by imino proton exchange catalysts analyzed by NMR technique, UV-Vis Spectrophotometry and Circular Dichroism.

October1998–September2000: Postdoctoral Fellow (supervisor: Prof Astrid Gräslund, Stockholms universitet, Institutionen för biokemi och biofysik, Arrheniuslaboratoriet, 106 91 Stockholm, Sweden).

Outline: DNA analyzed by Optical and Magnetic Spectroscopy.

Highlights:
Study on structure and dynamics of oligomeric triple helical DNAs.

Investigating the correlation between sequence-specific triplex-forming efficiency/stability of oligonucleotides and special conformational features of A-tracts.

Studying the base pair opening dynamics of oligonucleotide triple helix utilizing NMR technique.

• And also its conformational aspects using Optical Spectroscopies, namely, UV-Vis Spectrophotometry, Circular Dichroism.

October1998–September2000: Postdoctoral Fellow (supervisor: Prof Astrid Gräslund, Stockholms universitet, Institutionen för biokemi och biofysik, Arrheniuslaboratoriet, 106 91 Stockholm, Sweden).
Outline: DNA analyzed by Optical and Magnetic Spectroscopy.

Highlights:
Study on structure and dynamics of oligomeric triple helical DNAs.

Investigating the correlation between sequence-specific triplex-forming efficiency/stability of oligonucleotides and special conformational features of A-tracts.

Studying the base pair opening dynamics of oligonucleotide triple helix utilizing NMR technique.

• And also its conformational aspects using Optical Spectroscopies, namely, UV-Vis Spectrophotometry, Circular Dichroism.

October1998–September2000: Postdoctoral Fellow (supervisor: Prof Astrid Gräslund, Stockholms universitet, Institutionen för biokemi och biofysik, Arrheniuslaboratoriet, 106 91 Stockholm, Sweden).
Outline: DNA analyzed by Optical and Magnetic Spectroscopy.
Highlights:
Study on structure and dynamics of oligomeric triple helical DNAs.

Investigating the correlation between sequence-specific triplex-forming efficiency/stability of oligonucleotides and special conformational features of A-tracts.

Studying the base pair opening dynamics of oligonucleotide triple helix utilizing NMR technique.

• And also its conformational aspects using Optical Spectroscopies, namely, UV-Vis Spectrophotometry, Circular Dichroism.

October1998–September2000: Postdoctoral Fellow (supervisor: Prof Astrid Gräslund, Stockholms universitet, Institutionen för biokemi och biofysik, Arrheniuslaboratoriet, 106 91 Stockholm, Sweden).
Outline: DNA analyzed by Optical and Magnetic Spectroscopy.
Highlights:
Study on structure and dynamics of oligomeric triple helical DNAs.

Investigating the correlation between sequence-specific triplex-forming efficiency/stability of oligonucleotides and special conformational features of A-tracts.

Studying the base pair opening dynamics of oligonucleotide triple helix utilizing NMR technique.

• And also its conformational aspects using Optical Spectroscopies, namely, UV-Vis Spectrophotometry, Circular Dichroism.