Facile Preparation of Non-self-quenching Fluorescent DNA Strands with Degree of Labeling up to Theoretic Limit

A new aggregation-induced emission (AIE) compound 1,1-dimethyl-2,5-bis[4-(isothiocyanatemethyl)phenyl]-3,4-diphenylsilole (SITC) was synthesized and used to conjugate with aminoallyl-dUTP. The SITC-dUTP can be incorporated enzymatically into DNA strands with the degree of labeling (DOL) up to the theoretic limit.


Fig. S1.
DNA fragments with different bp numbers prepared by PCR.

Fig. S2.
Fluorescent DNA products with different sequences prepared by PCR method.

Fig. S3.
Plot of quantum yield of the FITC labeled DNA versus Fraction of FITC-dUTP.
Table S1.Sequence of DNA template used in nick translation and random priming.
Table S2.Sequence of DNA templates used in PCR.

Experimental Section
General information.Aminoallyl-dUTP (aa-dUTP), dNTPs, DNase I (RNase-free), DNA Polymerase I, Klenow Fragment exo-, Random Hexamer Primer, Long PCR Enzyme Mix and GeneJET TM PCR Purification Kit, GeneRuler TM 100 bp Plus DNA Ladder(ready-to-use, 100-3000 bp), 6× DNA Loading Dye were purchased from Fermentas (Thermo Scientific).Deep Vent R TM exo-DNA Polymerase was purchased from New England Biolabs (NEB).Synthesized oligonucleotides (primers) were purchased from Tech Drogon Limited.GelRed TM Nucleic Acid Gel Stain (10000× in water) was purchased from Biotium.Other chemicals, reagents and solvents were all purchased from Aldrich or Invitrogen.NMR spectra were taken on a Bruker ARX 400 NMR spectrometer using CDCl 3 as solvent.
HRMS spectra were recorded on a Finnigan TSQ 7000 triple quadrupole spectrometer operating in a MALDI-TOF mode.Photoluminescence (PL) spectra were recorded on a Perkin-Elmer LS 55 spectrofluorometer.UV spectra were measured on a Milton Roy Spectronic 3000 Array spectrophotometer.FL quantum yields (Φ F ) of amorphous powders of SITC was measured using C-701 Time-Resolved Spectrofluorometer with the integrating sphere as accessory.
Preparation of 4-bromobenzylazide (3).Into a flask equipped with a magnetic stirrer were added 4bromobenzyl bromide (7.5 g, 30 mmol), sodium azide (7.8 g, 120 mmol), and 40 mL of DMSO.After stirred at 70 o C for 12 h, the solution was poured into 150 mL of water and extracted with CH 2 Cl 2. The crude product was purified by silica-gel chromatography to give a colorless viscous liquid in 96.2% yield (6.12 g).temperature, 100 mL of 1 M HCl solution was added and the mixture was extracted with DCM.The combined organic layer was washed with brine and water and then dried over magnesium sulfate.

HRMS (MALDI
Gel electrophoresis.The DNA samples with equal volumes were mixed with 6× DNA loading dye and analyzed on 1.5% agarose gel.The running buffer contained 40 mM Tris acetate and 1 mM EDTA in water.All solutions were freshly prepared prior to use.Gel electrophoresis was carried out on a Thermo Scientific horizontal Owl B1A EasyCast Mini Gel system.Separation was performed at 100 V for 30 min.The gels were either prestained with 1×GelRed (10000× in water from Biotium) or poststained with 50 mL 3×GelRed water solution.
Detection of dsDNA.All stained gels were imaged with the Gel Doc XR+ documentation system (Bio-Rad).The images were analyzed by Quantity One gel image analysis software (Bio-Rad).

Fig. S1 .
Fig. S1.DNA fragments with different bp numbers prepared by PCR.(A) DNA fragments prepared by

Fig. S3 .
Fig. S3.Plot of quantum yield of the FITC labeled DNA versus Fraction of FITC-dUTP.The quantum (ESI)  for Chemical Communications This journal is © The Royal Society of Chemistry 2012
Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2012 with 1 μL of d A/G/CTP mixture (10 mM for each of them), 3.5 μL of SITC conjugated dUTP mixture (containing 1 μL of 10 mM conjugated dUTP), 10 μL of 10× DNA polymerase I reaction buffer (Fermentas), 10 μL of diluted DNase I and 4 μL of DNA polymerase I (10 units/ μL) and the adequate water was added to make a final volume of 100 μL.The reaction mixture was incubated at 15 o C for 2 hrs and then purified by GeneJET ™ PCR Purification Kit.General random priming process was carried out as follows: 2 μg of DNA template was denatured at 95 o C for 5 minutes and immediately cooled on ice.The denatured DNA template was on ice added with 1 μL of d A/G/CTP mixture (10 mM for each of them), 3.5 μL of SITC conjugated dUTP mixture (containing 1 μL of 10 mM conjugated dUTP), 10 μL of 10× random priming reaction buffer (Fermentas), 25 μL of random hexamer primer (0.2 μg/ μL), 2 μL of Klenow fragment exo-(5 units/ μL) and then adequate water was added to make a final volume of 100 μL.The reaction mixture was incubated at 37 o C for 4 hrs and then purified by GeneJET ™ PCR -TOF): m/z 556.1462 (M + , calcd 556.1463).Preparation of SITC conjugated dUTP.To a water solution of 5-(3-aminoallyl)-2'-deoxyuridine-5'triphosphate (aa-dUTP, 10 mM, 10 μL) was added SITC (5 mM in DMSO, 25 μL).The mixture was incubated at room temperature for 6 hrs with agitation and used directly without purification.HRMS (MALDI-TOF), m/z: 1077.2860([M+2H] + , calcd 1077.1465).Labeling DNA by PCR.PCR reactions were carried out in a total of 100 μL mix containing 10 μL of 10× Long PCR buffer with 15 mM MgCl 2 (for Long PCR Enzyme Mix, Fermentas) or 10× Thermopol reaction buffer (for Deep Vent R exo-DNA Polymerase, NEB), 2 ng of the template DNA, 0.2 μM of primers, 2 units of Long PCR Enzyme Mix or Deep Vent R ™ exo-DNA Polymerase, and 0.2 mM of each of the dNTPs.In the control reaction, 0.2 mM of all the four normal dNTPs and no SITC-dUTP was used.In other cases, dTTP was substituted with different amounts of SITC-dUTP.

Table S1 .
Sequence of DNA template used in nick translation and random priming.

Table S2 .
Sequence of DNA templates used in PCR.