Thymine Dimers - Dna Lesions Induced by Sunlight Cis-syn Thymine

8/7/2019 Thymine Dimers - Dna Lesions Induced by Sunlight Cis-syn Thymine

1/12

THYMI NE DI MERS - DNA LESI ONS I NDUCED BY SUNLI GHTCIS-SYNTHYMI NE DI MER PHOSPHORAMI DI TE NOW AVAI LABLE

VOLUME 16

NUMBER 2

NOVEMBER 2003

I SO BASES

LNA MONOMERS

TRIMER AMIDITES

NOVEL MONOMERS

DESTHI OBI OTI N

3900 COLUMNS

O ne of the major sources of DNAdamage in all organisms is the UVcomponent of sunlight . Thepredominant reaction induced by

UV light on DNA is dimerization ofadjacent pyrimidine bases leadingto cyclobutane dimers (CPDs) and6- 4 photoproducts. The dimersformed in the most significantquanti ty are the cis-syncyclobutane dimer of two thyminebases (1) and the corresponding 6-4 photoproduct (3). The trans-synthymine dimer (2) is formed at amuch lower level in single anddouble stranded DNA. In sunli ght ,the 6-4 photoproduct is converted

to its Dewar isomer (4) byabsorption of long-wave UV light ataround 325nm.1

Although formed routinely,these dimer products are,fortunately for us, eff ic ientlyexcised and repaired enzymatically(nucleotide excision repair) or thedimerization is reversed byphot olase enzymes. These lesionshave been connected to theformation of squamous cell carcinomas. In addition,humans who lack ability to repair CPD lesions withhigh efficiency may be genetically predisposed toXeroderma Pigmentosa (XP), a di sease characterizedby extreme sensit ivit y to sunlight and high frequencyof skin cancer. Polymerases encountering unrepairedCPD lesions are quit e error- prone, presumablyleading to incorrect base insert ions and subsequentmutations.2

The literature covering the chemistry ofthymidine dimers and other CPDs is replete withreferences from John- Stephen Taylors group atWashingt on Universit y in St . Louis. Any art icle on

O

O P O

NH

N

O

O

O

O

O

N

HNCH 3

CH 3H

HO

OH

O

OO

O P O

NH

N

O

O

O

O

O

N

HN

CH 3

HO

OH

O

O

CH 3

H

O

O P OO

O

O

N

HN

CH 3

H

O

OH

O

O

OH

N

N

H3C

OO

O P OO

O

O

N

HN

CH 3

H

O

OH

O

O

OH

NH3C

ON

H

FIGURE 1:PHOTO-INDUCED THYMINE DIMERS

(1) Cis-syn Thymine Dimer (2) Trans-syn Thymine Dimer

(3) 6-4 Thymine Dimer (4) Dewar Thymine Dimer

thymine dimer phosphoramidites has toacknowledge the work of Professor Taylor and hisco-workers. Their contribution3 to this field has beenoutstanding.

It has been clear to us for some time thatresearchers into DNA damage and repair would valuethe ability to produce oligonucleotides containingcis-synthymine dimer at specific locations withinthe sequence. Unfortunately, the chemical processesrequired to produce cis-syn thymine dimerphosphoramidite are very tortuous4,5 and it was onlyrecently that we were able to obtain this

(Cont inued on Back Page


2/12

ERAGEN BI OSCI ENCES TECHNOLOGY ( cour t esy of EraGen Biosciences)

In natural DNA, two complementary strandsare joined by a sequence of Watson-Crickbase pairs. These obey two rules ofcomplementarity: size (large purines pairwith smaller pyrimidines) and hydrogenbonding (hydrogen bond donors from onenucleobase pair with hydrogen bondacceptors from the other). The former is

necessary to permit the structure thatunderlies enzyme recognition. The latterachieves the specificity that gives rise to thesimple rules for base pairing (A pairs withT, G pairs wit h C) that underlie genetics andmolecular biology. No other class of naturalproducts has reactivity that obeys suchsimple rules. Nor is it obvious how onedesigns a class of chemical substances thatdoes so much so simply.

Some time ago, Professor StevenBenner noticed that the DNA alphabet neednot be l imited to the four standardnucleotides known in nature. Rather, twelvenucleobases forming six base pairs joinedby mutually exclusive hydrogen bondingpatterns might be possible within thegeometry of the Watson- Crick base pair.EraGen has exclusively li censed this idea andthe inventions to follow in order to buildmore robust recognition and analysissystems.

Today, EraGen technology consists oftwo additional bases (isoCytosine (iC) andisoGuanosine (iG) that form the third basepair, as shown in Figure 1. EraGen has spentyears optimizing the production of all

needed reagents to allow the technology toreach its full potential. EraGen technologyis being used in both solution and solid phasedetection as described below.

Details

Research has shown that thecomplementarity rules for EraGentechnology are analogous to t hose of naturalDNA. Therefore, any researcher can exploitthe rule-based molecular recognitionproperties to design molecules built fromEraGen components that will bind to other

molecules built from the correspondingbinding partner.

Since the recognit ion is specific, EraGenprovides a molecular recognition system thatis orthogonal to that provided by naturalDNA. The table shown in Figure 2 isconstructed so that the Tm value forduplexes obeying the extended Watson-Crick pairing rules are found along thediagonal (values are underlined). In eachcase, the underlined value is higher than anyof the Tm values associated with the

mismatches. Especially important is theobservation that the Tm values formismatches with standard nucleobases arelower than the Tm values for mismatcheswi th t he correct EraGen component.

State-Of-The-Art Products

EraGen is providing scientificprofessionals with new options to addresstheir research and molecular diagnosticneeds. Under license from EraGen, BayerDiagnostics has incorporated EraGenTechnology into their third generationbranched DNA assays (Versant) t o achievepreviously unattainable levels of sensit ivit yand specificity for quantitative viral loadtesting in clinical labs.

EraGen is currently commercializingtwo platforms with optimized reagent sets:

GENE-CODE, a real time PCR alternative toTaq-Man and SYBR Green; and MULTI-CODE, a multiplexed genotyping analysissystem. The platform technologies util ize theiC and iG amidites and newly developedtriphosphates to capture assay products andincorporate signaling molecules.

Figure 3 shows how these advanced

reagents are used to perform Gene-CodeOverall, t he advantages of t he Gene-Codereal- t ime PCR system are as follows:

Implemented with any current primerdesigns

High sensitivity and specificity Mult iplex capabilit y Implemented on most real time

instrumention Excellent reproducibility

Schematic of the natural pair Cytosine (C) and Guanosine (G) next to the EraGen pairisoCytosine (iC) and isoGuanosine (iG). Note the directionality of hydrogen bonding (depictedby arrows) is different betw een the tw o pairs.

Thermal melting points for 14-mer with single substitution of nucleotide at P (column) and Q(row) position. Correct match is shown on the diagonal in Red and underlined.

FIGURE 1:C-G AND iC-iG BASE PAIRS

FIGURE 2:EFFECT OF BASE PAIRING ON THERMAL STABILITY OF A 14MER DUPLEX

G C A T iG iC

C 58 46 49 44 54 44G 47 58 48 46 52 44

T 49 44 56 45 53 41

A 47 45 43 53 46 43

iC 45 44 42 41 62 44

iG 51 53 48 51 48 58

5'-CACPACTTTCTCCT-3'

3'-GTGQTGAAAGAGGA-5'

P = column, Q = row

Ribose

N N

N

NO

H

NHH

Ribose

N

N

NHH

O

Ribose

Ribose

N

N

O

N H

H

N NH

N

N

O

N

H

H

C

iG

iC

G

(Cont inued on Next Page)


3/12

Post PCR thermal melt Control forcorrect amplified sequence

Long shelf life

The unique characteristics of EraGentechnology were also used to develop Mult i-Code, a high throughput genotyping system(Figure 4). Overall, the advantages of theMulti-Code multiplexing platform are asfollows:

Flexibility No washing, no centrifugation, no

filtration High sensitivity and specificity High mult iplexing capabilit y Implemented on Luminex

instrumentation

Employing EraGen technology in thisfashion allows all Multi-Code assays to beperformed in a single reaction vesselrequiring less plastic and allowing for easierautomation.

Summary

The EraGen Technology is beingimplemented in some of t he most impressivenew platforms to hit the market recently.This new paradigm shift in fundamentalbiology will allow scientists to greatlysimplify genetic analysis. With these newreagents and their improved characteristics,EraGen Biosciences is proud to be working

with Glen Research to provide todaysresearcher wit h exciting new alternat ives inmolecular diagnostics and genetic analysistools. For more inf ormat ion please visitwww.eragen.com or reference recentpublications:

Nucleic Acids Research, 2003, 31(17),5048-5053, and Clinical Chemistry,2003, 49(3), 407-414.

The three steps of the Multi-Code system are shown below. Step one is standard mult iplexedPCR using a primer with an iC at the 5 end in the absence of iG-biotin, which results in a gated amplicon. Primer extenders and iG-biotin are added for the allelic-specific primerextension step, which results in extended primers with a label and specific capture code. In the

third and final step, SAPE and Luminex microparticles are added just prior to injection onto theLuminex instrument. Data analysis is performed on EraGens proprietary softw are.

Gene-Code assay system w ith a singlelabeled primer containing an iC with anatural reverse primer. During PCRamplification, iGTP-quencher is specificallyincorporated opposite the iC, causing adramatic and rapid decrease influorescence as shown in the bottompanel. Multiplexing is achieved by the useof multiple dyes that do not spectrallyoverlap.

FIGURE 3:GENE-CODE ASSAY FIGURE 4:MULTI-CODE ASSAY STEPS

Step 1

(Cont inued from Previous Page)

Step 2

Step 3


4/12

NN

NN

HN

N

NN

H

CH 3S NN

NN

HO2N

NN

NN

H

CH 3CH 2S N

N

H

CN

CNN

NNN

H

CH 2S

FIGURE 1:ACTIVATOR STRUCTURESBenzylthiotetrazole (BTT)

T he first activator described forphosphoramidite chemistry was 1H-tetrazole (1), which has served well over theyears. Nevertheless, more potent activators,5-methylthio-1 H-tetrazole (2) and 5-

nitrophenyl-1H-tetrazole (3), have poppedup but f allen down. More recent ly, 5-ethylthio-1H- tetrazole (ETT) (4) has gainedconsiderable acceptance as a more acidicactivat or. ETT has the added advantage ofbeing more soluble in acetonit rile than 1H-tetrazole (up to 0.75M versus 0. 5M solut ionin acetonit rile). Less acidic but much morenucleophilic is 4,5-dicyanoimidazole (DCI)(5). DCI is even more soluble in acetoni tri le(up to 1.2M solut ion in acetonit rile). ETTand DCI have proved popular for highthroughput synthesizers since they do not

tend to crystallize and block the fine outletnozzles.

The renewed interest in RNA synthesisdue to the explosion of siRNA technologyhas led us to evaluate 5-benzylthio-1H-tetrazole (BTT) (6), which was described1

several years ago as an ideal activator forRNA synthesis using TOM- protected RNAphosphoramidites2 and recently3 for TBDMS-protected monomers.

Our experiments with BTT have shownthat its maximum solubility is about 0.33M

in acetonit rile The optimal coupling t imefor TOM-protected RNA phosphoramiditeswas found t o be 90 seconds and for TBDMS-protected RNA phosphoramidites it was 3minutes. However, we were concerned thatthe increased acidity of BTT (pKa 4.08 vs ETTpKa 4.28 vs 1H-tetrazole pKa 4.89)3 couldpotentially detrit ylate the monomer duringthe coupling step leading to a dimeraddit ion. If t his process were signif icant,the full- length oligo would be contaminatedwith n+1 insertion mutations. However, byexamining oligonucleotides by ion- exchange

HPLC, we find that n+1 peaks are no moresignificant using BTT with lower couplingtimes than ETT with a 6 minute coupling or1H- tetrazole with a 12 minute coupling.

References:(1) X. Wu and S. Pitsch, Nucleic Acids Res,

1998, 26, 4315-23.(2) S. Pitsch, P.A. Weiss, L. Jenny, A. Stutz,

and X.L. Wu, Helv Chim Acta, 2001, 84,3773-3795.

(3) R. Welz and S. Muller, Tetrahedron Let t,2002, 43, 795-797.

Columns and Plates

W e are happy to announce the availabili tyof polystyrene columns fully compatiblewith the Applied Biosystems 3900synthesizer. Init ially, we are off ering theregular 4 nucleoside supports, Bz-dA, Bz-

dC, dmf- dG and dT. In the near fut ure, wewill be adding several of our most popularsupports, including RNA supports, for thisplatform.

We are also adding 96 well platescontaining both of our CPG-based universal

ORDERI NG I NFORMATI ON

I t e m C a t a log N o . P a ck P r ice ($ )

5- Benzylthio- 1H- tetrazole (BTT) 30- 3070- 10 1g 35.00(Dissolve 1g in 21.3mL anhydrous 30-3070-20 2g 60.00acetonit rile for a 0.25M solution) 30-3070-25 25g 500.00

0.25M 5-Benzylthio-1H-tetrazole in Acetonitrile 30-3170-45 45mL 40.00(Applied Biosystems) 30- 3170- 52 200mL 100.00

30- 3170- 57 450mL 200.0030-3170-62 2L 760.00

(Expedite) 30-3172-66 60mL 50.0030- 3172- 52 200mL 100.0030- 3170- 57 450mL 200.00

AB 3900dA PS 200 nmole columns 26- 2600- 62 Pack of 200 825.00

40 nmole columns 26- 2600- 65 Pack of 200 825.00

dC PS 200 nmole columns 26- 2610- 62 Pack of 200 825.0040 nmole columns 26- 2610- 65 Pack of 200 825.00

dmf - dG PS 200 nmole columns 26- 2629- 62 Pack of 200 825.0040 nmole columns 26- 2629- 65 Pack of 200 825.00

dT PS 200 nmole columns 26- 2630- 62 Pack of 200 825.0040 nmole columns 26- 2630- 65 Pack of 200 825.00

96 Well FormatUniversal Support 1000 96-5001-40 each 200.00

Universal Support II 96-5010-40 each 200.00

dT-CPG 1000 96-2031-40 each 200.00

1H-t etrazole (1) 5-methylthio-1H-t etrazole (2) 5-nit rophenyl-1H-t etrazole (3)

5-ethylthio- 1H-tetrazole (4) 4,5-dicyanoimidazole (5) 5-benzylthio-1H-t etrazole (6)

support s, as wel l as dT. Universal Support1000 is suitable for preparing unmodifiedoligonucleotides. Any regular or modif iedoligonucleotide, including RNA, can beprepared using Universal Support II. The dTsupport should f ind favor among researcherspreparing siRNA oligos. These plates are

offered initially filled at the 40 nmole levelThe support is held tightly in the wells withporous fr it s, which help to disperse the liquidflow more evenly through the support bedwhile avoiding the splashing of support thatis virtually inevitable in loosely filled wells

ACTI VATORS, COLUMNS AND PLATES


5/12

L ocked Nucleic Acid (LNA) was firstdescribed by Wengel and co-w orkers in19981 as a novel class of conformationallyrestrict ed oligonucleotide analogues. LNA isa bicyclic nucleic acid where aribonucleoside is linked between the 2-oxygen and the 4-carbon atoms with a

methylene unit. The structures are detailedin Figure 1. Under licence from Exiqon A/S(Denmark), and in association with LinkTechnologies Ltd (Scotland), we are now ableto offer the four standard LNAphosphoramidites.

Oligonucleotides containing LNAexhibit unprecedented thermal stabilitiestowards complementary DNA and RNA2,which allows excellent mismatchdiscrimination. In fact the high bindingaffinity of LNA oligos allows for the use ofshort probes in, for example, SNP

genotyping3, allele specific PCR and mRNAsample preparation. In fact, LNA isrecommended for use in any hybridizationassay that requires high specificity and/orreproducibility, e.g., dual labelled probes, insituhybridization probes, molecular beaconsand PCR primers. Furthermore, LNA offersthe possibil it y to adjust Tm values of primersand probes in multiplex assays.

As a result of these significantcharacteristics, the use of LNA-modifiedoligos in antisense drug development is now

coming under investigation

4

, and recentlythe therapeutic potential of LNA has beenreviewed.5

In general, LNA oligonucleot ides can besynthesized by standard phosphoramiditechemistry using automated DNAsynthesizers. The phosphoramidites can bedissolved in anhydrous acetonitrile tostandard concentrations, except for the 5-Me-C variant which requires the use of a25% THF/acetonitrile solution. They aremore sterically hindered compared tostandard DNA phosphoramidites and

therefore require a longer coupling t ime. 180seconds and 250 seconds coupling t imes arerecommended for ABI and Expeditesynthesizers, respectively.

The oxidation of the phosphite afterLNA coupling is slower compared to thesimilar DNA phosphite, and therefore alonger oxidation time is suggested. Usingstandard iodine oxidation procedures, 45seconds has been found to be the optimaloxidation time on both ABI and Expediteinstruments. LNA-containing oligo-

LOCKED NUCLEI C ACI D ( LNA) PHOSPHORAMI DI TES

nucleotides are deprotected followingstandard protocols. It is, however, advisableto avoid the use of methylamine whendeprotecting oligos containing Me-Bz-C-LNA since this can result in introduction ofan N4-methyl modification.

LNA-containing oligonucleotides canbe purified and analyzed using the samemethods employed for standard DNA. LNAcan be mixed with DNA and RNA, as well asother nucleic acid analogues, modifiers andlabels. LNA oligonucleotides are watersoluble, and can be separated by gelelectrophoresis and precipitated by ethanol.

Locked- nucleic Acid (LNA)phosphoramidites are protected by EP PatNo. 1013661, US Pat No. 6,268,490 andforeign applications and patents owned by

Exiqon A/S. Products are made and soldunder a license from Exiqon A/S. Productsare for research purposes only. Product s maynot be used for diagnostic, clinical,commercial or other use, including use inhumans. There is no implied license for

commercial use, including contract researchwith respect to the products and a licensemust be obtained directly from Exiqon A/Sfor such use.

References:(1a) A.A. Koshkin, S.K. Singh, P. Nielsen, V.K.

Rajwanshi, R. Kumar, M. Meldgaard, C.E.Olsen, and J. Wengel, Tetrahedron,1998,54, 3607-3630.

(1b) S.K. Singh, P. Nielsen, A.A. Koshkin, and JWengel, Chem. Comm., 1998, (4), 455-456.

(2) L. Kvrn and J. Wengel, Chem. Comm.,1999, (7), 657-658.

(3) P. Mouritzen, A.T. Nielsen, H.M.Pfundheller, Y. Choleva, L. Kongsbak, andS. Mller, Expert Review of MolecularDiagnostics, 2003, 3(1), 27-38.

(4a)J. Kurreck, E. Wyszko, C. Gillen, and V.A.Erdmann, Nucleic Acids Res., 2002, 30,1911-1918.

(4b)H. rum and J. Wengel, Curr. Opinion inMol. Therap., 2001, 3, 239-243.

(5a)M. Petersen and J. Wengel, Trends inBiotechnology, 2003, 21(2), 74-81.

(5b)D.A. Braasch, D.R. Corey, Biochemistry,2002, 41, 4503-4510.

FIGURE 1:LNA MONOMER STRUCTURES

O

DMTOO

NHBz

N

N

N

N

CNEtO

N(iPr)2P

OO

N

NO

NHBz

O

P N(iPr)2

O CNEt

ODMTO

CH 3

O

HN

N

N

N

O

N

DMTOO

CNEtO

N(iPr)2P

O

Me2N

O

HN

N

O

O

ODMTO

CH

O

CNEtO

N(iPr)2P

Bz-A-LNA 5-Me-Bz-C-LNA dmf-G-LNA T-LNA


I t e m C a t a log N o . Pa ck P r ice ($ )

Bz- A- LNA- CE Phosphoramidit e 10- 2000- 90 100 m 120.0010-2000-02 0.25g 225.00

10-2000-05 0.5g 450.00

5-Me-Bz-C-LNA-CE Phosphoramidi te 10-2011-90 100 m 120.0010- 2011- 02 0.25g 225.0010-2011-05 0.5g 450.00

dmf -G- LNA- CE Phosphoramidit e 10- 2029- 90 100 m 120.0010-2029-02 0.25g 225.0010-2029-05 0.5g 450.00

T- LNA- CE Phosphoramidite 10- 2030- 90 100 m 120.0010-2030-02 0.25g 225.0010-2030-05 0.5g 450.00


6/12


7/12



AAA Trimer Phosphoramidite 13- 1000- 95 50 m 350.0013-1000-90 100 m 700.00

AAC Trimer Phosphoramidite 13- 1001- 95 50 m 350.00

13-1001-90 100 m 700.00

ACT Trimer Phosphoramidite 13- 1013- 95 50 m 350.0013-1013-90 100 m 700.00

ATC Trimer Phosphoramidite 13- 1031- 95 50 m 350.0013-1031-90 100 m 700.00

ATG Trimer Phosphoramidite 13- 1032- 95 50 m 350.0013-1032-90 100 m 700.00

CAG Trimer Phosphoramidite 13- 1102- 95 50 m 350.0013-1102-90 100 m 700.00

CAT Trimer Phosphoramidite 13- 1103- 95 50 m 350.00

13-1103-90 100 m 700.00

CCG Trimer Phosphoramidite 13- 1112- 95 50 m 350.0013-1112-90 100 m 700.00

CGT Trimer Phosphoramidite 13- 1123- 95 50 m 350.0013-1123-90 100 m 700.00

CTG Trimer Phosphoramidite 13- 1132- 95 50 m 350.0013-1132-90 100 m 700.00

GAA Trimer Phosphoramidite 13- 1200- 95 50 m 350.0013-1200-90 100 m 700.00

GAC Trimer Phosphoramidite 13- 1201- 95 50 m 350.00

13-1201-90 100 m 700.00

GCT Trimer Phosphoramidite 13- 1213- 95 50 m 350.0013-1213-90 100 m 700.00

GGT Trimer Phosphoramidite 13- 1223- 95 50 m 350.0013-1223-90 100 m 700.00

GTT Trimer Phosphoramidite 13- 1233- 95 50 m 350.0013-1233-90 100 m 700.00

TAC Trimer Phosphoramidite 13- 1301- 95 50 m 350.0013-1301-90 100 m 700.00

TCT Trimer Phosphoramidite 13- 1313- 95 50 m 350.0013-1313-90 100 m 700.00

TGC Trimer Phosphoramidite 13- 1321- 95 50 m 350.0013-1321-90 100 m 700.00

TGG Trimer Phosphoramidite 13- 1322- 95 50 m 350.0013-1322-90 100 m 700.00

TTC Trimer Phosphoramidite 13- 1331- 95 50 m 350.0013-1331-90 100 m 700.00

Trimer Phosphoramidit e Mix 1 13- 1991- 95 50 m 375.00(Mix of above 20 trimers) 13-1991-90 100 m 750.00

for histidine, has to be differentiated fromTAC, coding for tyrosine. These two t rimershave virtually identical lipophilicit y and theiridentity cannot be clearly confirmed byHPLC. This problem has been solved10 usingHPLC electrospray mass spectrometricanalysis of the tr imers, which provides data

confirming molecular weight and sequence.In Table 1, the trimers, their codingamino acid and their reaction factor (RF) arelisted. The reaction factor is crit ical sincethe trimers wi ll likely be mixed and they havediffering react ivity in the coupling react ion.RF for AAC is 1.0 and for TAC is 1.6.Therefore, 1.6 equivalents of TAC are neededfor every 1.0 equivalent of AAC for equalcoupling. Mixtures can easily be made usingequimolar solut ions or the molecular weightof each trimer has to be used to generatethe appropriate weights of each trimer to

use if mixing by weight. An example of t hepreparation of a mixture of all 20 trimers isshown in the right column of Table 1 andcompleted in the footnotes.

All of the trimers are now availableindividually so that researchers can preparecustom mixtures. A mixture of all 20 trimersdesigned to produce equal coupling of all20 is also available. If you require customproduction of a specific mixture, please e-mail [email protected] for aquotat ion and projected delivery.

References:(1) J. Sondek and D. Shortle, Proc Natl Acad

Sci U S A, 1992, 89, 3581-3585.(2) P. Gaytan, J. Yanez, F. Sanchez, H. M ackie,

and X. Soberon, Chem Biol, 1998, 5, 519-527.

(3) P. Gaytan, J. Yanez, F. Sanchez, and X.Soberon, Nucleic Acids Res, 2001, 29, E9.

(4) B. Virnekas, L.M. Ge, A. Pluckthun, K.C.Schneider, G. Wellnhofer, and S.E.Moroney, Nucleic Acids Research, 1994,22, 5600-5607.

(5) M.H. Lyttle, E.W. Napolitano, B.L. Calio,and L.M . Kauvar, Biotechniques, 1995, 19,274.

(6) A. Ono, A. Matsuda, J. Zhao, and D.V.Santi, Nucleic Acids Research, 1995, 23,4677-4682.

(7) A.L. Kayushin, M.D. Korosteleva, A.I.Miroshnikov, W. Kosch, D. Zubov, and N.Piel, Nucleic Acids Research, 1996, 24,3748-3755.

(8) A. Kayushin, et al., Nucleos Nucleot, 1999,18, 1531-1533.

(9) A. Kayushin, M. Korosteleva, and A.Miroshnikov, Nucleos Nucleot NucleicAcids, 2000, 19, 1967-1976.

(10)T. M auriala, S. Auriola, A. Azhayev, A.Kayushin, M. Korosteleva, and A.Miroshnikov, J. Pharmaceutical andBiomedical Analysis, In Press.


8/12

PC Linker Phosphoramidit e

P hoto-triggered DNA cleavage is a majortool used for studying conformational

changes and strand breaks, as well as for

studying activation of nucleic-acid-targeted

drugs, such as antisense oligonucleotides.

A versatile photocleavable DNA buildingblock has been described by researchers in

Washington University, Missouri and used

in phototriggered hybridization.1 In

association with Link Technologies Ltd

(Scotland) this phosphoramidite is now

available f rom Glen Research.

This reagent has also been used in the

design of multifunctional DNAand RNA

conjugates2 for the in vitroselection of new

molecules catalyzing biomolecular reactions.

Researchers at Bruker Daltonik in Germany

have also developed genoSNIP, amethod forsingle-nucleotide polymorphism (SNP)

genot ypin g by M ALDI- TOF mass

spectrometry.3 This method uses size

reduction of primer extension products by

incorporation of the photocleavable linker

for phototriggering strand breaks near to the

3 end of the extension primer.

Similar to the 3 other available PC

monomers, the general design of the PC

Linker is based on an -substituted 2-

nit robenzyl group. The photo-reactive group

is derivatized as a -cyanoethylphosphoramidite and the non-nucleoside PC

Linker can be incorporated into

oligonucleotides at any posit ion by standard

automated DNA synthesis methodology.

Coupling efficiencies >95% are achieved

using an extended coupling time of 15

minutes. For ease of use, the product

contains a dimethoxytrityl group. The -

cyanoethyl group is removed under normal

synthesis conditions using 25% aqueous

ammonia soluti on.

Upon irradiating a PC-modified oligowith near-UV light, the phosphodiester bond

between the linker and the phosphate is

cleaved, resulting in the formation of a 5-

monophosphate on the released

oligonucleotide. Unlike other photocleavable

spacers, the PC Linker Phosphoramidite has

the added advantage in that photocleavage

result s in monophosphate fragments at both

the 3- and 5-termini (see Figure 2).

STI LL MORE NEW PRODUCTS

FIGURE 1:PHOTOCLEAVABLE MODIFIERS

NHDMTN

SNH

O

O

NH

O

NO2

H3C

CNEtO

N(iPr)2PO

TFAHNNH

O

NO2

H3C

CNEtO

N(iPr)2PO

NH

O

NO2

H3C

CNEtO

N(iPr)2PO

DMTO

NO2

CNEtO

N(iPr)2PODMTO

NO2

CNEtO

N(iPr)2PODMTO

O-oligo2

O-

O

oligo1-OP OO O P

O-

NO2

O-

O

oligo1-OP O

NO

O

O-oligo2

OO-

P

O-

NO

O

O-

O

oligo1-OP O

-

FIGURE 2:PHOTO-CLEAVAGE USING PC LINKER PHOSPHORAMIDITE



PC Linker Phosphoramidite 10- 4920- 90 100 m 170.0010- 4920- 02 0.25g 500.00

PC Biot in

PC Linker

PC Spacer

PC Amino-Modifier

DNA

Synthesis

photocleavage5'-monophosphate

3'-monophosphate

+

+

References:(1) P. Ordoukhanian and J-S. Taylor, J. Am.

Chem. Soc., 117, 9570-9571, 1995.(2a)F. Hausch and A. Jschke, Nucleic Acids

Research, 2000, 28, e35.(2b)F. Hausch and A. Jschke, Tetrahedron,

2001, 57, 1261-1268.

(3) T. Wenzel, T. Elssner, K. Fahr, J. Bimmler,S. Richter, I. Thomas, and M. Kostrzewa,Nucleosides, Nucleotides & Nucleic Acids,2003, 22, 1579-1581.

(Cont inued on Next Page)


9/12

DesthiobiotinTEG

A vidin, streptavidin and other biotin-binding proteins have the abili ty to form anintense association with biotin-containingmolecules. This association has been usedfor many years to develop systems designed

to capture biotinylated biomolecules. In theoligonucleotide field, probably the mostcommon modification is biotinylation andreagents are available to modify oligos atthe termini and within the sequence. A widevariety of tests and techniques are in routineuse to exploit the extraordinary affinity ofthese biotin-binding proteins forbiot inylated biomolecules. However, theintense affinity of biotin-binding proteinsfor biotin is also the biggest drawback inthat the association is essentiallyirreversible. Indeed, extremely low pH and

highly concentrated chaotropic reagents arerequired to break the association and theseconditions are not entirely compatible witholigonucleotides. 2- Iminobiot in has beenused as a reversibly binding biotin reagentsince its association with biotin-bindingproteins can be broken at pH4. However,2- iminobiotin is not stable to the conditionsof oligonucleotide deprotect ion. Anotherbiotin analogue that exhibits lower bindingto biotin- binding proteins like streptavidinis desthiobiotin (or dethiobiotin). This biotin

analogue is lacking the sulfur group fromthe molecule and has a dissociation constant(Kd) several orders of magnitude less thanbiotin/streptavidin. As a result, biomoleculescontaining desthiobiotin are dissociatedfrom streptavidin simply in t he presence ofbuffered solutions of biotin.1 We believethat t hese characteristics wi ll allow anotherbiotin product to prosper.

Our most versatile biotin products arethe biot inTEG products which can be addedsingly or in multiple additions anywherewithin an oligonucleotide. In addit ion, the

triethyleneglycol (TEG) section of thestructure separates the biotin from theoligonucleotide in such a way that i t is morereadily captured by streptavidin. We haveused the same structure to offerdesthiobiotinTEG phosphoramidite and thecorresponding CPG.

Reference:(1) J.D. Hirsch, L. Eslamizar, B.J. Filanoski, N.

Malekzadeh, R.P. Haugland, and J.M .Beechem, Anal Biochem, 2002, 308, 343-57.

FIGURE 1:DESTHIOBIOTIN MODIFIERS

NH OO

OO ODMT

O

CNEtO

N(iPr)2PO

O

NH NH

NH OO

OO ODMT

O O-succinyl-lcaa-CPG

O

NH NH

DesthiobiotinTEG Phosphoramidite

Desthiobiot inTEG-CPG



Dest hiobiot inTEG Phosphoramidi te 10- 1952- 95 50 m 185.00

10-1952-90 100 m 335.0010-1952-02 0.25g 775.00

Desthiobiot inTEG-CPG 20-2952-01 0.1g 140.0020- 2952- 10 1.0g 1150.00

0.2 mole columns 20-2952-42 Pack of 4 140.001 mole columns 20-2952-41 Pack of 4 230.0010 mole column (ABI) 20-2952-13 Pack of 1 345.0015 mole column (Expedit e) 20- 2952- 14 Pack of 1 520.00

5-Me-2'-deoxyZebularine-CE Phosphoramidite 10-1061-95 50 m 200.0010-1061-90 100 m 400.0010- 1061- 02 0.25g 975.00

N

NO

OH

HO O

OH

N

NO

OH

HO O

N

NO

DMTO

CNEtO

N(iPr)2PO

O

Zebularine (1) 5-Methylpyrimidin-2-one (2) 5-Me-2'- deoxyZebularine (3)

5-Me-2'-deoxyZebularine

Z ebularine (pyrimidin-2-one ribo-nucleoside) (1) is a cytidine analog that actsas a DNA demethylase inhibi tor, as well as a

cytidine deaminase inhibit or. This structureis very active biologically and Zebularine isnow used as a potent anti - cancer drug. A2- deoxynucleoside analogue of Zebularine,5-methyl-pyrimidin-2-one, 2-deoxy-nucleoside (2), has been used1 to probe theini t iat ion of t he cellular DNA repair process

by making use of its mildly fluorescentpropert ies. We believe that this combinationof biological activity and fluorescence

properties would make the phosphoramidit e(3) a strong addition to our array ofnucleoside analogue phosphoramidites.

Reference(1) S.F. Singlet on, et al., Org Lett, 2001, 3,

3919-22.

(Cont inued from Previous Page)


10/12

O ligonucleotide conjugat ion reactions arepredominantly carried out using anucleophilic group on the oligonucleotideto attach to an electrophilic group on a tagor support. This strategy presumablypredominates since oligonucleotidedeprotection schemes are carried out by

bases, which are inherently nucleophilic.Indeed, there are many strategies to produceoligonucleotides modified with amine andthiol groups at a variety of posit ions. Onthe other hand, there are situations whereresearchers wish to conjugate a variety ofnucleophil ic groups to oligonucleot ides. Inthis vein, convenient carboxy-modifiers havebeen descri bed recent ly. 5- Carboxy-Modifier C10 (1) which contains acarboxylate NHS ester (available from GlenResearch) allows convenient solid- phaseconjugation of amino compounds to

oligonucleotides on the solid support priorto deprotection.1 Simi larly, another reagentcontaining a protected carboxylic acid andsuitable for solid-phase conjugationreactions has been described2 recently. Aphosphoramidite containing an electrophilicchloroacetyl group has been described.3 Inthe latt er case, the oligonucleotide can alsobe derivatized on solid phase, but thechloroacetyl group is also stable todeprotection with potassium carbonate inmethanol and oligonucleotides modifiedwith this reagent can also be used for

solut ion-phase conjugations.Aldehyde modifiers would be att ractive

electrophil ic substitutions in oligo-nucleotides since they are able to react withamino groups to form a Schiffs base, withhydrazino groups to form hydrazones, andwith semicarbazides to form semi-carbazones. The Schiffs base is unstableand must be reduced with sodiumborohydride to form a stable linkage buthydrazones and semicarbazides are verystable li nkages. Simi larl y t o acti vatedcarboxylic acids, aldehydes are generallyunstable to oligonucleotide deprotectioncondit ions. Many strategies have used postsynthetic oxidation of a glycol with sodiumperiodate to generate an aldehyde group.However, a recent report describes4 the useof a formylindole nucleoside analog (2) togenerate an aldehyde group directly in anoligonucleotide without the need for aprotecting group on the aldehyde. Ourcollaboration with Epoch Biosciences hasallowed us to adopt the protected

NEW EPOCH PRODUCTS - 5 - ALDEHYDE- MODI FI ER C2 PHOSPHORAMI DI TE

SCHEME 1:IMMOBILIZATION OF 5'-ALDEHYDE-MODIFIED OLIGONUCLEOTIDE TO SC-GLASS

Si

Si

N NNH2

O

H H

N N

NH2O

H HO

POO

O

H

O-spacer-ODN

O-

N N

NH2

O

H H

N NN

O

H H

O-

O-spacer-ODNO

O P

O

Si

Si

Si

Si

N NN

O

H H

O-

O-spacer-ODNO

O P

O

N NN

O

H H

-O3S

SO-

3

SO-3

H

O

-O3S

SC-glass

benzaldehyde-modi fied ODN

Immobilizationacetate buff er

pH 5.0

acetat e buffer

pH 5.0

Blocking

benzaldehyde derivative (3) as our first 5-aldehyde modifier.5 The acetal protectinggroup is sufficiently hydrophobic for use inRP HPLC and cartridge purification and isreadily removed after oligonucleotidesynthesis under standard oligonucleotidedetritylation conditions with 80% aceticacid or 2% TFA after cart ridge purifi cation.

Scheme 1 illustrates the ut ilit y of t his5-aldehyde modifier in the preparation ofoligonucleot ide arrays on glass slides. First,glass slides were derivatized withsemicarbazidopropyltriethoxysilane, areagent readily prepared by the reaction ofisocyanopropyltriethoxysilane withhydrazine. The slides were washed, driedand cured at 110C to produce SC-glass

slides. The aldehyde- modif ied oli go-nucleot ides were prepared wit h a Spacer 18molecule between the benzaldehyde groupand the oligonucleotide to help separate theoligonucleotides from the glass surface foefficient hybridization with the targetArrays can be prepared by spotting the 5-aldehyde-modified oligonucleotides inacetate buffer at pH 5 onto the plates andmaintaining the slides in a humidenvironment for 3 hours at 37C. The slideswere washed to remove excessoligonucleotide and then excesssemicarbazide residues on the slide wereblocked with 5-formyl-1,3-benzene-disulfonic acid disodium salt . The slides werethen ready for hybridization experiments.


11/12

N

O

OO

O

O

P N(iPr)2

O CNEt

N

CHO

DMTO

CNEtO

N(iPr)2PO

O

CH 2CH 2CN

O

NPOO

O

O

(1) 5'-Carboxy-Modifier C10

(3) Epoch 5'- Aldehyde-Modif ier C2(2) 3-Formylindole

AND GI G HARBOR GREEN PHOSPHORAMI DI TE

O

O

O

OOO

O

NH

O

O P N

O

CH 2CH 2CN

FIGURE 1:ELECTROPHILIC MODIFIERS AND EPOCH GIG HARBOR GREEN

(4) Epoch Gig Harbor Green



5'-Aldehyde-Modif ier C2 Phosphoramidite 10-1933-90 100 m 85.0010-1933-02 0.25g 325.00

Epoch Gig Harbor Green Phosphoramidite 10-5922-95 50 m 165.0010-5922-90 100 m 325.0010-5922-02 0.25g 875.00

Epoch Gig Harbor Green (4) and 6-FAM (5)are based on the same fluorescein corestructure. The difference between the twomolecules is the linker strategy. Therefore,there should be lit t le difference between GigHarbor Green and FAM in terms ofabsorbance and emission spectra. It is well

known that the fluorescence of f luorescein-based dyes depends on the degree ofionization of the phenolic groups and,therefore, is pH dependent. Gig Harbor Greenis more pH dependent than FAM, whichmeans that at pH below 7.5 fluorescencewill drop slightly faster for Gig Harbor Greenthan for FAM. On the other hand, at higherpH such as 8.5 (PCR condit ions) both dyesare completely ionized and Gig Harbor Greenis 15-20% brighter than FAM.6

5'-Aldehyde-Modifier and Gig HarborGreen are offered in collaboration with

Epoch Biosciences, Inc. Please read thefol lowing statement before choosing to usethese products commercially.

"These Products are for researchpurposes only, and may not be used forcommercial, clinical, diagnostic or any otheruse. The Products are subject to proprietaryrights of Epoch Biosciences, Inc. and aremade and sold under license from EpochBiosciences, Inc. There is no impl ied licensefor commercial use with respect to theProducts and a license must be obtaineddirectly from Epoch Biosciences, Inc. with

respect to any proposed commercial use ofthe Products. Commercial use includes butis not limited to the sale, lease, license orother transfer of the Products or anymaterial derived or produced from them, thesale, lease, license or other grant of rightsto use the Products or any material derivedor produced from them, or the use of theProducts to perform services for a fee forthird part ies (including cont ract research)."

References:(1) RI Hogrefe and MM Vaghefi, 2001, US

Patent No. 6,320,041.(2) A.V. Kachalova, D.A. Stetsenko, E.A.

Romanova, V.N. Tashlitsky, M.J. Gait, andT.S. Oretskaya, Helv Chim Acta, 2002, 85,2409-2416.

(3) A. Guzaev and M. Manoharan, NucleosNucleot, 1999, 18, 1455-1456.

(4) A. Okamoto, K. Tainaka, and I. Saito,Tetrahedron Let t, 2002, 43, 4581-4583.

(5) M.A. Podyminogin, E.A. Lukhtanov, andM.W. Reed, Nucleic Acids Res, 2001, 29,5090-8.

(6) E. Lukhtanov, Epoch Biosciences, PersonalCommunication.

O

O

O

OO

O O

HN

OP

N

O

OCN

(5) 6-FAM


12/12

PRESORTED STANDARD

US POSTAGE

PAID

RESTON VA

PERMIT NO 536

(Continued from Front Page)



Cis-synThymine Dimer Phosphoramidi te 11-1330-95 50 m 2100.00(ABI septum vial i s standard vial. 11-1330-90 100 m 4200.00Add E to catalog no. for Expedite vial 11- 1330- 02 0.25g 10200.00or V to catalog no. for Expedite V vial)

ODMTO

O P O

NH

N

O

O

OCH 3

N(iPr)2PO

O

O

O

N

HNCH 3

CH 3H

HO

OCH 3

FIGURE 2:CIS-SYNTHYMINE DIMERphosphoramidite in sufficient quantity tooffer it f or sale. Due to the strategy used tosynthesize our version of thisphosphoramidite, it has methyl protectinggroups on phosphorus, rather than the moreusual cyanoethyl. This monomer exhibit shigh purity, performance and stability butrequires the use of thiophenoxide (oranother phosphate demethylati ng reagent)prior to regular deprotection.

Cis- syn thymine dimer phosphor-amidite (5) is by far the most expensiveproduct we have offered for sale. We arepackaging it initially in standard ABI andExpedite vials, as well as amber V vialscompatible with the Expedite synthesizer.Using t his V vial containing 50 micromoles,it is possible to prepare 6 oligos on a 0.2mole scale each containing a singleinsertion, or 4 oligos on a 1 mole scale.

Similar results should be possible using theLV cycles in Applied Biosystems instrument s.Given the high price of this phosphoramidite,if only one or two oligos are required, itmakes sense to have a custom oligo house

prepare them. A custom oligo house mayalso wish to group oli gos for several clientsfor most eficient use of thisphosphoramidit e. Contact us for the namesof custom oligo services active in preparingoligos containing cis-synthymine dimer.

References:(1) C.A. Smith and J.-S. Taylor, J Biol Chem,

1993, 268, 11143-51.(2) H. Ling, F. Boudsocq, B.S. Plosky, R.

Woodgate, and W. Yang, Nature, 2003,424, 1083-7.

(3) L. Sun, et al., Biochemistry, 2003, 42,9431-7, and references cited therein.

(4) J.-S. Taylor, I. Brockie, and C. ODay, J AmChem Soc, 1987, 109, 6735-6742.

(5) T. Murata, S. Iwai, and E. Ohtsuka, NucleicAcids Research, 1990, 18, 7279-86.

Cis-syn Thymine DimerPhosphoramidite (5)

Documents

Thymine Dimers - Dna Lesions Induced by Sunlight Cis-syn Thymine