IMPROVEMENT OF A SANDWICH ELISA ASSAY TO ENHANCE QUANTIFICATION CAPABILITIES OF LNA OLIGONUCLEOTIDES Nanna Albæk, 1* Jacob Ravn, 1 Henrik Frydenlund Hansen, 1 Troels Koch, 1 Christoph Rosenbohm 1 1 Santaris Pharma A/S, Kogle Allé 6, DK-2970 Hørsholm, Denmark. * Correspondence to: [email protected]ABSTRACT The refinement of an oligonucleotide sandwich ELISA assay to quantify a specific LNA oligonucleotide has been accomplished. The improvement of the ELISA as- say for the specific compound involved synthesis of modified nucleosides and a new design of the ELISA probes using the diaminopurine and 2-thiothymine nu- cleobases. INTRODUCTION In the development of drug candidates it is desirable to evaluate the distribution of the drug in tissue and serum. For the quantification of LNA oligonucleotides an oligonucleo- tide sandwich ELISA assay is used. The assay relies on the selective hybridization of the target oligonucleotide to a set of capture and detection probes. The probes are Biotin- and Digoxigenin-labelled full LNA phosphordiester oligonu- cleotides of approximately half the length of the target oli- gonucleotide. In some cases a high background has been observed. This is most likely due to affinity between the capture and the detection probe of the specific ELISA assay or due to selfcomplementarity of the target oligonucleotide resulting in a poor detection (Figure 1). Figure 1: A: Oligonucleotide sandwich ELISA assay, B: Selfcomplementarity of the gapmer LNA oligonucleotide or affini- ty between capture and detection probes can give a high back- ground signal. RESULTS AND DISCUSSION In order to break the recognition between the capture and the detection probes 2,6-diaminopurine (D) and 2- thiothymidine (T 2s ) monomers were introduced. This allows the capture and detection probes to bind to the LNA oli- gonucleotide but diminishes the affinity between the capture and detection probes (Figure 2). Figure 2: Diaminopurine (D) and 2-thiothymine (T 2S ) recognizes T and A respectively and have a lower affinity towards each other. We have used both DNA and LNA 2,6-diaminopurine and 2-thiothymidine monomers to screen sets of capture and detection probes. The DNA monomers are commercially available. The LNA monomers have previously been report- ed in the literature 1-3 and they were made via a slightly mod- ified synthesis route. A change of ion strength in the buffer systems used for the assay also added to the development of a more sensitive assay probably by disturbing the selfcomplementarity of the LNA oligonucleotide. CONCLUSION The modifications of this specific oligonucleotide ELISA assay resulted in a significant improvement of the detection limits for the LNA oligonucleotide. The results from this work provide a larger tool box when developing ELISA assays for other LNA oligonucleotides. REFERENCES 1. Koshkin, A. A. J. Org. Chem, 2004, 69, 3711-3718. 2. Rosenbohm, C., Pedersen, D. S., Frieden, M., Jensen, F.R., Arent, S., Larsen, S., Koch, T. Bioorg. Med. Chem., 2004, 12, 2385-2396. 3. WO2004024314 109
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IMPROVEMENT OF A SANDWICH ELISA ASSAY TO ENHANCE QUANTIFICATION CAPABILITIES OF LNA OLIGONUCLEOTIDES
Nanna Albæk,1* Jacob Ravn,1 Henrik Frydenlund Hansen,1 Troels Koch,1 Christoph Rosenbohm1
The refinement of an oligonucleotide sandwich ELISA
assay to quantify a specific LNA oligonucleotide has
been accomplished. The improvement of the ELISA as-
say for the specific compound involved synthesis of
modified nucleosides and a new design of the ELISA
probes using the diaminopurine and 2-thiothymine nu-
cleobases.
INTRODUCTION
In the development of drug candidates it is desirable to
evaluate the distribution of the drug in tissue and serum. For
the quantification of LNA oligonucleotides an oligonucleo-
tide sandwich ELISA assay is used. The assay relies on the
selective hybridization of the target oligonucleotide to a set
of capture and detection probes. The probes are Biotin- and
Digoxigenin-labelled full LNA phosphordiester oligonu-
cleotides of approximately half the length of the target oli-
gonucleotide.
In some cases a high background has been observed. This
is most likely due to affinity between the capture and the
detection probe of the specific ELISA assay or due to
selfcomplementarity of the target oligonucleotide resulting
in a poor detection (Figure 1).
Figure 1: A: Oligonucleotide sandwich ELISA assay, B: Selfcomplementarity of the gapmer LNA oligonucleotide or affini-ty between capture and detection probes can give a high back-ground signal.
RESULTS AND DISCUSSION
In order to break the recognition between the capture and
the detection probes 2,6-diaminopurine (D) and 2-
thiothymidine (T2s
) monomers were introduced. This allows
the capture and detection probes to bind to the LNA oli-
gonucleotide but diminishes the affinity between the capture
and detection probes (Figure 2).
Figure 2: Diaminopurine (D) and 2-thiothymine (T2S) recognizes T and A respectively and have a lower affinity towards each other.
We have used both DNA and LNA 2,6-diaminopurine
and 2-thiothymidine monomers to screen sets of capture and
detection probes. The DNA monomers are commercially
available. The LNA monomers have previously been report-
ed in the literature1-3
and they were made via a slightly mod-
ified synthesis route.
A change of ion strength in the buffer systems used for
the assay also added to the development of a more sensitive
assay probably by disturbing the selfcomplementarity of the
LNA oligonucleotide.
CONCLUSION
The modifications of this specific oligonucleotide ELISA
assay resulted in a significant improvement of the detection
limits for the LNA oligonucleotide. The results from this
work provide a larger tool box when developing ELISA
assays for other LNA oligonucleotides.
REFERENCES
1. Koshkin, A. A. J. Org. Chem, 2004, 69, 3711-3718.
2. Rosenbohm, C., Pedersen, D. S., Frieden, M., Jensen,
F.R., Arent, S., Larsen, S., Koch, T. Bioorg. Med.
Chem., 2004, 12, 2385-2396.
3. WO2004024314
109
PHOTOPHYSICAL PROPERTIES OF SINGLE AND DOUBLE STRANDED DNA CONTAINING 6-PHENYLPYRROLOCYTOSINE (PHPC)
Fereshteh Azizi and Masad Damha*
1Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, Canada H3A 0B8
The effect of surrounding base pairs on the fluores-cent properties of phenylpyrrolocytosine (PhpC) was studied in both single- and double-stranded structures containing all 16 possible neighbouring XY pair combi-nations (i.e., X-PhpC-Y). The data indicate that the nature and orientation of nucleoside neighbours sur-rounding dPhpC affect its fluorescent properties.
INTRODUCTION
The possibility to detect emission at the single molecule
level makes fluorescence one of the most sensitive analyti-
cal techniques available today.[1] In this regard fluorescent
base analogues are particularly interesting. Of the common-
ly used fluorescent nucleoside analogues, those containing
6-Phenylpyrrolocytosine (PhpC)[2] are of interest due to
their ability to form stable Watson-Crick base pairs with
guanine with minimal disturbance to the overall duplex
structure.[2, 3] Due to its red-shifted absorbance, PhpC can
be selectively excited in the presence of natural nucleosides
[3]. Incorporation of PhpC into an oligonucleotide results in
a significant reduction of fluorescence intensity, relative to
the free ribonucleoside [3]. Oligonucleotides containing
PhpC have shown fluorescence quenching upon duplex
formation. These properties, together with its high quantum
yield and sensitivity to its micro environment makes PhpC a
potential site-specific probe for studying nucleic acid struc-
ture and dynamics.
In this study, we exam-
ine the properties of
PhpC when incorporated
into different DNA se-
quence environments.
Specifically, we investi-
gated the effect of sur-
rounding nucleotides on
the quantum yield of
PhpC within 16 single-
and double-stranded
DNA sequences. This library covers all possible nearest
neighbour variations around the PhpC residues (Table 1).
RESULTS AND DISCUSSION
The data indicates that (a) the average fluorescence quan-
tum yield for single-strands is greater than for their corre-
sponding double-strands; (b) in a single strand, a neighbor-
ing adenine causes a significant increase in PhpC fluores-
cent intensity, whereas guanine most effectively quenches
the fluorescence; c) the orientation of neighbors around
PhpC (X-PhpC-Y vs Y-PhpC-X) significantly affects PhpC
quantum yield, e.g. the quenching effect of guanine is high-
er when it is placed at the 3'-side of PhpC.
Table 1. Oligonucleotides synthesized for this study. C = PhpC
CONCLUSION
In conclusion, the fluorescence quantum yield of PhpC
within DNA structures is highly affected by the nature of
nearest neighbours and the orientation of these bases around
Figure 1. Scheme of AptaVISens-V. (a) An anti-vaccinia
aptamer on a gold microelectrode surface. (b) Binding of the
viable virus to the immobilized aptamer causes a decrease in
impedance. (c) However, binding of the nonviable virus causes
a negligible change in impedance.
111
REACTIVITY STUDIES OF 2,6-DITRIAZOLYLPURINE NUCLEOSIDES WITH
NUCLEOPHILES
Armands Kovaļovs, Irina Novosjolova, Ērika Bizdēna* and Māris Turks
Faculty of Material Science and Applied Chemistry, Riga Technical University, 14/24 Azenes str., Riga LV1007, Latvia. *Correspondence to: Email address [email protected]
ABSTRACT
Reaction of 2,6-ditriazolylpurine nucleosides with nu-
cleophiles is mild and efficient route to C6 derivatization
of purine base. To explore scope and limitations of the
method, we studied reactivity of various N- and S-
nucleophiles as well as kinetics for selected reactions.
INTRODUCTION, RESULTS AND DISCUSSION,
CONCLUSION
New methods for the synthesis of C6 purine derivatives have been intensively developed for decades. Search for new anticancer and antiviral agents, adenosine receptors agonists and antagonists prompted an renewed interest in purine chemistry, resulting in numerous synthetic method-ologies [1,2]. Purines are excellent scaffolds for construc-tion of bioprobes. To the best of our knowledge, di-(1,2,3-triazol-1-yl)purines were unexploited for the synthesis of C6 substituted purines.
We used click chemistry for the synthesis of series of 2,6-ditriazolylpurine nucleosides 1 from corresponding 2,6-diazidopurine derivatives. The observation that treatment of 1 with ammonia and amines gave fluorescent compounds, promted us to more detailed investigation of this reaction.
sides were prepared earlier by a different method and inves-tigated as selective adenosine A3 receptor agonists or antag-onists [2].
We report here reactions of 2,6-ditriazolylpurine nucleo-sides 1 with primary and secondary amines and hydrazines. The nucleophilic aromatic substitutions at C6 with amines, such as methyl- and dimethylamine, pyrrolidine, piperidine and other low molecular weight amines proceed smoothly at ambient temperature in water, water-THF or water-MeCN. Reaction times are from 30 min to 2 h. During these reac-tion conditions acetyl protecting groups were simultaneous-ly removed from sugar moiety. Dipropylamine, dibenzyla-
mine, morpholine required longer reaction time and/or ele-vated (40-50 0C) temperatures, and deprotection of sugar was carried out separately with NH3/EtOH or CH3NH2/H2O. All obtained N6-modified 2-triazolylpurine derivatives 2 possess fluorescent properties. The only exception is 6-hydrazino derivative.
Further, we extended investigation to the reactions of amino acid esters as nucleophiles. Reaction of 1 with pro-line methyl ester is rather slow, however, we obtained prod-uct with bright blue fluorescence in UV light. Reactivity studies of 1 with amino acids are still in progress.
A number of 6-thioalkylated purines have interesting bio-logical activity. For example, 6-mercaptopurine ribonucleo-side is inhibitor of de novo purine biosynthesis.
To explore suitability of 2,6-ditriazolpurines as substrates for the synthesis of 6-thioalkylated purine nucleosides, we investigated reactions of 1 with thiols. Dodecanethiol was chosen for reaction kinetics studies. 6-Dodecylthio-2-triazolylpurine ribonucleoside 3 was obtained in 60-65% yield.
In conclusion, we have demonstrated the versatility of
2,6-ditriazolylpurines as reactive intermediates suitable for C6 modifications.
REFERENCES
1. a) Lagisetty, P., Russon, L.M., Lakshman, M.K. Angew.Chem. Int.Ed., 2006, 45, 3660-3663 and refer-ences therein; b) Guo, H-M., Wu, J., Niu, H-Y., Wang, D.C., Zhang, F., Qu, G-R. Bioorg.Med.Chem.Lett., 2010, 20, 3098-3102; c) Veliz, E.A., Beal, P. J.Org.Chem., 2001, 66, 8592-8598; d) Lakshman, M. K., Choudhury, A., Bae, S., Rochttis, E., Pradhan, P., Kumar, A. Eur. J. Org. Chem., 2009, 152-159.
2. Cosyn, L., Palaniappan, K.K., Kim, S-K., Duong, H.T., Gao, Z-G., Jacobson, K.A., Van Calenbergh, S. J. Med.
Chem. 2006, 49, 7373-7383.
112
ELABORATION OF NEW NITROGEN MUSTARDS ANALOGUES
Benjamin Boëns,* Tan-Sothea Ouk, Rachida Zerrouki.
Laboratoire de Chimie des Substances Naturelles, EA1069, 123, Avenue Albert Thomas, 87060, Limoges, France
The synthesis of benzopC and deoxybenzopC nucleoside
analogues has been achieved and their photophysical prop-
erties described.
REFERENCES
1. (a)Hudson, R. H. E.; Dambenieks, A. K.; Viirre, R. D.
Synlett 2004, 13, 2400–2402. (b) Hudson, R. H. E.;
Ghorbani-Choghamarani, A. Synlett, 2007, 6, 870–873.
2. Lin, K. Y.; Matteucci, M. D. J. Am. Chem. Soc. 1998,
120, 8531-8532.
116
Investigation of Ire1p endonuclease activity in the S. cerevisiae unfolded protein response using a fluorescent oligonucleotide.
Andrew Frazer,1* John Zhao,2 James Tucker,2 David Timson3 and Joseph Vyle1
1School of Chemistry and Chemical Engineering, Queen's University Belfast, David Keir Building, Stranmillis Road,
Belfast BT9 5AG, Northern Ireland, United Kingdom, 2 School of Chemistry, The University of Birmingham, Edgbaston,
Birmingham B15 2TT, United Kingdom and 3School of Biological Sciences, Queen’s University Belfast, Medical Biolo-
gy Centre, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland, United Kingdom. * Correspondence to:[email protected]
ABSTRACT
The unfolded protein response (UPR) is a eukaryotic stress response that mediates the response to unfavoura-ble protein folding conditions in the endoplasmic reticu-lum (ER). In Saccharomyces cerevisiae Ire1p is the main signal transduction protein in the UPR. Here we de-scribe the development of a fluorescence-based approach to the in vivo monitoring of Ire1p activity.
The Saccharomyces cerevisiae inositol-requiring enzyme
1 (Ire1p) is an ER transmembrane signalling protein that
monitors the protein folding environment in the ER in a
pathway known as the unfolded protein response. Misfold-
ed proteins in the lumen of the ER interact with the luminal
domain of Ire1p to promote oligomerisation of the protein.
The cytoplasmic domain of Ire1p contains a Ser/Thr kinase
and a kinase extension nuclease that possesses endonuclease
activity. Oligomerisation of Ire1p initiates signal transduc-
tion by allowing the cytoplasmic domain kinase to trans-
autophosporylate its neighbouring subunit. Phosphorylation
subsequently activates the endonuclease activity of the pro-
tein.1
Upon activation, the endonuclease activity of Ire1p
leads to the splicing of an intron from the Hac1 pre-mRNA,
lifting the translational repression caused by the long-range
interaction between the intron and the 5'-UTR.2 The Ire1p
cytoplasmic domain targets splice sites in the Hac1 pre-
mRNA that are both located within the loop of a stem-loop
secondary structure and contain the consensus motif 5'-
CXGXXGX-3'.3 Following ligation of the exons, the ma-
ture Hac1 mRNA is translated, generating a bZip transcrip-
tion factor (Hac1p) that binds to the UPR elements present
in the promoter regions of the target genes. The genes that
are under the regulation of Hac1p encode a wide range of
proteins, including chaperones and those involved in ER-
associated protein degradation, cell growth and differentia-
tion.4
In order to monitor the activation of Ire1p directly
in vivo, we have developed an approach that utilises a fluo-
rescently-tagged short oligonucleotide analogue of the target
Hac1 pre-mRNA stem-loop. The fluorescent probe has a
similar design to that of a molecular beacon, with a 3'-
quencher moiety positioned proximally to a 5'-fluorophore
(fig. 1). Upon splicing by Ire1p, the fluorophore is liberat-
ed from the quencher and a fluorescence signal is emitted.
This approach has been used previously in in vitro to inves-
tigate small molecule activators and inhibitors of Ire1p and
its homologues.5,6
This in vivo approach will allow for the
investigation of UPR activation under a wide range of cellu-
lar stress conditions.
In this poster we will describe the optimisation of
the transfection of yeast with the short oligonucleotide
probe through the analysis of several different techniques,
alongside complementary in vitro biochemical studies in-
vestigating Ire1p activation.
Figure 1. Activation of the fluorescent probe by the endonuclease
activity of Ire1p.
REFERENCES
1. Ron, D., Walter, P. Nature Rev, 2007, 8, 519-29.
2. Ruegsegger, U., Leber, J. H. and Walter, P. Cell, 2001,
107, 103-14.
3. Kawahara I., Haruta K., Kojima C., Tanaka Y. Nucleic
Acids Symp Ser, 2009, 53, 269-70.
4. Mori K., Ogawa N., Kawahara T., Yanagi H., Yura T.B.
which contain a single ‘thiazole orange base’ as reporter
dye. The so-called DNA FIT-probes facilitate the
detection of DNA and RNA in complex biological
samples.
INTRODUCTION
Fluorogenic hybridization probes enable the real-time
detection of DNA during PCR and allow the imaging of
RNA in biological samples like cell lysate or live cells.
Conventional probes such as the Molecular Beacons rely on
the distant-dependent interaction of two chromophores.
Fluorescence signalling is observed not only upon
hybridization but any change in distance like unspecific
binding or degradation. We have introduced the single
labelled PNA FIT-probes, in which the intercalator dye
thiazole orange (TO) occupies the position of a ‘canonical’
nucleobase. [1]
The ‘TO base’ serves as a local probe, which
reports hybridization by increases of TO emission. PNA
FIT-probes have been successfully used in qPCR analysis
and in the imaging of viral mRNA in live infected cells.[2]
However, PNA probes are, at current; more costly than
DNA probes. Furthermore, PNA is not adapted to molecular
biology methods commonly used to modify DNA. To
overcome these disadvantages we set out to develop DNA
FIT-probes.
Figure 1. Concept of forced intercalation DNA-probes.
RESULTS AND DISCUSSION
Previous studies on PNA-based FIT-probes have shown the
critical influence of the linker that connects the ‘TO base’
with the PNA scaffold. A hybridization induced turn-on of
TO emission can only be achieved when very short linkers
are used. In the development of DNA-based FIT-probes we
explored the acyclic serinol-TO (1L) and a new, bioisosteric
carbacyclic TO-nucleoside (1).[3]
We found that the duplex
stability is not significantly disturbed by the introduction of
the TO-nucleotides. Most importantly, DNA FIT-probes
provide enhancements of TO emission upon hybridization
with both complementary DNA and RNA (Fig. 2A). Exper-
iments with cell lysates revealed that DNA FIT-probes
overcome two major drawbacks pertinent to molecular bea-
con probes: 1. false positive signaling caused by degrada-
tion; 2. false positive signaling due to unspecific binding in
cell media. To enable the simultaneous, yet spectrally re-
solved detection of two different RNA targets we developed
a BO-containing nucleoside. The combined use of TO and
BO probes allowed the simultaneous, sequence specific de-
tection of two different RNA-targets (Fig. 2B).
Figure 2. A) Fluorescence spectra of 1L-TO labelled FIT-probe
(ex. 485 nm). B) Simultaneous fluorescence readout of BO and TO
probes in cell lysate upon addition of neuraminidase RNA (7min)
and actin RNA (14 min).
For sequence specific DNA detection during real-time
PCR we introduced a 3’-cap on DNA FIT-probes. The
probes did not perturb the PCR process as inferred by the up
to 91% PCR-efficiency. A H1N1-neuraminidase-specific
DNA FIT-probe enabled the sensitive and unambiguous
detection of viral transcripts in cell lysate over a linear range
of at least 7 orders of magnitude.
REFERENCES
1. O. Köhler, D. V. Jarikote, and O. Seitz, Chembiochem,
2005, 6, 69-77.
2. a) S. Kummer, A. Knoll, E. Socher, L. Bethge, A.
Herrmann, and O. Seitz, Angew. Chem. Int. Ed., 2011,
50, 1931-1934.; b) A. G. Torres, M. M. Fabani, E.
Vigorito, D. Williams, N. Al-Obaidi, F. Wojciechowski,
R. H. E. Hudson, O. Seitz, and M. J. Gait, Nucleic
Acids Res., 2012, 40, 2152-2167.
3. L. Bethge, I. Singh, and O. Seitz, Org. & Biomol.
Chem., 2010, 8, 2439-2448.
120
ISOLATION OF NEW DNA APTAMERS FOR PANCREATIC CANCER CELLS
Mamoru Hyodo,1* Mst Naznin Ara1, Yusuke Takaya1 and Hideyoshi Harashima1,2
1Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-Ku, Sapporo, Japan and 2 Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences,
Hokkaido University, Kita 12, Nishi 6, Kita-Ku, Sapporo, Japan. * Correspondence to: Email address for [email protected]
ABSTRACT
We applied cell-SELEX method to pancreatic cancer cells to generate DNA aptamers. We completed the selec-tion and identified sequences and confirmed that one of these aptamers showed good affinity and selectivity to pancreatic cells.
INTRODUCTION, RESULTS AND DISCUSSION, CONCLUSION
Pancreatic cancer ranked fourth among cancer-caused death. It is difficult to detect at early stages and 5-years sur-vival rate of pancreatic cancer is less than 5% [1]. For the treatment of pancreatic cancer, the device to detect and tar-get pancreatic cancer cells is highly demanded. Aptamers are new class of molecules comparable to antibody in terms of its binding affinity and selectivity. Aptamers were firstly reported by Ellington and Turck group, indipendently [2,3]. Aptamers have the potential for the use of diagnosis, therapy, biomarker identification, biosensor and the ligand for drug delivery system.
We applied cell-SELEX method that was explored by Tan group [4]. Cell-SELEX is the method to investigate aptamers using cells directly as a target. It has beneficial points about the representation of membrane proteins as a native form and the identification of new biomarkers. For this research, we selected PANC-1 cells as the model of pancreatic cancer cells. We employed one modification through this selection, the use of temperature-detachable culture dishes named RepCell available from CellSeed. It coated with special polymer and could detach cells by cool-ing dishes. Compared to trypsin, we could reduce the risk to degrade cell surface proteins.
Figure 1. Schematic representation of cell-based SELEX.
After 5 rounds of selection, random ssDNA pool was ap-plied for cloning, transformed to E. coli and sequenced each random sequence. We picked some sequence up and checked the binding affinity with PANC-1 cells via flow cytometry. Fortunately, one of the sequence 'PANC-20' showed good binding affinity. Next, we tried the binding assay of PANC-20 aptamer with NIH3T3 which is the nega-tive target cell in our selection and we could show that it didn't bind.
Figure 2. Binding assay of PNAC-aptamer 20 to PANC-
We have investigated aptamers to pancreatic cancer cells
via cell-SELEX method. We employed the temperature-detachable culture dishes to keep cell surface proteins intact. We applied 5 rounds of selection and checked the enrich-ment of binding affinity. We confirmed the binding affinity and specificity of PNAC-20 aptamer to PANC-1 and other cells.
REFERENCES
1. Hidalgo. M. N. Engl. J. Med., 2010, 362, 1065-1078.
2. Ellington, A. D., Szostak, J. W. Nature, 1990, 346, 818-822.
3. Tuerk, C., Gold, L. Science, 1990, 249, 505-510.
4. Shagguan, D., Li, Y., Tang, Z., Cao, Z. C., Chen, H. W., Mallikaratchy, P., Sefah, K., Yang, C. J., Tan, W. Proc. Natl. Acad. Sci. USA, 2006, 103, 11838-11848.
121
FUNCTIONALIZED MRNA CAP ANALOGUES AND THEIR CONJUGATES – MOLECULAR
TOOLS FOR INVESTIGATION OF PROTEINS INVOLVED IN MRNA METABOLISM
Marcin Warminski, Zofia Tomasiewicz, Krystian Ubych, Joanna Kowalska, Edward Darzynkiewicz, Jacek Jemielity*
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland *Correspondence to: Email address [email protected]
ABSTRACT
Synthetic mRNA cap analogues functionalized with an
amino- or carboxy- group attached via linker to various
positions of the dinucleoside 5’, 5’ triphosphate structure
have been synthesized. An efficient method for their con-
jugation with biotin, fluorescent dyes, or nanoparticles is
also presented. The resulting conjugates have been de-
signed for investigation of several gene expression pro-
cesses, in which the mRNA cap structure is involved.
INTRODUCTION,
The 5’ end of eukaryotic mRNA is modified by a
distinctive structure called 5’ cap. It consists of 7-
methylguanine attached by an unusual 5’,5’-triphosphate
linkage to the first transcribed nucleotide. In cells cap is
bound by numerous cap-binding proteins and thus plays an
important role in a variety of cellular processes associated
with mRNA metabolism and regulation of gene expression,
including maturation, transport, degradation and initiation of
translation. Thus synthetic mRNA cap analogues are invalu-
able tools for investigation of cap dependent processes [1].
RESULTS AND DISCUSSION
We synthesized a series of cap analogues containing
linkers varying in length and hydrophobicity and functional-
ized with either amine or carboxylic group. Some of them
have been additionally modified in the triphosphate bridge
with either bisphosphonate or imidodiphosphate moiety to
stabilize them against specific cap related pyrophosphatases
[2-4]. The analogues are suitable for labelling with biotin,
fluorophores and for covalent binding to macromolecules
and nanomaterials. Hence, they are intended to serve as pre-
cursors of versatile tools for studying mRNA fate in the
cells, localization of cap molecules in vivo or for medical
diagnostic applications.
The linkers are attached at one of three different po-
sitions: 2’/3’-OH groups of 7-methylguanosine (m7Guo),
2’/3’-OH groups of second nucleoside (Guo) via carbamate
moiety or N6 exocyclic amine group of adenosine as a se-
cond nucleoside (Ado). The selection of those modification
positions is based on the finding that their effect on associa-
tion with particular cap-binding proteins is generally slight.
The analogs containing an amino group have been labelled
with biotin, carboxyfluorescein or carboxy-X-rhodamine.
For this purposes we developed a method for efficient con-
jugation of in situ generated NHS active esters of appropri-
ate labels with aliphatic amino functionalized dinucleotide
cap. We believe that the adenine-labelled derivatives are
especially useful for studies on Decapping Scavenger
(DcpS), whereas the m7G-carbamates should be appropria-
ble for studying interactions between cap and eukaryotic
Initiation Factor 4E (eIF4E), crucial for cap-dependent initi-
ation of translation and hence for the total rate of protein
synthesis. The latter analogues are incorporated into mRNA
during transcription in vitro, and hence, pave the way for
obtaining variously 5’ end labelled, yet biologically active
mRNAs. Linkers attached at the ribose of the second nucle-
oside enabled us to prepare conjugates with macromolecules
and nanomaterials without affecting association with cap-
binding proteins to a significant extent. We believe that
modified caps and their conjugates described herein will
benefit a wide range of biotechnological applications.
REFERENCES
1. Jemielity, J., Kowalska, J., Rydzik, A.M.
Darzynkiewicz, E. New J. Chem., 2010, 34, 829-844.
2. Kalek, M., Jemielity, J., Darzynkiewicz, Z.M., Bojarska,
E., Stepinski, J., Stolarski, R., Davis, R.E.,
Darzynkiewicz, E.; Bioorg. Med. Chem. 2006, 14,
3223-3230.
3. Rydzik, A.M., Kulis, M., Lukaszewicz, M., Kowalska,
J, Zuberek, J., Darzynkiewicz, Z.M., Darzynkiewicz, E.,
Jemielity, J. Bioorg. Med. Chem. 2012, 20, 1699-1710.
4. Su, W., Slepenkov, S., Grudzien-Nogalska, E.,
Kowalska, J., Kulis, M., Zuberek, J., Lukaszewicz, M.,
Darzynkiewicz, E., Jemielity, J., Rhoads, R.E. RNA
2011, 17, 978-988.
n=1, 3 or 5
linkers =
X=Y=O, CH2 or NH
linker
linker linker
= dyes, biotin, nanoparticle, protein
m=1, 2 or 5
Figure 1. General structure of mRNA cap analogue conjugates
122
FLUORESCENCE TURN-ON SENSOR FOR TRIPLEX FORMATION UTILIZING BENZOFURAN-MODIFIED PYRIMIDINES
Takashi Kanamori1, Hiroki Ohzeki
2, Hirosuke Tsunoda
2, Akihiro Ohkubo
2, Mitsuo Sekine
1,2* and
Kohji Seio2*
1Educational Academy of Computational Life Sciences, Tokyo Institute of Technology and
2Department of Life Science,
Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama, 226-8501, Japan. *Correspondence to: Email address [email protected], [email protected]
Figure 2. Chemical structures of fluorescent pyrimidine deriva-
tives used in this study.
Figure 1. Structure of G-PPIR•C3 triad.
123
QUENCHER FREE MOLECULAR BEACON DESIGN BASED ON PYRENE-UNA EXCIMER
FLUORESCENCE EMMISION
Kasper K. Karlsen* and Jesper Wengel
Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark. *Correspondence to: [email protected]
ABSTRACT
A pyrene-UNA (monomer X, Figure 1) modified ON,
capable of generating pyrene excimer fluorescence when
single stranded, was used in the development of a
quencher-free molecular beacon (QF-MB). Based on
explorations concerning the optimal placement and
number of incorporations of monomer X, molecular
beacons was synthesized, and their ability to detect a
target was investigated.
INTRODUCTION, RESULTS AND DISCUSSION,
CONCLUSION
Recently, we discovered the occurrence of pyrene
excimer formation in a 21-mer DNA oligonucleotide (ON)
modified with three pyrene labeled 2´-piperazino UNA
monomers (monomer X, Figure 1).[1]
Interestingly, no
excimer emission was present for when ONX was
hybridized to its complementary DNA strand (Figure 1). We
have investigated the possibility of applying this unforeseen
feature of ONX in the construction of a novel molecular
beacon (MB), as will be described in the following.
Figure 1. Steady-state fluorescence spectra of single strand and
duplex ONX: 5´-TGCACXGTAXGTCTGXACCAT.
MBs were introduced in 1996 by Tyagi and Kramer as
nucleic acid hairpin structures used for detection of nucleic
acid sequences.[2]
Classical MBs are ONs with terminal
conjugations of a fluorophore and a quencher, one at each
end of the strand.[3]
Two drawbacks are associated with this
construction, one being increased background noise or false
positive signals upon degradation, and another being that
further terminal functionalization is very difficult. In this
light, quencher-free molecular beacons (QF-MBs) have
emerged, and as the name indicates, the QF-MBs contains
no quencher and therefore are often not associated with the
same drawback as classical MBs.[4]
Two pyrene molecules, one being in an exited state and one
in the ground state, are able to form a complex called an
excimer.[5]
The relaxation of pyrene excimer is accompanied
by fluorescence emission with a λmax around 480 nm and a
relatively long fluorescence lifetime (30-60 ns) compared to
the autofluorescence of cellular extracts (~7 ns).[6]
Pyrene
excimer fluorescence can therefore be selectively detected
in biological assays if fluorescence is measured after the
cellular autofluorescence has decayed. Due to these
photophysical properties, pyrene excimer emission is a
valuable tool within diagnostic applications, and therefore
we decided to investigate the possibilities of creating a QF-
MB based on ONX (Figure 2).
The pyrene excimer forming sequence was studied with
respect to the number and specific placement of the pyrene-
UNA modification X. We found that the excimer formation
was sequence dependent, and thus decided to use the
original sequence (ONX) as part of the stem sequence for
new QF-MB (UNA-MB). As a target for the UNA-MB,
swine origin influenza A virus (H1N1) was chosen, since
this target is both highly relevant and the sequence had
previously been detected by a MB as reported by Ge et al.[7]
The current version of UNA-MB can detect the H1N1 target
sequence with a ~7-fold fluorescence intensity increase at
492 nm, but we hope to improve this by further modifying
the construction of the MB.
Figure 2. Schematic representation of the UNA-MB based on
the sequence of ONX.
REFERENCES
1. Karlsen, K. K., Pasternak, A., Jensen, T. B., Wengel,
J., ChemBioChem 2012, 13, 590-601.
2. Tyagi, S., Kramer, F. R., Nat. Biotechnol. 1996, 14,
303-308.
3. Wang, K. M., Tang, Z. W., Yang, C. Y. J., Kim, Y.
M., Fang, X. H., Li, W., Wu, Y. R., Medley, C. D.,
Cao, Z. H., Li, J., Colon, P., Lin, H., Tan, W. H.,
Angew. Chem. Int. Ed. 2009, 48, 856-870.
4. Venkatesan, N., Seo, Y. J., Kim, B. H., Chem. Soc.
Rev. 2008, 37, 648-663.
5. Winnik, F. M., Chem. Rev. 1993, 93, 587-614.
6. Kolpashchikov, D. M., Chem. Rev. 2010, 110, 4709-
4723.
7. Ge, Y. Y., Cui, L. B., Qi, X., Shan, J., Shan, Y. F., Qi,
Y. H., Wu, B., Wang, H., Shi, Z. Y., J. Virol. Methods
2010, 163, 495-497.
124
HIGH-AFFINITY DNA-TARGETING USING SIMPLE MIMICS OF N2’-INTERCALATOR-FUNCTIONALIZED 2’-AMINO-α-L-LNA
Saswata Karmakar,1* Dale C. Guenther,
1 Sujay P. Sau,
1 Brooke A. Anderson,
1 Rie L. Rathje,
1 Sanne
Andersen1 and Patrick J. Hrdlicka
1
1Department of Chemistry, University of Idaho, PO Box 442343, Moscow, ID 83844-2343, USA. * Correspondence to:
CHARGE TRANSFER DYNAMICS IN DNA AT THE SINGLE MOLECULE LEVEL
Kiyohiko Kawai,1* Atsushi Maruyama,
2 Tetsuro Majima
1*
11The Institute of Scientific and Industrial Research (SANKEN), Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-
0047, Japan, 2Institute for Materials Chemistry and Engineering, Kyushu University, Motooka 744-CE11, Nishi-ku, Fu-kuoka 819-0395, Japan. [email protected], [email protected]
ABSTRACT
Charge separation, charge transfer, and charge re-
combination process in DNA was monitored by blinking
of the fluorescence emitted from each single fluorophore.
INTRODUCTION
Photo-induced charge-transfer quenching results in the
formation of a non-emissive radical-ion state of the fluoro-
phore and charge injection into a bio-molecule. An injected
charge can migrate along the biomolecule, and subsequent
charge-recombination makes the reaction reversible. The
charge-migration dynamics along the biomolecule are re-
flected in a change in the lifetime of the charge separated
state (), thus the measurement of would provide us with
additional unique and fruitful information around the fluo-
rescent dye. However, the is usually determined by the
transient absorption measurement, which is labor-intensive
and requires a significant amount of sample (>1 nmol); thus
it is essentially incompatible with live-cell imaging and
high-throughput applications. Looking at each single fluor-
ophore, charge-separation and -recombination causes a
blinking of the fluorescence, and here the duration of the
dark state “off time” is supposed to correlate with the (Fig.
1). In this study, we clearly demonstrated that charge-
separation and -recombination dynamics in DNA in the time
range of microsecond to tens of microseconds can be probed
by fluorescence correlation spectroscopy (FCS).
Figure 1. A schematic representation for charge separation, charge migration, and charge recombination in DNA.
RESULTS AND DISCUSSION
ATTO 655 (ATTO) was selected as a fluorophore to
measure the charge-transfer dynamics in DNA by FCS. One
G was replaced with deazaguanine (Z) to behave as a posi-
tive-charge trap. Charge-recombination dynamics were in-
vestigated by conventional transient absorption measure-
ment as well as FCS. In addition to the diffusion component,
an additional relaxation process was observed by FCS (FCS)
which correlated well with the measured from the transient
absorption measurements (TA) and was attributed to the
on/off dynamics of the single ATTO due to the formation of
the non-emissive charge-separated state. FCS varied reflect-
ing the DNA sequence and the presence of a mismatch
which allows the read-out of DNA sequence information
including data on single-nucleotide polymorphisms (SNPs).
The present method enables the automatic measurement and
analysis of more than 100 samples within 1 h using less than
10 fmol (0.5 nM 20 L) of sample, which is over 100,000
times less than that typically required for transient absorp-
tion measurements. This makes possible the high-
throughput screening needed in DNA diagnosis.
CONCLUSION
The present report clearly demonstrated that single-
molecule-level fluorescent measurement is a powerful tool
for examining the charge-transfer dynamics in the time
range of microsecond to milliseconds. Since FCS can be
measured in living cells, the investigation of the charge-
transfer dynamics may provide unique information around
the fluorescent dye in living cells.
REFERENCES
1. Kawai, K., Hayashi, M., Majima, T. J. Am. Chem. Soc.
2012, 134, 4806-4811.
2. Kawai, K., Matsutani, E., Maruyama, A., Majima, T. J.
Am. Chem. Soc. 2011, 133, 15568-15577.
3. Kawai, K., Kodera, H., Majima, T. J. Am. Chem. Soc.
2010, 132, 14216-14220.
4. Kawai, K., Kodera, H., Majima, T. J. Am. Chem. Soc.
2010, 132, 627-630.
5. Kawai, K., Kodera, H., Osakada, Y., Majima, T. Nature
Chem. 2009, 1, 156-159.
Off-time
FCS
~
3'
X
ATTO 655
Chargeseparation
Charge migration& recombination
ATTO 655
•
hn
Single-molecule-levelfluorescencemeasurement
•+
•+
•+
on!
off
on!
~5'
127
SULFUR-CONTAINING PHOSPHONATE MONOMERS FOR OLIGONUCLEOTIDE SYNTHESIS
Ondřej Kostov, Eva Zborníková and Ivan Rosenberg *
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, 166 10, Prague 6, Czech Republic. * Correspondence to: Email address [email protected]
ABSTRACT
A series of novel compounds, suitably protected (i)
nucleoside-5'-S-methylphosphonates and (ii) nucleoside-
-5'-O-methylphosphonothioates, was prepared as
monomers for solid phase synthesis of modified
oligonucleotides. In addition we have examined the
synthetic potential of nucleoside-5'-O-methyl-(H)-
phosphinates for phosphonate oligonucleotide assembly.
INTRODUCTION, RESULTS AND DISCUSSION,
CONCLUSION
A partial replacement of the phosphodiester C3'-O-P-O-
-C5´ bonds in oligodeoxynucleotides with the isopolar
SYNTHESIS OF NUCLEOTIDES BEARING FLUOROPHOSPHATE MOIETY AND THEIR NON-HYDROLYZABLE ANALOGS - USEFUL PROBES FOR NMR STUDIES
Joanna Kowalska, Marek R. Baranowski, Renata Kasprzyk, Agnieszka Osowniak, Jacek Jemielity
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, Warsaw, Poland, e-mail: [email protected]
ABSTRACT
A comprehensive method for the synthesis of nucleo-
side mono-, di-, tri- and teraphosphates bearing a termi-
nal fluorophosphate moiety and their non-hydrolyzable
analogs is reported.
INTRODUCTION
Fluorophosphate analogs of nucleotides are known for their
use in studying substrate properties of various phosphotrans-
ferases and phosphohydrolases. Replacing one of the oxy-
gen atoms within the phosphate moiety by fluorine elimi-
nates the negative charge, but the produced analogue, due to
the small size and high electronegativity of fluorine, may
putatively still be involved in hydrophilic interactions im-
portant for biomolecules.1
Hence, nucleoside fluorophos-
phates have been identified as unnatural enzyme substrates,
receptor agonists, and, in the cases they proved to be enzy-
matically resistant, as enzymatic inhibitors. This, in combi-
nation with the fact that fluorine atom allows selective and
sensitive studies by means of 19
F NMR spectroscopy, makes
nucleoside fluorophosphates an interesting target for effi-
cient chemical synthesis.
RESULTS AND DISCUSSION
Here, we report a method enabling straightforward access
to fluorophosphate analogs of nucleoside mono-, di- and tri-
and even tetraphosphates (Fig. 1). The method consists of
three complementary synthetic approaches, each based on
phosphorimidazolide chemistry. First approach, which is
particularly useful for the synthesis of nucleoside fluo-
romonophosphates, but could be also applied for fluorodi-
phosphates and their analogs, employs substituting the im-
idazole leaving group of an activated nucleotide with fluo-
ride anion (Fig. 1A). Second approach, applicable mainly
for the synthesis of nucleoside β-fluorodiphosphates and γ-
fluorotriphosphates, encompasses coupling an appropriate
nucleoside phosphorimidazolide with triethylammonium
monofluorophosphate (MPF) as an nucleophile (Fig. 1B).
The third approach is particularly useful for the synthesis of
either highly polar nucleotides (e.g. tetraphosphate analogs)
or those bearing a modified oligophosphate bridge (Fig. 3C).
In this case a “reverse” strategy has been employed, in
which the fluorophosphate unit is activated as an electro-
phile and an appropriate nucleotide is used as an nucleo-
philic agent. In each case the pyrophosphate bond formation
is highly accelerated by the presence of excess divalent met-
al chloride, either ZnCl2 or MgCl2.
Based on the presented methodology we have synthe-
sized, with good to excellent yields, over 20 different nucle-
5. Xu, Y.Z., Swann, P.F. Nucleic Acids Res., 1990, 18,
4061-4065.
6. McManus, F.P., O'Flaherty, D.K., Noronha, A.M,
Wilds, C.J., Org. Biomol. Chem., 2012, submitted.
Figure 1.Chemical structures of the (a) O6-dG-alkyl-O6-dG and (b) O4-dT-alkyl-O4-dT ICLs, where n=1 or 4.
133
CLICK, SUBSTITUTE AND FLUORESCE: SYNTHESIS AND APPLICATIONS OF 2,6-DI-(1,2,3-TRIAZOLYL)-PURINE NUCLEOSIDES
Irina Novosjolova*, Armands Kovaļovs, Inga Bižāne, Ērika Bizdēna and Māris Turks
Faculty of Material Science and Applied Chemistry, Riga Technical University, 14/24 Azenes str., Riga LV1007, Latvia. *Correspondence to: [email protected]
ABSTRACT
A novel class of ditriazolylpurine nucleosides were ob-
tained from 2,6-diazido precursors via copper catalyzed
azide-alkyne cycloaddition. These intermediates ap-
peared to be very reactive towards N- and S-
nucleophiles and thus selectively gave C(6)-substituted
analogs with triazolyl moiety at C(2)-position. The latter
Shaughnessy, K.H. J. Org. Chem. 2003, 68, 6767-6774.
5. Elmquist, C.E., Stover, J.S., Wang, Z., Rizzo, C.J. J.
Am. Chem. Soc. 2004, 126, 11189-11201.
6. Omumi, A., Beach, D.G., Baker, M., Gabryelski, W.,
Manderville, R.A. J. Am. Chem. Soc. 2011, 133, 42-50.
Figure 1. C8-heteroaryl-dG derivatives.
136
OXIDATION OF H-PHOSPHONATES WITH IODINE BY INTRAMOLECULAR SUPPORT
OF A 2-PYRIDYL THERMOLABILE PROTECTING GROUP
Tomasz Ratajczak and Marcin K. Chmielewski*
Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14, Poznań, Poland * Correspondence to: [email protected]
ABSTRACT
Acceleration of H-phosphonate diester oxidation with
iodine accompanied by a thermolabile protecting group
(TPG) is presented. It is shown that the intermediate
product of this reaction is an oxazaphospholidine oxide
which forms a phosphate diester only when a 2-pyridyl
TPG is applied. 31
P NMR spectroscopy was used to eval-
uate the relationship between chemical shift and abso-
lute configuration at the phosphorus center of H-
phosphonate diesters, oxazaphospholidine oxides and
phosphorothioate diesters.
INTRODUCTION
Thermolabile protecting groups (TPGs) have been
introduced to enhance effective protection of a phosphate,
hydroxyl or amine center. Removal of these groups is based
on intramolecular cyclization depending on temperature [1].
However, phosphate triesters with some TPGs are very
unstable [2] and to enhance their stability a “click-clack”
approach has been applied [3]. This approach increases 2-
pyridyl TPG stability by forming a five-membered
oxazaphospholidine ring. A linear form of TPG may be
easily recovered by acid hydrolysis, during which also an H-
phosphonate diester is formed. H-phosphonate diesters also
get oxidized to the corresponding phosphates by iodine in a
basic environment like pyridine. In this case oxidation of an
H-phosphonate diester involves a multi-step mechanism
where an phosphoroiodidate derivative and further
pyridinium cation is formed [4,5]. We discovered that H-
phosphonate diesters which contain a 2-pyridyl moiety get
easily oxidized to phosphate by using the iodine only.
RESULTS AND DISCUSSION
Our study shows a possibility of using N-(2-pyridyl)ethyl
moiety as a thermolabile protecting group as well as
intramolecular catalyst of the oxidation reaction. We take
advantage of the nucleophilic and basic properties of exo-
cyclic nitrogen atom (3, 5), which is involved in a five-
membered ring with a phosphate center after substituting the
hydrogen atom by iodine (Fig. 1). The oxazaphospholidine
oxide 9 formed from the 2-pyridyl TPG (3) opens very fast
(less then 1 min) in the presence of water giving 11 while 10
is very stable under these conditions. While studying the
mechanisms of phosphate cyclization it is important to
assign absolute configuration to H-phosphonate center. We
have enzymatically determined the absolute configuration of
thiophosphate 12 while configurations of 3, 5, 9, 10 were
assigned using correlation methods (Fig. 2).
CONCLUSION
Our work presents the application of a 2-pyridyl TPG which
intramolecularly catalyzes oxidation of H-phosphonate
diesters with iodine only. However, forming a phosphate
diester in this reaction is possible only when a 2-pyridyl
TPG is applied. Moreover, the process is very fast and quan-
titatively completed within 15 minutes.
REFERENCES
1. Grajkowski, A., Wilk, A., Chmielewski, M.K., Phillips,
L.R., Beaucage, S.L. Org. Lett. 2001, 3, 1287-1290.
2. Cieślak, J., Beaucage, S.L. J. Org. Chem. 2003, 68,
10123-10129.
3. Chmielewski, M.K. Org. Lett. 2009, 11, 3742-3745.
4. Stawiński, J., Stromberg, R., Zain, R. Tetrahedron Lett.
1992, 33, 3185-88.
5. Garegg, P.J., Regberg, T., Stawiński, J., Stromberg, R. J.
Chem. Soc. Perkin Trans. I 1987, 6, 1269-1274.
NH
O
ON
O
OAc
OPH
OON
HX
NH
O
ON
O
OAc
OPN
O
X
NH
O
ON
O
OAc
OPN
O
X
O
NH
O
ON
O
OAc
OPOH
OON
HN
NH
O
ON
O
OAc
OPS-
OON
H
S8/Py
BTT
MeCN Iodine/MeCN
H2O 11
9 X=N10 X=CH2
X=N
X=CH2
3 X=N5 X=CH2
12
1 X=N2 X=CH2
PyH+
Figure 1. Oxidation of H-phosphonate diesters to phosphate by
the intramolecular oxidative coupling.
Figure 2. Correlation between chemical shifts and absolute con-figuration of 5, 10 and 12.
137
SOLID PHASE SYNTHESIS OF NUCLEOSIDE- AND OLIGONUCLEOTIDE 5’-TRIPHOSPHATES WITH CYCLOSALIGENYL PHOSPHITYLATING REAGENTS
Ivo Sarac1* and Chris Meier1
1Organic Chemistry, Department of Chemistry, Faculty of Sciences, University of Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany. * Correspondence to: [email protected]
ABSTRACT
Starting from immobilized nucleosides or oligo-nucleotides the corresponding 5’-cycloSal-phosphate triesters were directly synthesized with cycloSaligenyl phosphitylating reagents. These compounds were re-acted with pyrophosphate yielding nucleoside- and oligo-nucleotide 5’-triphosphates after cleavage from various solid supports.
INTRODUCTION
2’-Deoxyribo- and ribonucleoside 5’-triphosphates are the building blocks for enzymatic synthesis of DNA and RNA in vivo and in vitro.[1] While DNA 5’-triphosphates are mostly used in the biotechnology industry to obtain syn-thetic genes, RNA 5’-triphosphates have a broader spectrum of applications. For example, RNA 5’-triphosphates are used in the induction of antiviral immunity and for the ligation of RNA fragments.[2]
Nucleoside- as well as oligonucleotide 5’-triphosphates are very important compounds in biological systems with therapeutic applications. Although there have been a num-ber of different approaches reported, the general access to these important classes of compounds is still a challenge.
Therefore, we attempted to develop a generally applic-able route to both nucleoside- and oligonucleotide 5’-tri-phosphates.
RESULTS AND DISCUSSION
In an earlier approach 5’-cycloSal-nucleotides were con-verted to a wide range of different biomolecules such as nucleoside 5’-triphosphates using an in-solution method.[3]
Later we reported on a solid-phase synthesis route to nucleoside 5’-triphosphates by first synthesizing 5’-cycloSal-nucleotides along with the linker unit and attaching these building blocks to polystyrene. The sub-sequent reaction with pyrophosphate led to nucleoside 5’-triphosphates in high yields after cleavage from the resin, which facilitated the purification.[4]
With regard to a more versatile and modular approach not only for immobilized nucleotides but also oligo-nucleotides these compounds were directly converted to 5’-cycloSal-phosphate triesters using cycloSaligenyl phosphitylating reagents and oxidation. The advantage of this new approach is a faster, more flexible and more con-venient way (Scheme 1) to nucleoside- and oligonucleotide
5’-triphosphates compared to our first published method in which protection as well as purification steps of the 5’-cycloSal-nucleotides were involved.
YOP
OX
2. oxidation
1.
phosphorylation
B
O
O
PO
HO
O B
O
O
ONC
n
O
NH
O
OPG/H
OPG/H
B
O
O
PO
O
O B
O
O
ONC
n
O
NH
O
OPG/H
OPG/H
OP
OX
O
B
O
O
PO
O
O B
O
O
ONC
n
O
NH
O
OPG/H
OPG/H
POPOPO
O O O
O O O
B
O
O
PO
O
O B
O
OH
O
n
OH/H
OH/H
POPOPO
O O O
O O O
deprotectionand cleavage
PG: protecting group
--B: nucleobasex.X: Cl, NO2, Ac
x.Y: Cl, phosphoramidite
x.--: for n=0 aminomethyl polystyrene, for n>0 controlled pore glass
2
Scheme 1. General synthesis of immobilized nucleoside and oligo-nucleotide 5’-triphosphates
CONCLUSION
With our new method both nucleoside- and oligo-nucleotide 5’-triphosphates were synthesized in a more convenient way. This approach opens a general way to a variety of complex biomolecules.
REFERENCES
1. Burgess, K., Cook, D. Chem Rev. 2000, 100, 2047-2059.
2. Zlatev, I., Lavergne, T., Debart, F., Vasseur, J-J., Manoharan, M., Morvan, F. Org. Lett. 2010, 12, 2190-2193.
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta,Midori-ku, Yokohama, Japan.*Kohji Seio: Email address for [email protected]; Mitsuo Sekine: Email address for [email protected]
ABSTRACT
We have reported the oligodeoxynucleotide containing
bulky and anionic modification (dAChcmp
) at its 5´-
terminus which selectively hybridized with the short
complementary RNA. The position of dAChcmp
was fixed
by incorporating a propylene bridge between the 5´-
phosphate the 5-position of the 3´-downstream uridine
residue. Short-RNA selective binding abilities of the
modified oligodeoxynucleotides were assessed by the Tm
measurements.
INTRODUCTION, RESULTS AND DISCUSSION,
CONCLUSION
There are a lot of studies to introduce conformational re-straint in the nucleoside moiety and the phosphodiester backbones in order to modify the structures and the hybridi-zation properties of oligonucleotides.
1 For example, the
fixation of the sugar puckering by the introduction of addi-tional ring structure has been proved to be effective to in-crease the hybridization stability of oligonucleotides. In addition, there are several examples of the conformational fixation of the sugar-phosphate backbones. We have also reported the incorporation of the cyclic nucleoside residue having a propylene linkage between the uracil moiety and the phosphorous atom forming the phosphotriester structure, and their hybridization properties.
2
This time, we studied the synthesis of oligonucleotides hav-
ing conformationally fixed phosphotriester backbone previ-
ously reported2 and the dA
ChcmP residue
3 at the 5'-end of the
oligonucleotide aiming the development of oligonucleotide
probes capable of short RNA selective binding.3
The Rp and Sp isomers of the dAChcmP
-c3T dimer unit (Figure
1) was synthesized from deoxyadenosine and deoxyuridine
and purified by silica gel column chromatography. The oli-
godeoxynucleotides incorporating the dimer unit at the 5’-
termini was performed by the conventional solid-phase syn-
thesis. The Tm analyses revealed the short RNA selective
binding properties of the conformation fixed probes which
are useful for the development of miRNA detection methods.
REFERENCES
1. a) Veedu, R. N., Wengel, J. Chem. Biodivers. 2010, 7,
536-542. b) Leumann, C. J. Bioorg. Med. Chem. 2002,
10, 841-854. c) Dupouy, C., Iché-Tarrat, N., Durrieu, M.
P., Vigroux, A., Escudier, J. M. Org. Biomol. Chem.
2008, 6, 2849-2851.
2. Sekine, M., Kurasawa, O., Shohda, K., Seio, K., Wada,
T. J. Org. Chem. 2000, 65, 6515-6524.
3. Seio, K., Kurohagi, S., Kodama, E., Masaki, Y., Tsu-
noda, H., Ohkubo, A., Sekine, M. Org. Biomol. Chem.
2012, 10, 994-1006.
Figure 1. Structure of backbone fixed and terminally modified oli-gonucleotides.
ENZYME-LINKED SMALL-MOLECULE DETECTION USING SPLIT APTAMER LIGATION
Ashwani K. Sharma*, Alexandra D. Kent, and Jennifer M. Heemstra
Department of Chemistry and the Center for Cell and Genome Science, University of Utah, Salt Lake City, Utah 84112, United States. * Correspondence: [email protected]
ABSTRACT
A split aptamer based analogue of the widely-used
ELISA diagnostic assay is described for the detection of
cocaine. This assay utilizes the ligation of two split ap-
tamer fragments in the presence of cocaine, providing a
colorimetric output that can be quantified using an ab-
sorbance plate reader.
INTRODUCTION, RESULTS AND DISCUSSION,
CONCLUSION
The detection of small molecules is an active area of re-
search as these molecules play a critical role in disease pro-
gression, environmental health, and clinical diagnostics.
Antibodies have long been the first choice for use in molec-
ular recognition due to their high affinity and broad sub-
strate scope. However, nucleic acid aptamers have emerged
as a promising alternative to antibodies, as they are easily
obtained through in vitro selection and benefit from higher
chemical stability relative to antibodies. Moreover, aptamers
can be generated for a broad range of targets ranging from
small molecules to proteins and enzymes.
Figure 1. Enzyme-linked cocaine detection using split aptamer ligation.
We have recently demonstrated novel Split Aptamer
Proximity Ligation (StAPL) technology1 in which attach-
ment of reactive groups to the termini of split aptamer frag-
ments2 enables translation of a small molecule signal into
the output of DNA ligation. Herein, we describe the use of
this technology for the development of an enzyme-linked
assay for detecting cocaine. The capture and detection anti-
bodies used in the standard ELISA assay were replaced by
chemically modified fragments of the cocaine split aptamer.
In brief, a capture strand having an azide at one terminus
and an amine at the opposite terminus was attached to an N-
hydroxysuccinimide (NHS)-functionalized microplate via
amide bond formation (Figure 1). The detection strand, hav-
ing a cyclooctyne at one terminus and a biotin at the oppo-
site terminus, is added to the DNA functionalized micro-
plate well along with a test sample containing varying con-
centrations of cocaine. If present, cocaine directs assembly
of the aptamer fragments, bringing the azide and cy-
clooctyne groups into close proximity and thus promoting a
cycloaddition to ligate the two fragments3. The resulting
ligation yield is dependent upon cocaine concentration, and
is measured as a colorimetric signal by adding streptavidin-
horseradish peroxidase (SA-HRP) conjugate that binds to
biotin and converts a colorless tetramethylbenzidine (TMB)
substrate into an optically observable blue product. This
assay is capable of detecting cocaine at concentrations of
100 nM-100 µM in buffer and 1-100 µM in human blood
serum. The detection limit of 1 µM in serum is two orders
of magnitude better than previously reported split aptamer-
based sensors.
In conclusion, we demonstrate use of the cocaine split ap-
tamer to construct the first DNA-based analogue of sand-
wich ELISA capable of small molecule detection. The total
assay time is less than two hours. Moreover, the enzyme-
linked format provides a convenient colorimetric output and
functions analogously to antibody-based ELISA, the current
standard in clinical diagnostic laboratories.
REFERENCES
1. Sharma, A. K.; Heemstra, J. M. J. Am. Chem. Soc. 2011,
133, 12426-12429.
2. (a) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J.
Figure 1. Noncovalent and site-directed spin-labeling. A. Structure of the spin label ç and its base-pairing scheme with G. B. A model of a duplex DNA containing an abasic site (grey) and spin label ç (black), EPR spectra of ç in free state and in DNA containing an abasic site.
143
SYNTHESIS AND INCORPORATION OF DIAZIRINE-MODIFIED URIDINE PHOSPHORAMIDITE
Christine Smith1* and Christian J Leumann
1
1Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3008 Bern, Switzerland
We have synthesized novel push-pull-type fluorescent
punine- and 7-deazapurine 2'-deoxynucleosides, contain-
ing a 1,6-disubstituted pyrene chromophores. Among
them, 7-deazaadenosine derivatives, CNZ
A, was found to
exhibit a remarkable solvatofluorochromicity.
INTRODUCTION, RESULTS AND DISCUSSION,
CONCLUSION
Environmentally sensitive fluorescence nucleosides in which emission spectra and quantum yields change sensi-tively according to solvent polarity are of greatest interest owing to their wide range of applications [1,2]. They are used as fluorescence sensors in various fields, as for exam-ple, incorporation of such solvatofluorochromic nucleosides into oligonucleotides provides powerful tools for the detec-tion of target DNA and SNP genotyping [1].
Recently, we reported C8-substituted push–pull-type flu-orescent guanosines, Ac
G and CNG, which contain a cova-
lently linked electron donor–acceptor system consisting of guanosine as electron donor and pyrene fluorophore as ac-ceptor (Figure 1a)[3]. Although these guanosine derivatives exhibited interesting solvatofluorochromic properties, the steric bulk of the C8-substituents in both Ac
G and CNG caus-
es considerable destabilization of the DNA duplex structure due to their syn-conformation, and thus, these molecules may not be suitable for the use as fluorescent DNA probes. Thus, we have designed novel push-pull-type 7-deazapurine 2'-deoxynucleosides. An electron-withdrawing 4-cyano-phenyl group (acceptor) and 7-deazapurine nucleoside (do-nor) are directly attached to pyrene chromophore via triple bonds in order to construct an intramolecular donor–acceptor system. We report herein the synthesis and photo-
physical properties of novel push–pull-type 7-deaza-2'-deoxyadenosine (CNZ
A) and 7-deaza-2'-deoxyguanosine (CNZ
G) containing a 1,6-disubstituted pyrene chromophore (Figure 1b).
CNZA was synthesized from 7-iodo-7-deaza-2'-deoxy-
adenosine prepared according to protocol Seela et al. [4]. CNZ
G was also synthesized through a similar reaction route. The photophysical properties of newly synthesized push-
pull-type 7-deazapurine derivatives, CNZA and CNZ
G, were examined. Initially, we measured the fluorescence spectra of CNZ
A in various solvents of different polarity. Upon excita-tion of CNZ
A at 416 nm in chloroform, strong fluorescence emission was observed at 470 nm (Φfl = 0.448). Upon exci-tation of CNZ
A in THF, we observed moderate emission at 510 nm (Φfl = 0.305). In contrast, very weak fluorescence emission was observed at 530 nm in a polar solvent such as DMF (Φfl = 0.089). As expected, push–pull-type 7-deaza-2'-deoxyadenosine derivative CNZ
A exhibited a considerable solvatofluorochromicity (Δλfl.max = 60 nm).
The photophysical properties of 7-deaza-2'-deoxy-guanosine derivative CNZ
G were also examined. While the fluorescence intensity of CNZ
G is strong in low-polarity sol-vents such as chloroform, weak fluorescence was observed in polar solvents. In the case of CNZ
G, no remarkable red-shift of fluorescence emission was observed by changing solvent polarity, unlike 7-deazapurine derivative, CNZ
A
which exhibited a redshift with increasing solvent polarity. In summary, we have synthesized novel push-pull-type fluorescent 7-deazapurine nucleosides CNZ
A and CNZG, the
first highly fluorescent pyrene containing 7-deaza-2'-deoxypurine nucleosides. Among them, CNZ
A exhibited strong fluorescence at long wavelength and a remarkable solvatofluorochromicity (Δλfl.max = 60 nm). Such environ-mentally sensitive and strongly fluorescent nucleosides may be used as a fluorescence sensor for structural studies of nucleic acids and as a building block for constructing a fluo-rescent DNA nanostructure. REFERENCES 1. Saito, Y., Miyauchi, Y., Okamoto, A., Saito, I. Tetra-
hedron Lett. 2004, 45, 7827-7831. 2. Tainaka, K., Tanaka, K., Ikeda, S., Nishiza, K., Unzai,
T., Fujiwara, Y., Saito, I., Okamoto, A. J. Am. Chem.
Soc. 2007, 129, 4776-4784. 3. Saito, Y., Suzuki, A., Imai, K., Nemoto, N., Saito, I.
Tetrahedron Lett. 2010, 51, 2606-2609. 4. Seela, F., Zulauf, M. synthesis, 1996, 726-730.
Figure 1. Strucure of push-pull-type fluorescent nucleosides containing a disubstituted pyrene chromophore.
147
RECOGNITION AND FLUORESCENCE DETECTION OF 8-OXO-2’-DEOXYGUANOSINE IN DNA BY ADENOSINE-1,3-DIAZAPHENOXAZINE DERIVATIVE
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 Ja-pan, * Correspondence to: Email address for. [email protected]
ABSTRACT
The sequence specific detection of 8-oxo-2’-deoxyguanosine (8-oxodG) in DNA without chemical or enzymatic treatment is an attractive tool for genomic research. We designed and synthesized the non-natural nucleoside analogue, the adenosine-1,3-diazaphenoxazine derivative (Adap), for selective recog-nition of 8-oxodG in DNA. This study has clearly shown that Adap has a highly selective stabilizing effect of the duplex having the Adap-8-oxodG base pair and an abil-ity to detect the 8-oxodG in DNA.
INTRODUCTION, RESULTS AND DISCUSSION, CONCLUSION
Cellular DNA is continuously exposed to a variety of chemically reactive species such as alkylating agents, reac-tive oxygen species (ROS), etc., resulting in DNA damage that may increase the risk of developing diseases. 8-Hydroxy-2’-deoxy-guanosine (or 8-oxo-2’-deoxyguanosine, 8-oxodG) is the representative damaged nucleoside, which is generated by the oxidation of 2’-deoxyguanosine triphos-phate (dGTP) in the triphosphate nucleotide pool or 2’-deoxyguanosine (dG) in DNA [1]. There are several meth-ods to detect the 8-oxodG nucleoside using the degradation products of DNA. Therefore, the selective detection of 8-oxo-2’-deoxyguanosine (8-oxodG) in DNA without chemi-cal or enzymatic treatment is an attractive tool for genomic research.
We designed and synthesized the non-natural nucleoside analogue, the adenosine-1,3-diazaphenoxazine derivative
(Adap), for selective recognition of 8-oxodG in DNA with a focus on the glycocil conformation of it. We have success-fully synthesized the Adap phosphoramidite, and it was in-corporated into oligodeoxynucleotide (ODN). From the re-sults of Tm analysis, it is clearly shown that Adap has a high-ly selective stabilizing effect of the duplex having the Adap-8-oxodG base pair. Moreover, the fluorescent property of Adap has been shown to be useful for the selective detection of 8-oxodG in the duplex DNA. To the best of our knowledge, this is the first successful demonstration of the non-natural nucleoside with a high selectivity for 8-oxodG in DNA [2].
Furthermore, we developed a OFF-to-ON type FRET probe, in which one strand contains Adap and another con-tains natural nucleoside for the formation of a less stale double strand. Each strand was labeled with Cy3 or BHQ2 at the 5’-end or 3’-end, respectively. It was expected in this system that fluorescence of the duplex probe was first quenched by FRET, but the target DNA strand containing 8-oxodG at the complementary site of Adap would enhance the displacement reaction of the less stable duplex probe that results in the fluorescence recovery. The results showed that the duplex probe containing the Adap-T base pair ex-hibited a complete discrimination between 8-oxodG and dG in DNA by fluorescence enhancement [3].
In conclusion, we have shown that Adap has a highly se-lective stabilizing effect on the duplex containing the Adap-8-oxodG base pair. And the fluorescent property of Adap has been shown to be useful for the selective detection of 8-oxodG in DNA. Furthermore, we have designed the off-to-on type fluorescent probe for the detection of 8-oxodG. The Adap-T pair masks the base recognition ability of Adap and shows that the effective strand displacement reaction only occurs in the presence of 8-oxodG in the target DNA.
REFERENCES
1. (a) Risom, L.; Moller, P.; Loft, S. Mutat. Res. 2005, 592, 119–137. (b) Knaapen, A. M.; Güngör, N.; Schins, R. P. F.; Borm, P. J. A,; van Schooten, F. J. Mutagenesis 2006, 21, 225–236. (c) Valavanidis, A.; Vlahogianni, T.; Dassenakis, M.; Scoullos, M. Ecotoxicol. Environ. Saf. 2006, 64, 178–189. (d) Halliwell, B. Biochem. J. 2007, 401, 1–11.
2. Taniguchi, Y., Kawaguchi, R., Sasaki, S. J. Am. Chem.Soc., 2011, 133, 7272-7275.
3. Taniguchi, Y., Koga, Y., Fukabori, K., Kawaguchi, R., Sasaki, S., Bioorg. Med. Chem. Lett., 2012, 22, 543-546.
Figure 1. Speculated recognition structure of Adap-oxodG pair.
Adenosine 1,3-diazaphenoxadine
(Adap)
8-oxo-2’-deoxyguanosine(8-oxodG)
148
INVESTIGATION OF RNA APTAMERS TARGETING MITOCHONDRIA BY MITOCHONDRIA-BASED SELEX
Yuri Tawaraya,1* Mamoru Hyodo,
2 Yuma Yamada,
1 and Hideyoshi Harashima
1,2
1 Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-Ku, Sapporo, Japan.
2Laboratory of Innovative Nanomedicine, Faculty of Pharmaceutical Sciences, Hok-
kaido University, Kita 12, Nishi 6, Kita-Ku, Sapporo, Japan * Correspondence to: [email protected]
ABSTRACT
We have investigated two RNA aptamers (mitomer1,
2) which bind isolated mitochondria identified via mito-
chondoria based SELEX method. We confirmed that
mitomer2 binds to rat liver mitochondria well and short-
mitomer2, which is truncated form of mitomer2, binds
stronger than mitomer2.
INTRODUCTION, RESULTS AND DISCUSSION,
CONCLUSION
Mitochondria are essential organelle that produce ATP
and metabolize lipids. It has been reported that functional
abnormalities of mitochondria is related to various diseases
such as diabetes, obesity and so on (1). Exploring drug de-
livery system to mitochondria can open the gate to cure such
diseases. In our research, we chose RNA aptamers as a lig-
and for mitochondria. We selected aptamers by mitochon-
dria-based SELEX (Systematic Evolution of Ligands by
Exponential enrichment) method which is modified from
cell-SELEX (2).
First, we established mitochondria-based SELEX method
(Figure 1). In this method, we applied RNA library that con-
tains random sequences to mitochondria isolated from rat
affinity than mitomer2 and random sequence. It was close to
the same level as D-arm which is the special sequence taken
from Leishmania tRNA which is already known as the tar-
geting device to mitochondria (4). We proved that the stem-
loop structure of mitomer2 is essential to bind mitochondria.
We have established mitochondria-based SELEX method
and obtained two candidates for mitochondria-binding ap-
tamers. We truncated the better candidate, mitomer2 and
short-mitomer2 showed higher binding affinity to isolated
mitochondria.
REFERENCES
1. Johannsen, D. L., Ravussin, E. Curr. Opin. Pharmacol.,
2009, 9, 780-786.
2. Sefah, K., Shangguan, D., Xiong, X. et al., Nat. Protoc.,
2010, 5, 1169-1185.
3. Zhou, J., Battig, M. R., Wang, Y. Anal. Bioanal. Chem.,
2010, 398, 2471-2480.
4. Mahapatra, S., Ghosh, S., Bera, S. K., Ghosh, T., Das,
A., Adhya, S. Nucleic Acids Res., 1998, 26, 2037-2041.
Figure 1. Schematic representation of mitochondria-based SELEX.
Figure 2. Binding assay of mitomer2 and short-mitomer2.
149
HYPOXANTHINE-CONTAINING PNA PROBES FOR IMAGING THE MUTATIONS OF KRAS2 ONCOGENE MRNA
Chang-Po Chen1, Dalip Sethi1, Mathew E. Wampole1, Jeffrey M. Sanders1, Yuan-Yuan Jin1, Mathew L. Thakur2, 3, and Eric Wickstrom1,3*
1Department of Biochemistry & Molecular Biology, 2Radiology; 3Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107 *Correspondence to: Eric Wickstrom. email: [email protected]
ABSTRACT
We hypothesize that determining KRAS2 cancer gene mutation status by external PET imaging of mutant KRAS2 mRNA overexpression will inform decisions on EGFR-targeted cancer therapy. We synthesized a wobble base hypoxanthine PNA monomer to incorporate into radionuclide-chelator-spacer-peptide nucleic acid (PNA)-spacer-insulin-like growth factor 1 (IGF1) analogs for radioimaging of mutant KRAS2 mRNA in suspect masses.
INTRODUCTION
Cancer cell growth depends on high expression of cancer genes. Overexpression of epithelial growth factor receptor (EGFR) stimulates lung cancer cell growth. Anti-EGFR antibodies or tyrosine kinase inhibitors (TKIs) fail in most lung cancer patients. KRAS2 activation makes cancer cell proliferation independent of EGFR activity. As a result, treating lung cancer with anti-EGFR antibodies or TKIs fails when KRAS2 has been mutated. We hypothesize that determining KRAS2 cancer gene mutation status by external genetic PET imaging of mutant KRAS2 mRNA overexpression, as an adjunct to biopsy, will enable physicians to decide on alternatives to EGFR-directed therapies. We designed radionuclide-chelator-spacer-peptide nucleic acid (PNA)-spacer-insulin-like growth factor 1 (IGF1) analogs to enable IGF1R-mediated cellular uptake and radioimaging of mutant KRAS2 mRNA in suspect masses. We demonstrated sequence-specific radioimaging and quantitation of high levels of mutant KRAS2 mRNA in pancreas cancer xenografts after tail vein injection of 99mTc (SPECT) [1] or 64Cu (PET) [2] PNA-spacer-IGF1 probes. However, imaging several specific 12th codon mutants of KRAS2 mRNA would require administration of a cocktail of specific agents. As an alternative, we synthesized a hypoxanthine PNA monomer to enable wobble basepairing to the most frequent base mutations. The monomer was inserted at one or two positions in a chelator-spacer-PNA-spacer-peptide.
RESULTS AND DISCUSSION
In order to develop a general probe for imaging of multi-mutations in KRAS2 mRNA, hypoxanthine-containing PNA monomer was prepared (Scheme 1). Chelator-PNA-peptide sequences were designed (Table 1), assembled, purified, and
characterized. PNA/RNA duplex stabilities were predicted by molecular dynamics. The melting temperatures of the PNA/RNA duplexes were determined.
Scheme 1
OHN
NH
O
O O
N
N N
N
O
COOCH2CH3
N
NH N
N
ON
N N
N
O
COOH
ONH
NO
OO
N
N N
N
OPhe
O
ONH
N
OO
N
N N
N
OH
O
HO
Phe PhePhe
CONCLUSION
In vivo imaging of KRAS2 mRNA mutants in lung cancer is possible. Supported by NIH CA148565; IP owned by EW/MLT, licensed to MTTI.
REFERENCES
1. Amirkhanov, N.V., Zhang, K., Aruva, M.R., Thakur, M.L., and Wickstrom, E. (2010) Bioconj. Chem. 21(4):731-740. PMID: 20232877.
2. Chakrabarti, A., Zhang, K., Aruva, M.R., Cardi, C.A., Opitz, A.W., Wagner, N.J., Thakur, M.L., and Wickstrom, E. (2007) Cancer Biol. Ther. 6(6):948-956. PMID: 17611392.
PCR-FREE TELOMERASE ASSAY WITH CYCLING PROBE TECHNOLOGY TOWARD VALID EVALUATION OF TELOMERASE INHIBITOR
Hidenobu Yaku,1,2,3
Takashi Murashima,1,2
Daisuke Miyoshi 1,2*
and Naoki Sugimoto 1,2*
1Faculty of Frontiers of Innovative Research in Science and Technology (FIRST) and
2Frontier Institute for Biomolecu-
lar Engineering Research (FIBER), Konan Univ., 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan, 3Advanced Technology Research Laboratories, Panasonic Corp., 3-4 Hikaridai, Seika-cho, Soraku-gun, Kyoto 619-0237, Japan. * Correspondence to: (D. M.) [email protected] and (N. S.) [email protected]
ABSTRACT
Novel PCR-free assay for telomerase activity was de-
veloped. The assay is based on a catalytic hydrolysis by
RNase H. Detection limit of this assay was 50 HeLa
cells/L.
INTRODUCTION
Telomerase is responsible for an elongation of telomere
DNA, which is composed of a repeated sequence of 5’-
TTAGGG-3’, at chromosome ends (1). In 85-90% human
tumor cells, telomerase is highly activated, which plays a
role in carcinogenesis (2). Since it was found that G-
quadruplex of telomere DNA inhibits telomerase activity,
development of G-quadruplex ligands as a telomerase inhib-
itor has been attracting attention. For the development, ap-
propriate method for evaluation of telomerase activity is