rationally-designed fluorescent lysine riboswitch probes

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Supplementary information

Rationally-designed fluorescent lysine riboswitch probes

Pradeep Budhathoki, Lina F. Bernal-Perez, Onofrio Annunziata and Youngha Ryu*

Department of Chemistry, Texas Christian University, 2800 S. University Dr., Fort Worth, TX 76129, USA

MATERIALS AND METHODS

General synthetic methods. All commercial chemicals were used without further purification. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian INOVA 400 spectrometer. Chemical shifts are reported in ppm and coupling constants J are reported in Hz. Assignments of 1H resonances were assisted by the COSY spectral data. High resolution mass spectrometry (ESI-HRMS) data were obtained by using the Fourier-transform ion cyclotron resonance (FTICR) operating in tandem with an LTQ linear ion trap mass spectrometer equipped with an electrospray ionization (ESI) source (LTQ-FT, Thermo). Synthesis of LLD. A solution of Boc-Lys(Boc)-OH (0.524 g, 1.51 mmol), N,N-diisopropylethylamine (1 mL, 5.7 mmol) and 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (0.455 g, 1.51 mmol) in DMF (5 mL) was stirred at room temperature for 4 h. To this solution at 0 °C was added dropwise 4,7,10-trioxa-1,13-tridecanediamine (0.331 µL, 1.51 mmol) in DMF (3 mL). The reaction mixture was stirred at room temperature overnight and evaporated to give an oily residue, which was purified by silica-gel column chromatography with 1-4% methanol in chloroform to give LL (0.554 g, 69 %). 1H NMR (300 MHz, CDCl3) 8.17 (bs, 1H), 7.11 (bs, 2H), 5.59 (bd, J=7.2, 1H), 4.88 (bs, 1H), 4.02 (1H, bs), 3.75 (m, 2H), 3.65 (m, 12H), 3.51 (m, 2H), 3.30 (m, 2H), 3.10 (m, 2H), 2.65 (m, 2H), 1.98 (m, 2H), 1.76 (m, 4H), 1.52 (m, 2H), and 1.58-1.30 (m, 22H). MS (ESI) m/z= 549.3 [M+H]+.

A mixture of LL (50 mg, 0.09 mmol), triethylamine (53 µl, 38 mmol) and dansyl chloride (24.3 mg, 0.09 mmol) in CH2Cl2 (0.5 ml) was stirred overnight at room temperature and evaporated. The residue was purified using preparative TLC with 10% methanol in chloroform to give the fully-protected intermediate (35 mg, 50%). A mixture of the intermediate (17 mg, 0.022 mmol) in 1.25 M HCl in methanol (1 mL) was stirred and evaporated to give LLD (10.6 mg, 82%). 1H NMR (400 MHz, D2O) 8.73 (d, J = 8.9, 1H, CArH), 8.44 (d, J= 8.8, 1H, CArH), 8.36 (d, J= 7.4, 1H, CArH), 8.05 (d, J= 8.1, 1H, CArH), 7.89 (dd, J=7.4, 8.9, 1H, CArH), 7.89 (dd, J= 8.1, 8.8, 1H, CArH), 3.92 (t, J= 6.7, 1H, CH), 3.58 (s, 6H, NMe2), 3.51 (t, J= 6.5, 2H, CH2O), 3.30 (m, 12H, 5 CH2O and NCH2), 2.99 (m, 4H, NCH2 and CH2), 1.88 (m, 2H, CH2), 1.76 (m, 2H, CH2), 1.66 (m, 2H, CH2), 1.57 (m, 2H, CH2), and 1.41 (m, 2H, CH2).

13C NMR (100 MHz, D2O) 169.3 (C=O), 139.0, 134.9, 130.5, 128.6, 128.0, 126.7, 126.0, 125.8, 125.7, 119.2 (10 naphtyl C), 69.4, 69.3, 69.2, 68.9, 68.1, 67.4 (6 CH2O), 53.0 (CH), 46.5 (2 CH3), 39.4, 38.8, 36.4 (2 NCH2 and C

), 30.2 (C), 28.2, 28.0 (2 CH2), 26.2 (C and 21.2 (C. The other four aromatic carbons of the NBD group were not shown in the 13C NMR spectrum even

Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2012

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with a good signal to noise ratio after 25,000 scans probably due to very fast quadrupole relaxation caused by neighboring nitrogen atoms. HRMS (ESI) calculated for C28H48N5O6S

+ [M+H]+: 582.3320; found 582.3318.

Synthesis of LLN. A mixture of LL (44 mg, 0.08 mmol), triethylamine (48 µl, 34 mmol) and 4-chloro-7-nitrobenzofurazan (15.9 mg, 0.08 mmol) in CH2Cl2 (0.5 ml) was stirred overnight at room temperature and evaporated. The residue was purified using preparative TLC with 10% methanol in chloroform to give the fully-protected intermediate (17 mg, 30%). A mixture of the intermediate (8 mg, 0.011 mmol) in 1.25 M HCl in methanol (1 mL) was stirred and evaporated to give LLN (5.3 mg, 91%). 1H NMR (400 MHz, D2O) 8.42 (d, J= 9.2, 1H, C6H ), 6.33 (d, J= 9.2, 1H, C5H), 3.91 (t, J= 6.8, 1H, CH ), 3.68-3.58 (m, 12H, 6 CH2O), 3.49 (t, J= 6.4, 2H, NCH2), 3.24 (t, J= 7.0, 2H, NCH2), 2.97 (t, J= 7.6, 2H, CH2), 2.03 (m, 2H, CH2), 1.86 (m, 2H, CH2), 1.76 (m, 2H, CH2), 1.66 (m, 2H, CH2), and 1.41 (m, 2H, CH2).

13C NMR (100 MHz, D2O) 169.7 (C=O), 139.2 (C6), 99.8 (C5), 69.5, 69.5, 69.3, 69.2, 68.1, 68.1 (6 CH2O), 53.0 (CH), 40.7, 38.8, 36.3 (2 NCH2 and C

, 30.4 (C), 28.0, 27.3 (2 CH2), 26.2 (C and 21.2 (C. HRMS (ESI) calculated for C22H38N7O7

+ [M+H]+: 512.2827; found 512.2820. General biological methods. XL1-Blue E. coli cells (Stratagene) were used for cloning and maintaining plasmids. All enzymes and biological reagents were purchased from New England Biolabs unless indicated otherwise. Taq DNA polymerase was used for polymerase chain reaction (PCR). Synthetic oligonucleotides were obtained from Integrated DNA Technologies. Preparation of the E. coli lysC riboswitch DNA. The PCR of the E. coli genomic DNA with the primer pair LF (5’-AGC TGA ATT CTA ATA CGA CTC ACT ATA GGT ACT ACC TGC GCT AGC GC-3’) and LR (5’-CAT GAA GCT TGG ATG GAT CAC CTG GGC ACA AGG GAA GAG CGG-3’) generated the 271-base E. coli lysC riboswitch gene under control of T7 promoter (italic in the primer LF) along with the EcoRI and HindIII restriction sites (underlined in the primers LF and LR, respectively) at the 5’ and 3’-ends, respectively. The PCR product was inserted between the EcoRI and HindIII sites of pUC19 to generate pUC-lysC. The plasmid was verified by sequencing. The linear DNA template for in vitro transcription was prepared by PCR of pUC-lysC using two primers: 5’-TAA TAC GAC TCA CTA TAG GG-3’ and 5’-GGC ACA AGG GAA GAG CGG-3’. Preparation and purification of the lysC riboswitch. The transcription reaction (200 μL) was carried out in a buffer containing 40 mM Tris-HCl (pH 7.9), 2 mM spermidine, 14 mM MgCl2, 10 mM dithiothreitol, 4 mM each of ATP, UTP, GTP and CTP, 160 U RNase inhibitor, 2 U yeast inorganic pyrophosphatase (Sigma), 3.6 g DNA template and 200 U T7 RNA polymerase. The reaction mixture was incubated at 37 °C for 7 h and quenched by the addition of 16 U RNAse-free DNAse I and 80 mM CaCl2. After the incubation at 37 °C for 1h, the reaction was stopped with the addition of 100 mM EDTA. The crude transcript was obtained by ethanol precipitation and purified on 8% preparative denaturing polyacrylamide gel. The RNA band was visualized by UV-shadowing and cut from the gel. The finely minced gel slices were extracted with 6 mL 100 mM sodium acetate (pH 4.5) containing 1 mM EDTA and 0.1% SDS overnight and filtered. The filtrate was concentrated by successive extraction with n-butanol. Ethanol precipitation provided the pure lysC riboswitch (82 g) which was dissolved in DEPC-treated water and stored at -20 °C.


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Fluorescence measurements. The lysC riboswitch RNA was folded in the binding buffer (50 mM HEPES pH 7.5, 20 mM MgCl2, and 100 mM NaCl and 100 mM KCl) by incubating at 90 °C for 1 min, 4 °C for 5 min and 37 °C for 30 min. The molar extinction coefficients () of LLD and LLN were determined by measuring absorbance at different known concentrations of the dye solutions. They were determined to be 200 M-1cm-1 at 328 nm and 2000 M-1cm-1 at 478 nm, respectively. For titration, a stock solution of the folded RNA containing 200 nM dye in the binding buffer were added to a 200 nM dye solution in the binding buffer. Fluorescent intensity measurements were performed on a Shimadzu RF-5301PC fluorescence spectrometer using quartz cuvettes. The emission intensities were recorded in 5 min after each addition. The LLD and LLN samples were excited at 328 nm and 480 nm, respectively, and the emission was monitored at 550 nm for both samples. Determination of dissociation constants. The binding constant between RNA (R) and Dye (D) is defined as

R D⇋R∙D

K R∙DR D

1

R∙D K R D 2

The fluorescence emission intensity of the sample solution is defined by

i αUio αBi∞ αUio 1‐αU i∞ 3 where and are the fraction of the unbound and bound dye, respectively, and and are the fluorescence emission intensity of the unbound and bound dye, respectively. The fraction of the unbound dye is defined by

αUD

R∙D D 4

Equations (2) and (4) give

αU1

1 K R 5

Equations (3) and (5) give

iio

1 K R1 K R

6

where i∞

io , the ratio of the fluorescence intensity in the presence of an infinite concentration

of RNA and in the absence of RNA.


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The initial concentrations and are defined by

R 0 R R∙D 7

L 0 L R∙D 8 Equations (2), (7), and (8) give the free RNA concentrations as

R ‐ K D 0‐K R 0 1 K D 0‐K R 0 1 2 4K R 0

2K 9

Then the following equation is obtained from Equations (6) and (9):

iio

1 0.5 ‐ K D 0‐K R 0 1 K D 0‐K R 0 1 2 4K R 0

1 0.5 ‐ K D 0‐K R 0 1 K D 0‐K R 0 1 2 4K R 0

10

The nonlinear curve fitting by Equation (10) was performed using Origin Pro 8.0 and provided the binding constant K. The dissociation constant Kd is the reciprocal of K.


Fig. S1. (the thiM increasin

Fig. S2. (the thiM increasin

(a) Fluorescriboswitch

ng concentra

(a) Fluorescriboswitch

ng concentra

cence intensRNA. (b) R

ations of the

cence intensRNA. (b) R

ations of the

sity of 200 nRelative fluoe thiM ribow

sity of 200 nRelative fluoe thiM ribow

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nM LLD in orescence inwitch RNA.

nM LLN in orescence inwitch RNA.

the presencntensity of 2

the presencntensity of 2

ce of the incr00 nM LLD

ce of the incr00 nM LLN

reasing conD as a functi

reasing conN as a functi

ncentration oion of

ncentration oion of

of

of


Fig. S3.

Fig. S4.

. 1H NMR o

. 13C NMR o

of LLD

of LLD

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Fig. S5.

Fig. S6.

2012051T: FTMS

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

. ESI-HRMS

. 1H NMR o

14_LLD_FT #1 RTS + p NSI Full ms [15

579

578.8664

S of LLD

of LLN

T: 0.00 AV: 1 NL:50.00-2000.00]

580

579.8573 5

: 5.66E6

581

581.30580.3192

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582m/z

582.331

030

582

582.1706

583

8

583.33542.4654

583.4

583.0629

584

584.33924729 584.57

585 5

585.3333 585779

586

5.8387


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Fig. S7. 13C NMR of LLN

Fig. S8. ESI-HRMS of LLN

20120514_LLN_FT #1 RT: 0.00 AV: 1 NL: 3.79E6T: FTMS + p NSI Full ms [150.00-2000.00]

509 510 511 512 513 514 515m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

512.2820

513.2858

514.2897509.4020 515.1875510.3943 511.2513 513.8478

512.4041

512.1570

512.8543511.8312

508.8508


rationally-designed fluorescent lysine riboswitch probes

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