Q-D) 1994 Oxford University Press
`
`Nucleic Acids Research, 1994, Vol. 22, No. 20 4259-4267
`
`Termination of DNA synthesis by novel 3'-modified-
`deoxyribonucleoside 5'-triphosphates
`
`Michael L.Metzker*, Ramesh Raghavachari1 +, Stephen Richards, Swanee E.Jacutin1,
`Andrew Civitello, Kevin Burgess1 and Richard A.Gibbs
`Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza,
`Houston, TX 77030 and 1Department of Chemistry, Texas A & M University, College Station,
`TX 77843, USA
`
`Received June 3, 1994; Revised and Accepted July 25, 1994
`
`ABSTRACT
`Eight 3'-modified-dNTPs were synthesized and tested
`in two different DNA template assays for incorporation
`activity. From this enzymatic screen, two 3'-0-methyl-
`dNTPs were shown to terminate DNA syntheses
`mediated by a number of polymerases and may be used
`as alternative terminators in Sanger sequencing. 3'-0-
`(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and
`was shown to be incorporated by several thermostable
`DNA polymerases. Base specific termination and
`efficient photolytic removal of the 3'-protecting group
`was demonstrated. Following deprotection, DNA
`synthesis was reinitiated by the incorporation of natural
`nucleotides into DNA. The identification of this labile
`terminator and the demonstration of a one cycle stop-
`start DNA synthesis are initial steps in the development
`of a novel sequencing strategy.
`
`INTRODUCTION
`2'-Deoxyribonucleoside-5'-triphosphates (dNTPs) modified at
`their 3'-hydroxyl position can act as terminators of enzyme-
`directed DNA synthesis (1-12). These nucleotide analogs are
`probes,
`mechanistic
`as DNA sequencing tools,
`useful
`antimetabolites, and as antiviral agents. Consequently, such
`compounds have been used for analytical and therapeutic studies
`(2). Overall, however, the number of compounds that are well
`characterized is small, and there is considerable scope for new
`combinations of terminators and polymerases to be identified.
`Among the most familiar terminators of DNA synthesis are
`the 2', 3 '-dideoxyribonucleoside-5 '-triphosphates (ddNTPs) that
`are the basis for Sanger DNA sequencing (13). In that method
`oligonucleotide-primed DNA or RNA templates are enzymati-
`cally extended in a 5' - 3' direction in the presence of a mixture
`of dNTPs and ddNTPs to generate a population of molecules that
`are terminated at specific base positions. DNA fragments of
`different lengths are resolved by denaturing polyacrylamide gel
`
`electrophoresis and detected either by radioactive or fluorescent
`labels to reveal the underlying base sequence. Despite the obvious
`limitations of gel electrophoresis for sequencing long DNA
`strands, this method has been the favored approach for more than
`ten years (13,14).
`Improvements to the Sanger protocols are being sought to meet
`the increasing demands of large scale sequencing of whole
`genomes (14). We and others (15-18) have independently
`conceived a radically different, gel-free alternative to the Sanger
`scheme for DNA sequencing. This method, called the Base
`Addition Sequencing Scheme (BASS), is based on novel
`nucleotide analogs that terminate DNA synthesis. BASS involves
`repetitive cycles of incorporation of each successive nucleotide,
`in situ monitoring to identify the incorporated base, and
`deprotection to allow the next cycle of DNA synthesis, (Figure
`1). Compared to Sanger sequencing, BASS has two major
`advantages: base resolution would not require gel electrophoresis
`and there is a tremendous capacity for simultaneous analyses of
`multiple samples. The complete scheme demands nucleotide
`analogs that are tolerated by polymerases, spectroscopically
`distinct for each base, stable during the polymerization phase,
`and deprotected efficiently under mild conditions in aqueous
`solution. These stringent requirements are formidable obstacles
`for the design and synthesis of the requisite analogs.
`The investigation of the interactive patterns between various
`terminating analogs and different enzymes is an important
`preliminary phase in the development of the BASS method.
`Consequently, eight 3'-modified-dNTPs were synthesized and
`examined for their ability to terminate DNA synthesis mediated
`by a variety of polymerases. The majority of 3'-modified analogs
`have labile protecting groups that have the potential to be
`incorporated into BASS. Active combinations of terminators and
`enzymes were identified using two different primer-template gel
`assays. One of these compounds, 3'-0-(2-nitrobenzyl)-dATP [7],
`was used to demonstrate one complete cycle of termination,
`deprotection, and reinitiation of DNA synthesis.
`
`*To whom correspondence should be addressed
`+Present address: LI-COR Inc., Biotechnology Division, Lincoln, NE 68504-5000, USA
`
`Illumina Ex. 1039
`IPR Petition - USP 10,435,742
`
`

`

`4260 Nucleic Acids Research, 1994, Vol. 22, No. 20
`
`=UmHE
`
`Solid support
`
`'OPA
`
`3' OPC
`
`3' OPG
`
`3' OPT
`
`Base Addition
`and
`Termination
`
`/
`
`/ 3' OPA
`/lllllll
`A
`
`I
`
`k
`
`l~~~~~~IIIIIImIIII A/3OH
`
`Base
`Identification
`
`Base Protecting
`Group Removal
`
`Imaging of
`Reporter Group
`
`HO
`
`R
`O-
`
`9
`9
`O-V -O--Adenine
`O2- I
`0-
`
`where P' =
`
`Op
`
`/CH3
`
`1
`0
`
`>,jSCH3 /.CH2
`0
`2
`
`3
`
`H2N0
`
`H2N>:
`
`NO2
`5
`
`6
`9
`9
`R
`HO-{) PV-O-I
`0-
`0-
`
`OCH3
`8
`
`4
`
`7
`
`Figure 1. Cartoon of the Base Addition Sequencing Scheme (BASS). A primer
`is annealed to a biotinylated-labeled template bound to a solid support. Four
`deoxynucleotides triphosphates that have spectroscopically unique blocking groups
`attached to the 3'-position are added. Polymerase extension is terminated after
`the addition of one base. Upon imaging of the reporter group, the protecting group
`is removed resulting in a 'free' 3'-OH group, allowing the addition of the next
`complement base.
`
`MATERIALS AND METHODS
`General
`High field Nuclear Magnetic Resonance (NMR) spectra were
`recorded on a Bruker AC250 (1H at 250 MHz, 13C at 62.9
`MHz, 31P at 101.26 MHz) or a Varian XL 200 (1H at 200
`MHz, 13C at 50 MHz, 31P at 81 MHz). Ultraviolet (UV) spectra
`were recorded on a Hewlett-Parkard model 8452A diode array
`spectrophotometer. Thin layer chromatography was performed
`on Whatman silica gel 60 A F254 plates. Flash chromatography
`was performed on SP silica gel 60 (230-600 mesh ASTM). Ion-
`exchange chromatography was performed on Fluka DEAE
`cellulose C451 (HCO3- form). Photodecomposition of 3'-O-
`(2-nitrobenzyl)-dATP [7] was performed using a FisherBiotech
`transilluminator.
`
`Organic syntheses
`The chemical structures of compounds [1] -[8] are shown in
`Figure 2. Compounds [1]- [4], [6], [8] were prepared according
`to the general scheme:
`
`HO
`
`Base
`
`t-BuPh2SiC1
`
`TBDPSO
`
`Base
`
`OH
`
`imidazole, DMF
`
`add 3-group
`
`TBDPSO
`
`Base
`
`Bu4NF
`
`OH
`
`9
`
`HO s
`
`Bjlase
`
`OH
`OH
`The 5'-hydroxyl was protected with a tert-butyldiphenylsilyl
`(TBDPS) group, and the specific addition of the 3'-protecting
`
`THF
`
`Figure 2. Chemical structures of the 3'-modified-nucleotides. Details of the
`chemical syntheses are described in the Materials and Methods section.
`
`groups (P') are described below. Desilylations were performed
`by the addition of 1.0 equiv. of tetrabutylammonium fluoride
`(Bu4NF) to the 3'-protected-5'-silyl-adenosine or thymidine
`derivatives. The reactions were monitored by TLC; after
`completion (ca. 15 min.), the reactions were quenched with 1.0
`equiv. of glacial acetic acid. The solvent was removed, and the
`residues were purified by silica column chromatography (10%
`methanol/ethyl acetate).
`
`[1]. To 2'-deoxy-5 '-tert-
`2 '-Deoxy-3 '-0-methyladenosine
`butyldiphenylsilyladenosine [9] (200 mg, 0.4 mmol) in benzene
`(5 mL), methyl iodide (568 mg, 4.0 mmol, 10 equiv.),
`tetrabutylammonium hydroxide (TBAH) (40% solution, 325 ,uL),
`and 1 M NaOH (5 mL) were vigorously stirred at 25°C for 16
`h. The organic layer was extracted with ethyl acetate and washed
`with deionized (D.I.) water, saturated NaCl, dried over Na2SO4
`and purified by flash chromatography using a stepwise gradient
`(0% methanol/ethyl acetate to 5% methanol/ethyl acetate in 2%
`intervals) (180 mg, 89%) (19).
`The 0-methyl derivative from the above procedure (80 mg,
`0.16 mmol), after desilylation and flash chromatography gave
`compound [1] as colorless crystals (30 mg, 70%). High resolution
`mass spectrometry (HRMS) m/e calculated for C11H15N503:
`265.1172, observed 265.1154.
`
`2'-Deoxy-3'-O-acyladenosine
`[2].
`2'-Deoxy-5'-tert-butyldi-
`phenylsilyladenosine [9] (100 mg, 0.2 mmol), acetic anhydride
`(28 mg, 0.27 mmol), and 4-dimethylaminopyridine (DMAP) (5
`mg, 0.05 mmol) in dry pyridine were stirred at 25°C for 6 h.
`After removing pyridine under vacuum, the residue was dissolved
`in D.I. water, extracted in chloroform, washed with D.I. water,
`10% HCI, saturated NaHCO3, saturated NaCl, dried over
`Na2SO4 and flash chromatographed (96 mg, 90%).
`The 3'-O-acyl derivative (100 mg, 0.19 mmol) following
`desilylation and flash chromatography afforded compoud [2] (44
`
`

`

`mg, 80%). HRMS mie calculated for C12H15N504: 293.1121,
`observed 293.1107.
`
`2'-Deoxy-5 '-tert-butyldi-
`[3].
`2 '-Deoxy-3 '-0-allyladenosine
`phenylsilyladenosine [9] (200 mg, 0.4 mmol) in benzene (5 mL),
`allyl bromide (484 mg, 4.0 mmol, 10 equiv.), TBAH (40%
`solution, 390 MLL), and 1 M NaOH (5 mL) were stirred at 25°C
`for 15 h. Following ethyl acetate extraction, the organic phase
`was washed with D.I. water, saturated NaCl, dried over
`Na2SO4, and flash chromatographed (157 mg, 74%).
`The O-allyl derivative (198 mg, 0.37 mmol) following
`desilylation and flash chromatography gave compound [3] (106
`mg, 98.5%). HRMS mie calculated for C13H17N503: 291.1328,
`observed 291.1318.
`
`2 '-Deoxy-3 '-O-tetrahydropyranyladenosine [4]. 2'-Deoxy-5'-tert-
`butyldiphenylsilyladenosine [9] (2.90 g, 5.92 mmol), dihydro-
`pyran (4.89 g, 59.2 mmol, 10 equiv.) and pyridinium nitro-
`benzenesulfonate (1.67 g, 5.92 mmol) were dissolved in
`methylene chloride (20 mL) and stirred at 40°C for 20 h. The
`reaction mixture was washed with D.I. water, saturated NaCl,
`dried over Na2SO4 and flash chromatographed to give a
`diastereomeric mixture of 2'-deoxy-3'-O-tetrahydropyranyl-5'-
`tert-butyldiphenylsilyladenosine (0.4 g, 12%).
`The tetrahydropyran derivative formed above, after desilylation
`and flash chromatography yielded compound [4] (147 mg, 84%)
`as a mixture of diastereomers. HRMS m/e calculated for
`C15H21N504: 335.1589, observed 335.1581.
`
`2 '-Deoxy-3 '-O-(2-aminobenzoyl)adenosine-5 '-triphosphate [5].
`This compound was prepared according to the procedure of
`Hiratsuka et al. (20) directly from the 2'-deoxyadenosine-5'-tri-
`phosphate sodium salt.
`2'-Deoxy-3'-O-(4-nitrobenzoyl)adenosine [6]. 2'-Deoxy-5'-tert-
`butyldiphenylsilyladenosine [9] (100 mg, 0.2 mmol), 4-nitro-
`benzoyl chloride (89 mg, 0.48 mmol), DMAP (5 mg, 0.04 mmol)
`were dissolved in pyridine and stirred for 8 h. Following solvent
`removal, the residue was dissolved in chloroform and was washed
`D.I. water, saturated NaHCO3, D.I. water, saturated NaCl,
`dried over Na2SO4, and flash chromatographed giving a
`colorless solid (73 mg, 57%).
`The 4-nitrobenzoyl derivative (66 mg, 0.1 mmol) following
`desilylation and flash chromatography gave compound [6] (22
`mg, 57%). HRMS mie calculated for C17H16N606: 400.1128,
`observed 400.1140.
`
`2'-Deoxy-3'-0-(2-nitrobenzyl)adenosine [7]. 2'-Deoxyadenosine
`(100 mg, 0.4 mmol) [dried by repeated coevaporation with
`pyridine] was dissolved in hot DMF and cooled to 0°C in an
`ice bath. To the above solution, NaH (26 mg, 0.52 mmol [50%
`in mineral oil] in DMF after washing with dry benzene was added
`and stirred for 45 min. 2-Nitrobenzyl bromide (95 mg, 0.44
`mmol) in DMF was added, and the reaction stirred for 3 h. The
`reaction was quenched with cold D.I. water and stirred overnight.
`The solid obtained was filtered, dried, and recrystalized in ethanol
`(122 mg, 79%). HRMS mie calculated for C17H18N605:
`386.1335, No mie was observed. Fast atom bombardment MS,
`nitrobenzyl alcohol (NBA) mle 387.1 (M +1).
`2'-Deoxy-3'-O-methylthymidine [8]. 2'-Deoxy-5'-tert-butyldi-
`phenylsilylthymidine [91 (100 mg, 0.21 mmol) in benzene (5 mL),
`
`Nucleic Acids Research, 1994, Vol. 22, No. 20 4261
`
`methyl iodide (43 mg, 0.3 mmol), TBAH (40% solution, 325
`ML), and 1 M NaOH (5 mL) were vigorously stirred at 25°C
`for 6 h. The organic layer was extracted with ethyl acetate and
`washed with D.I. water, saturated NaCl, and dried over Na2SO4
`(100 mg, 98%).
`The above sample, following desilylation and purification by
`flash chromatography, gave compound [8] (36 mg, 88%). HRMS
`mie calculated for C1IH16N205: 256.1059, observed 256.1082.
`Syntheses of nucleoside 5'-triphosphates
`In general, the 3'-modified nucleoside (1.0 equiv.) was dissolved
`in trimethylphosphate under nitrogen atmosphere. Phosphorus
`oxychloride (POCl3) (3.0 equiv.) was added, and the reaction
`stirred at - 10°C for 4 h. The reaction was quenched with a
`solution of tributylammonium pyrophosphate (5.0 equiv.) in
`DMF and tributylamine (0.2 mL) (21). After stirring vigorously
`for 10 min., the reaction was quenched with 2 mL of 2 M TEAB,
`pH 7.5. The solution was concentrated, and the triphosphate
`derivative was isolated by linear gradient (0.01 M to 0.5 M
`TEAB) using a DEAE cellulose (HCO3- form) column.
`Reverse-phase high performance liquid chromatography
`(RP-HPLC)
`The RP-HPLC hardware system consisted of a Beckman
`controller and model 100A pumps, a Rheodyne model 7125
`injector, an Applied Biosystems (ABI) model 759A absorbance
`detector, and a Spectra-Physics model SP4600 DataJet integrator.
`Gradient RP-HPLC was performed using an ABI aquapore
`OD-300 column (4.6 mmx250 mm) where 'Buffer A' is 100
`Buffer
`mM triethylammonium acetate (TEAA), pH 7.0 and
`B' is 100 mM TEAA, 70 % (v/v) acetonitrile. Compounds
`[1]-[6] and [8] were purified using the following gradient
`conditions: 0% B, 5 min.; 0% B - 40% B, 60 min.; 40% B
`- 100% B, 18 min.; 100% B, 5 min. at a flow rate of 0.5 mL
`per min. Compound [7] was purified using the following gradient
`conditions: 30% B, 5 min.; 30% B - 70% B, 60 min.; 70%
`B -100% B, 15 min.; 100% B, 5 min. The gradient conditions
`used to analyze individual nucleotides were: 0% B, 5 min.; 0%
`B - 40% B, 30 min.; 40% B - 100% B, 18 min.; 100% B,
`5 min.
`
`Polymerases
`Avian Myeloblastosis Virus (AMV) and Moloney Murine
`Leukemia Virus (M-MuLV) reverse transcriptases, Klenow
`fragment of DNA polymerase I, and T4 polynucleotide kinase
`were purchased from Pharmacia. Bst DNA polymerase was
`purchased from Bio-Rad Laboratories. AmpliTaqe DNA
`polymerase and rTth DNA polymerase were purchased from
`Perkin Elmer. Sequenase® was purchased from United States
`Biochemical. VentR®
`(exo-) DNA polymerase was kindly
`provided by New England Biolabs. Pfu (exo-) DNA
`polymerase was purchased from Stratagene.
`DNA templates
`M13mpl9 DNA was obtained from a 250 mL culture by
`polyethylene glycol precipitation and purified by a QIAGEN-tip
`100 column according to the manufacture's protocol. Universal
`primer (5'-TGTAAAACGACGGCCAGT), biotinylated and
`unbiotinylated oligonucleotide template (5 '-TACGGAGGTGG-
`ACTGGCCGTCGTTTTACA) and biotinylated oligonucleotide
`template (5'-TACGGAGGTTTTTGGACTGGCCGTCGTTT-
`
`

`

`4262 Nucleic Acids Research, 1994, Vol. 22, No. 20
`
`ACA) were synthesized using an ABI model 380B DNA
`synthesizer and purified by trityl-on RP-HPLC. All
`nonradioactive nucleotides were purchased from Pharmacia, and
`['y-32P]ATP was purchased from Amersham.
`Polymerase incorporation assays
`Two different template assays were used to test for 3 '-modified
`nucleotide incorporation. In the first, designated the 'M13mpl9-
`template assay', [32P]-labeled universal primer was annealed to
`single-stranded M13mpl9 DNA (0.1 pmol to 0.45 ,ug respect-
`ively, per 5 AL) in the specific enzyme buffer by heating to 80°C
`for 5 min. and cooling slowly to 25°C. The subsequent enzymatic
`extension of the primer-template complex was performed under
`conditions that are analogous to Sanger sequencing, where the
`natural nucleotides were mixed with either a dideoxynucleotide
`or 3'-modified nucleotide terminator to generate a sequencing
`ladder. For the second assay, designated the 'Oligo-template
`[32P]-labeled universal primer was annealed to an
`assay',
`oligonucleotide template (0.05 pmol to 0.1 pmol respectively,
`per 5 AL) in the same fashion. Subsequent extensions were
`performed in the absence of the natural nucleotide when either
`a dideoxynucleotide or 3'-modified nucleotide was tested.
`For each reaction, 5 ,uL aliquots of the annealed primer-
`template samples were dispensed into separate tubes containing
`5 liL mixtures of each enzyme and nucleotides in their specific
`buffers. The final buffer conditions, concentrations of nucleotides,
`enzymatic units, and incubation temperatures are given in Table
`1. The reactions were incubated for 10 min. and then stopped
`by the addition of 5 /sL of stop solution containing 98% D.I.
`
`formamide, 10 mM EDTA, pH 8.0, 0.025% bromophenol blue,
`and 0.025% xylene cyanol. The samples were heated to 85°C
`for 3 min., chilled on ice, and either 4 ,uL (M13mpl9-template
`Assay) or 3 izL (Oligo-template assay) were loaded on a 10%
`or 20% polyacrylamide gel,
`respectively. Following
`electrophoresis, the gel was fixed in an aqueous 10% acetic acid,
`10 % methanolic solution (v/v), dried, and autoradiographed on
`HyperfilmTM-MP (Amersham).
`Biotinylated Oligo-template assay
`The conditions of this assay are similar to the Oligo-template
`assay except prior to primer annealing, 2.0 pmol of biotinylated
`template was captured on 10 mL streptavidin coated magnetic
`M-280) in 1 M NaCl for 15 min.
`beads (Dynal Dynabeads®
`After washing the bound template in the specific enzyme buffer,
`0.1 pmol of [32P]-labeled universal primer was annealed to an
`oligonucleotide template and extension reactions were performed
`as described in the Oligo-template assay.
`
`RESULTS
`Syntheses and purification
`Compounds [1] through [8] were synthesized, desilylated,
`phosphorylated and purified as described above. In general, the
`yields of 3'-protection reactions ranged from 57% to 98% with
`the exception of 3'-O-THP-dATP [4]. This low yield (12%) is
`believed to be due to the acidic properties of the silica gel
`hydrolyzing the linkage to the THP group. For the desilylation
`reactions, the yields ranged from 70% to 98%, except for 3'-O-
`
`Table 1. Specific enzymatic conditions for both the M13mpl9-template and Oligo-template assays
`Bst DNA AmpliTaqe
`Klenow
`AMV-RT
`M-MuLV-RT
`Enzymatic
`SequenaseQD
`polymerase
`fragment
`DNA
`Conditions
`polymerase
`
`Pfu(exo-)
`DNA
`polymerase
`
`rTth DNA
`polymerase
`
`Vent(exo-)G
`DNA
`polymerase
`
`Buffers
`[Tris-HCl]
`[MgC12I
`
`50 mML pH 8.3 50 nmML pH 8.3
`8 mm
`8 mM
`10 mM DTT
`30 mM KCI
`
`10mM, pH 8.5 40nmL pH 7.5 10 mh,pH 8.5 10nML pH 8.5 20 niML pH 8.75
`5mM
`5mM
`10mM
`10omM
`2 mM MgSO4
`50 mM NaC
`50 mM NaCI
`10 mM KCI
`10 mM (NH)2SO4
`0.1% Triton X-100
`0.1 mg/mL BSA
`
`50 mhL pH 8.3 20mM, pH&8
`8mM
`2 ml MgSO4
`10 mM KG
`10rnMDTT
`10 mM (NH4)2SO4
`0.1% Triton X-100
`
`Incubation
`Temperature ('C)
`
`Units
`
`M13mpl9-Templ.
`[dATPi (pM)
`[dCTPI (uM)
`[dGTPl (,M)
`IdTTPl (o)
`[ddATPI (W)
`[ddCTPI (riM)
`[ddGTPI (pM)
`[ddTTPI (&tM
`
`37
`
`1.3
`
`40
`40
`40
`40
`2
`2
`2
`2
`
`37
`
`1.9
`
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`
`37
`
`1.0
`
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`
`37
`
`1.3
`
`10
`10
`10
`20
`0.25
`0.25
`0.25
`0.5
`
`65
`
`0.1
`
`1
`1
`1
`1
`10
`10
`10
`10
`
`68
`
`0.3
`
`2.5
`2.5
`2.5
`2.5
`150
`75
`15
`150
`
`Oligo-T*mpl.
`[dATPJ (MM)
`0.5
`2.5
`0.5
`2.5
`0.025
`0.25
`[dCTPJ (pM)
`0.5
`2.5
`2.5
`0.5
`0.025
`0.25
`[dTTPJ (ps)
`2.5
`2.5
`0.5
`2.5
`0.025
`0.25
`[ddATP] (M)
`250
`2.5
`0.05
`2.5
`0.25
`2.5
`[ddGTPJ (g)
`0.5
`250
`0.05
`2.5
`0.25
`0.25
`[ddTTPl (,)
`5
`5
`0.5
`2.5
`25
`2.5
`N/D means the assay conditions were not determined and I/T means ddNTP termination was incomplete.
`
`75
`
`0.1
`
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`
`2.5
`2.5
`2.5
`I/T
`I/T
`I/T
`
`74
`
`0.3
`
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`
`0.5
`2.5
`0.5
`125
`25
`100
`
`72
`
`0.1
`
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`N/D
`
`0.5
`0.5
`0.5
`125
`25
`100
`
`

`

`(4-nitrobenzoyl)-dATP [6]. The yield was reduced in that case
`to 57% due probably to a rearrangement of the 4-nitrobenzoyl
`group from the 3'-position to the 5'-position (data not shown).
`The phosphorylation yields of compounds [1]- [4] and [6]- [8]
`ranged from 25% to 40%. Thymidine analogs including 3'-O-
`methyl-dTTP [8], however, had to be handled more cautiously
`since they were more rapidly degraded by tributylammonium
`pyrophosphate than were the adenosine analogs.
`The 3'-modified-dNTPs were further purified by RP-HPLC
`to 2 99% prior to the polymerase assay. Each nucleotide
`synthesis initially contained several major peaks that were
`individually tested in the Oligo-template assay to determine the
`active species. In general, the adenosine analogs contained both
`the natural dATP and 3'-modified-dATP.
`
`Termination assays
`A series of polymerases were chosen to test the candidate
`based on their broad template
`terminators,
`3 '-modified
`specificities and their commercial availability. The conditions for
`screening compounds [1]- [8] using each enzyme were first
`defined by a series of control polymerization experiments. For
`the M13mpl9-template assay, a range of dNTP and ddNTP
`concentrations was identified that gave a clear sequencing ladder.
`Each test gel subsequently contained constant dNTP/ddNTP ratios
`in control lanes for three bases, while the concentrations of the
`test compound and its corresponding ddNTP were varied.
`The Oligo-template assay was also standardized before testing
`each 3'-modified-dNTP for termination. The synthetic template
`contained all four bases to allow for the incorporation of the
`remaining natural nucleotides, so that other aspects of the enzyme
`performance could be identified. We found that
`all the
`polymerases misincorporated other dNTPs in the absence of the
`complement dNTP, and this nucleotide readthrough was
`concentration dependent. Thus, minimum dNTP concentrations
`that gave efficient incorporation, but no apparent misincorporation
`were first defined in this assay. These dNTP concentrations were
`then used to determine the minimum ddNTP concentration that
`yielded complete termination.
`(exo-) DNA polymerase was
`excluded from the Oligo-template assay since a ddNTP
`concentration that yielded complete termination for this enzyme
`
`Nucleic Acids Research, 1994, Vol. 22, No. 20 4263
`
`could not be identified. In each of the Oligo-template gels, the
`reactions contained all the required nucleotides except the natural
`nucleotide corresponding to the analog tested. The samples
`routinely used in this assay were a blank control (absence of
`corresponding dNTP or ddNTP), a titration of the corresponding
`ddNTP, a readthrough control (presence of corresponding
`dNTP), and a titration of the corresponding 3'-modified-dNTP.
`
`Terminator screen
`Table 2 summarizes the data from the enzymatic screen of
`compounds [1] - [8]. Three main classes of activity were defined:
`termination, inhibition, and inactive. Termination was apparent
`when the reaction containing the test compound mimicked the
`migration pattern of the ddNTP control. Inhibition was revealed
`when the presence of the test compound prevented the polymerase
`from incorporating the natural nucleotides. No activity was
`recorded when the 3'-modified-dNTPs mimicked either the blank
`or readthrough controls. In addition, a fourth effect related to
`an alteration in enzymatic fidelity is discussed below. Compounds
`[1], [8], and [7] showed specific termination and were further
`evaluated with respect to their concentration dependent effects.
`3'-O-methyl-dATP [1] incorporation
`in Figure 3A shows the
`The M13mpl9-template assay
`incorporation of 3'-O-methyl-dATP [1] by AMV-RT. The
`termination of DNA synthesis by 3'-O-methyl-dATP [1] mimics
`the ddATP controls in a concentration dependent manner,
`although each band appears to migrate slightly slower. From the
`comparison of termination band intensities, it can be estimated
`that 3'-O-methyl-dATP [1] is approximately 200 to 250-fold less
`efficiently incorporated by AMV-RT than ddATP (compare lanes
`6 and 8: 5 mM and 1 mM, respectively). In Figure 3B, the Oligo-
`template gel also shows the incorporation of 3 '-O-methyl-dATP
`[1] by AMV-RT. In addition to termination, some readthrough
`was also observed due to the presence of contaminating dATP.
`All RP-HPLC purified 3'-modified-dATPs (compounds [1]- [7])
`showed approximately 1% dATP contamination, and these trace
`levels could not be removed by subsequent RP-HPLC.
`3'-O-Methyl-dATP [1] was also incorporated by M-MuLV-
`RT and inhibited DNA syntheses by rTth and VentR® (exo-)
`
`Table 2. Activity matrix of RP-HPLC purified 3'-protecting dNTPs challenged against commercially available polymerases
`AmpliTaq@ VentR(exo-)P
`Kienow
`3'-modified-dATP
`%qumase9
`rTth DNA
`Bst DNA
`AMV-RT
`M-MuLV-RT
`(except compound [81)
`polymerase
`polymerase
`fragment
`DNA
`DNA
`polymerase
`polymerase
`
`[11 0-methyl
`[21 0-acyl
`
`[3] 0-allyl
`
`[4] 0-tetrahydropyran
`[5l 0-(4-nitrobenzoyl)
`[6] 0-(2-anmnobenzoyl)
`
`[7] 0-(2-nitrobenzyl)
`[8] 3'-0-methyl-dTTP
`
`Termination Termination*|
`
`|
`
`|
`
`Ihibition
`
`Inhibition*
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`hihibition
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`Inhibition
`
`Termination*
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`hIhibition
`
`Termination Termination* Termination
`
`-
`
`Inhibition
`
`Temiination Termination Termination Termination
`
`All compounds were assayed at a final concentration of 250 AM according to the conditions specified in Table 1. '-' means
`no activity was detected, 'Termination' means that the termination bands mimic ddNTP termination bands, and 'Inhibition'
`means the rate of DNA synthesis is reduced in a nonspecific manner. '*' means the activity was incomplete at a final concentration
`of 250 AM .
`
`

`

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`4264 Nucleic Acids Research, 1994, Vol. 22, No. 20
`
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`obtained by using the AMV-RT M13mpl9-template assay (data
`not shown). However, 3'-O-methyl-dTTP [8] was efficiently
`incorporated by AmpliTaq®
`DNA polymerase in the
`M13mpl9-template assay, and the termination pattern mimicked
`the ddTTP DNA ladders in a concentration dependent manner,
`(Figure 4A). Additional bands, however, were observed in the
`3'-O-methyl-dTTP [8] ladders that were generated by both
`AmpliTaqe DNA polymerase (see arrows) and Bst DNA
`polymerase (data not shown) assays. The position of these
`additional bands corresponded to ddATP termination bands
`suggesting misincorporation of the thymidine analog in place of
`the deoxyadenosine analog. The efficiency of incorporation
`relative to ddTTP was, therefore, not determined in the
`Ml3mp 19-template assays because of the additional bands.
`Figure 4B shows the results of challenging 3'-O-methyl-dTTP
`[8] against four thermostable DNA polymerases in the Oligo-
`template assay. 3'-O-Methyl-dTTP [8] terminates all four
`polymerases in a concentration dependent manner. From the
`comparison of band intensities of 3'-0-methyl-dTTP [8] to ddTTP
`termination products, it can be estimated that 3'-O-methyl-dTTP
`[8] is approximately 50-fold less efficiently incorporated by Bst
`efficiently
`DNA polymerase than ddTTP, 20-fold
`less
`incorporated by AmpliTaqe DNA polymerase than ddTTP,
`2-fold less efficiently incorporated by rTth DNA polymerase than
`ddTTP, and 10-fold less efficiently incorporated by VentR®
`(exo-) DNA polymerase than ddTTP.
`Further investigation of a different nucleotide composition in
`the Oligo-template assay revealed altered base specificity of 3'-0-
`methyl-dTTP [8]. In the absence of dATP, 3'-O-met1Vl-dTTP
`[8] was incorporated by both Bst and AmpliTaq
`DNA
`polymerases, and the termination bands mimicked the ddATP
`controls, (Figure 4C). It is noteworthy that in addition to 3'-O-
`methyl-dTTP [8] incorporation in Figure 4C, significant levels
`of readthrough were observed. Since the Oligo-template assay
`was performed in the absence of a deoxyadenosine analog, the
`readthrough must reflect the misincorporation of dNTPs in the
`noncomplement base position. This result suggests that 3'-O-
`methyl-dTTP [8] alters the base specific properties of DNA
`polymerases, not only in its incorporation, but in the incorporation
`of other natural nucleotides present in the reaction. This result
`also highlights the importance of the precise conditions of
`incorporation assays.
`
`3'-0-(2-nitrobenzyl)-dATP [7] incorporation by Bst DNA
`polymerase
`3'-O-(2-Nitrobenzyl)-dATP [7] terminated Bst DNA synthesis
`in a base specific manner. In the Bst M13mpl9-template assay,
`however, the termination did not correspond to any of the ddNTP
`controls, (Figure SA). This result made it difficult to assign a
`specific mode of action to 3'-O-(2-nitrobenzyl)-dATP [7]. The
`assignment was resolved using the biotinylated Oligo-template
`assay that gave strong evidence for termination. As shown in
`Figure 5B, 3'-O-(2-nitrobenzyl)-dATP [7] terminates Bst DNA
`synthesis where the DNA fragment migrates slower than the
`ddATP control, (compare lanes 3 and 7). 3'-O-(2-Nitrobenzyl)-
`dATP [7] is a UV sensitive compound that undergoes an
`intramolecular rearrangement by irradiation to give dATP and
`nitrosobenzaldehyde (22). Following UV exposure, the DNA
`termination fragment migrated at the same rate as the ddATP
`control, (compare lanes 3 and 8). This observation suggests that
`the 2-nitrobenzyl group can significantly alter the mobility of
`DNA in the gel and may provide a possible reason for the
`
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`
`Incorporation of 3'-O-methyl-dATP by AMV-RT. (A)
`Figure 3.
`Ml3mpl9-template assay: In addition to the conditions specified in Table 1, lane
`1 contained no ddNTPs, and lanes 2-5 contained ddTTP, ddGTP, ddCTP, and
`ddATP, respectively. Lanes 6 and 7 contained 5 /AM and 10 zM of ddATP,
`respectively. Lanes 8-10 contained 1 mM, 5 mM, or 10 mM of 3'-O-methyl-
`dATP, respectively. (B) Conditions for the AMV-RT Oligo-template assay were
`used where lanes 1 and 6 contained no dNTPs or ddNTPs. Lanes 2-5 contained
`dATP, dCTP and ddGTP. In addition, lane 3 contained ddTTP, lane 4 contained
`dTTP, and lane 5 contained 500 itM of 3'-O-methyl-dTTP. Lanes 7-14 contained
`dCTP, dTTP and ddGTP. In addition, lane 8-10 contained 0.1 M, 0.5 ZM,
`and 2.5 ,sM of ddATP; lane 11 contained dATP; and lanes 12-14 contained
`10 ItM, 50 IM, and 250 jAM of 3'-O-methyl-dATP.
`
`DNA polymerases. In the M-MuLV-RT Oligo-template assay,
`we observed a batch-to-batch difference in assaying both 3'-O-
`methyl-dATP [1] and 3'-O-acyl-dATP [2], (data not shown).
`While, the ddNTP controls gave similar results in both M-MuLV-
`RT batch assays, 3'-O-methyl-dATP [1] showed minor
`termination, and both 3'-O-dATP analogs ([1] and [2]) showed
`partial inhibition of DNA synthesis in M-MuLV-RT batch 1. In
`contrast, 3'-O-methyl-dATP [1] showed significant termination
`of DNA synthesis, and both 3'-O-dATP analogs ([1] and [2])
`showed significant readthrough in M-MuLV-RT batch 2. This
`observation illustrates the importance of assaying candidate
`compounds with multiple enzyme batches.
`3'-O-methyl-dTTP [8] incorporation
`Unlike 3'-O-methyl-dATP [1], 3'-0-methyl-dTTP [8] was not
`incorporated by AMV-RT, (Figure 3B). A similar result was
`
`

`

`A.
`
`B.
`
`Bst DNA polymerase
`I4-.--.
`*
`
`AmpliTaqg DNA polymerase
`1 -
`4....
`r.
`r.
`
`234567 9a
`4
`
`..
`
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`
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`
`Termination
`
`0
`
`Z
`
`f'W _
`
`1
`
`2 3 4 5 6
`
`7 8 9
`
`ddll P
`
`3 -0-methyl-dITI
`
`1 2 34 5 6 7 89
`ddTTP
`3 -D-methyl-dTl'
`
`I
`
`VentRr Cexoo) DNA polymerase
`
`rT1h DNA polymerase
`
`a-a_
`
`150
`
`4.s
`
`4.
`
`_
`
`1 2 3 4 5 6 7 8 9
`lT
`l'
`ddTTP
`3-O-methvy-dTTP
`
`1 2 3 4 5 6 7 8 9
`L llt
`l-
`ddTTP
`3'-O-methyl-dTTP
`
`C.
`
`Bst DNA polymnerase
`
`AmpliTaqg DNA polymeras
`
`...Readthrouph
`
`Ternination
`
`b
`
`.4.*
`
`-
`
`_
`681
`
`-3
`
`50
`
`1 2 3 4567 89
`ddA-T-P LT
`3'-D-methyl-dTTP
`
`12 34 56 7 89
`
`ddATP
`3'-O-methyl-dT1P
`
`Figure 4. Incorporation of 3'-O-methyl-dTTP by Bst, AmpliTaq®, rTth, and
`(exo-) DNA polymerases. (A) M13mpl9-template assay (AmpliTaq® ):
`VentR®
`Enzymatic conditions in Table 1 were used where lane 1 contained no ddNTPs,
`and lanes 2 -5 contained ddATP, ddCTP, ddGTP, and ddTTP, respectively. Lanes
`6 and 7 contained 450 ,AM and 750 of /oM ddTTP, respectively. Lanes 8-10
`contained 1 mM, 2.5 mM, or 5 mM of 3'-O-methyl-dTTP, respectively. Arrows
`correspond to termination bands that are observed in the ddATP control (compare
`lanes 2 with 8-10). (B) Conditions for the Oligo-template assay were used for
`Bst, AmpliTaq®, rTth , and VentR®
`(exo-) DNA polymerases. Lane 1
`contained no dNTPs or ddNTPs. Lanes 2-7 contained dATP, dCTP and ddGTP.
`In addition, lanes 3-5 contained (Bst ) 0.1 MM, 0.5 sAM and 2.5 IM ddTTP;
`(AmpliTaq® ) 1.0 MM, 5.0 AM and 25 AM ddTTP; (rth ) and (VentR®
`(exo-))
`4 IAM, 20 MM, and 100 AM ddTTP, respectively; lane 6 contained dTTP; and
`lanes 7-9 contained (Bst ) 4 MM, 20 MM, and 100 AM of 3'-O-methyl-dTTP;
`(exo-)) 20 AM, 100 AM, and 500 AM of
`(Ampl