General Method for Post-Synthetic Modification of Oligonucleotides Based on Oxidative Amination of 4-Thio-2′-deoxyuridine

Jingyi Wang, Jiachen Shang, Yu Xiang, and Aijun Tong*

Article

General Method for Post-Synthetic Modiﬁcation of Oligonucleotides

Based on Oxidative Amination of 4 ‑Thio-2′-deoxyuridine

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sı Supporting Information

ABSTRACT: Functionalized oligonucleotides (ONs) are widely applied as

target binding molecules for biosensing and regulators for gene expression.

Numerous eﬀorts have been focused on developing facile methods for

preparing these useful ONs carrying diverse modiﬁcations. Herein, we

present a general method for postsynthetic modiﬁcation of ONs via oxidative

amination of 4-thio-2′-deoxyuridine (4SdU). 4SdU-containing ON can be

derived by both alkyl and aromatic amines. Using this approach, ONs are

successfully attached with alkyne/azide, biotin and dansylamide moieties, and

these as-prepared ONs possess the expected biorthogonal reactivity,

streptavidin aﬃnity and ﬂuorescent property, respectively. Furthermore, we

also directly install ﬂuorophores to the ON nucleobase based on oxidative

amination of 4SdU, and these ﬂuorophores exhibit distinct luminescence

behaviors before and after conjugation. We believe our method will be a

versatile strategy for constructing various functionalized ONs used in a wide range of nucleic acid applications.

19,20

INTRODUCTION

groups through this approach.

Postsynthetic modiﬁcations

■

utilize the easy-to-obtained ONs bearing reactive handles for

conjugation of desired labels under mild conditions, providing

a simpler, more cost-eﬀective and universal method for

functionalization of ONs compared to solid-phase syn-

Oligonucleotides (ONs) are chemically synthesized short

nucleic acids, which can speciﬁcally recognize DNA or RNA

sequences via complementary Watson−Crick base pairing.

ONs can serve as recognition units for identifying the nucleic

acid analytes with certain sequences through hybridization, and

usually modiﬁed with reporter units to construct the

biosensors for nucleic acid quantiﬁcation and imaging analysis,

19,21

thesis.

Several reactions have been applied for post-

synthetic modiﬁcation of ONs, for instance, condensation of

2,23

24,25

amine with activated esters,

thiol−ene addition,

and inverse electron demand

Although able to produce ONs with

26,27

including real-time PCR,

array,

RNA imaging.

rationally designed as drugs according to the sequences of

target genes and used for treatment of cancer, neuropathy,

musculoskeletal disorder, ophthalmopathy, and other diseases

complementary DNA micro-

azide−alkyne cycloaddition,

6,7

28,29

ﬂuorescence in situ hybridization,

and live-cell

Diels−Alder reaction.

−10

On the other hand, ONs can also be

the required functions, these strategies hardly yield the ONs

with extended pi-system, for example, ﬂuorescent nucleobase-

containing ONs, which usually exhibit unique photophysical

0,31

characteristics.

In recent years, the Suzuki-Miyaura

by speciﬁcally regulating the gene expression. ON-based

reaction has emerged for postsynthetic arylation of ONs,

reacting aryl boronic acids/esters with halogenated nucleo-

2,13

pharmaceuticals, such as antisense ONs

and small

are extensively modiﬁed to improve

nuclease resistance, delivery eﬃciency, and cell targeting

14,15

2,33

interfering RNAs,

bases mediated by palladium.

Using this strategy,

ﬂuorogenic ON conjugates are prepared and found sensitive

6,17

ability.

Consequently, bioconjugation of ONs for desired

to the environment of neighboring bases, serving as probes for

functions is essential to develop analytical tools for nucleic

acids and improve the therapeutic eﬃcacy of ONs-based

medicines.

A variety of functional groups can be introduced into ONs

through either solid-phase synthesis or postsynthetic mod-

iﬁcation. Solid-phase synthesis functionalizes ONs by using

34,35

studying nucleic acid interactions and conformations.

However, the transition metal catalysts used in Suzuki-Miyaura

coupling make it tough for removal of the potentially toxic

Received: January 12, 2021

Revised: March 7, 2021

Published: March 17, 2021

16,18

noncanonical phosphoramidite monomers.

Nevertheless,

the procedures of solid-phase synthesis usually need case-by-

case optimization for incorporating the modiﬁed building

blocks, as well as the harsh conditions like strong acids, alkalis,

and oxidants, make it challenging to modify ONs with labile

2021 American Chemical Society

Bioconjugate Chem. 2021, 32, 721−728

Bioconjugate Chemistry

Article

Scheme 1. General Method for Post-Synthetic Modiﬁcation of 4-Thio-2′-deoxyuridine-Containing Oligonucleotide (4SdU

ON) with NaIO and Functional Amines via Oxidative Amination Reaction

metals from the reaction solution. Therefore, a general

reaction still remain in need for postsynthetic modiﬁcation of

ONs, especially for postsynthetic construction of arylated

nucleobases on ONs in transition metal-free manner.

biorthogonal chemistry, biotin for streptavidin pull-down,

and dansylamide for ﬂuorescent signal generation. We also

created arylated cytosine in ONs attached with two

ﬂuorophores, ﬂuorescein and pyrene, respectively at the

nitrogen of cytosine 4-position, showing diﬀerent ﬂuorescence

intensity changes before and after the derivatization.

In this work, we developed a general method for

postsynthetic modiﬁcation of ONs through oxidative amina-

tion of 4-thio-2′-deoxyuridine (4SdU), converting the 4SdU

on ONs into deoxycytidine (dC) derivates by sodium

periodate (NaIO ) and multiple amines (Scheme 1). We

found both alkyl and aromatic amines could be used to derive

SdU on ONs, thereby allowing a series of functional groups

introduced into ONs. Using this versatile method, we

successfully synthesized ONs carrying alkyne/azide for

RESULTS AND DISCUSSION

■

Design of General Method for Post-Synthetic

Modiﬁcation of 4SdU ON through Oxidative Amination.

The 4-thiouracil is a noncanonical nucleobase replacing the

oxygen at the uracil 4-position with a sulfur, which acts as both

37,38

a naturally occurring base in prokaryotic tRNAs

and

Bioconjugate Chem. 2021, 32, 721−728

Bioconjugate Chemistry

Article

Figure 1. Modiﬁcation of 4SdU ON with alkyl amines through oxidative amination. (a) MALDI-TOF MS analysis of 4SdU ON before (left, 4SdU

ON, calc. 5139.4, found 5139.0) and after (right, TFE-dC ON, calc. 5204.4, found 5204.7) the treatment with NaIO and TFEA. (b) PAGE

analysis of ONs treated with ﬂuorescent alkyne or azide through CuAAC reaction. The gel was imaged without staining (left) and subsequently

stained by SYBR Gold (right). (c) Absorbance at 260 nm of 4SdU ON and Bio-dC ON before and after SA-MB adsorption. (d) Fluorescence

intensity at 515 nm of 10 μM Dan-NH , 10 μM dU ON, and 10 μM 4SdU ON treated with NaIO and Dan-NH . Condition: 90% DMF + 10% 10

mM sodium phosphate buﬀer (pH 7.0), λ_e_x= 343 nm.

39,40

metabolic label for studying RNA dynamics.

The aryl thiol,

which condition has been utilized to derive s U in RNAs.

a resonance form of aromatic thioketone in 4-thiouracil, can be

oxidized to sulfonate intermediates and further substituted by

various nucleophiles such as alcohols, amines, and mercap-

Matrix-assisted laser desorption ionization-time-of-ﬂight mass

spectrometry (MALDI-TOF MS) and high performance liquid

chromatography coupled with ultraviolet detector (HPLC-

UV) analysis revealed the eﬀective amination of 4SdU ON to

give the product, TFE-dC ON with small amount of hydrolysis

byproduct (Figures 1a, S1, and S2 and Table 1). Meanwhile, to

understand the necessary role of 4SdU for the oxidative

amination, we also carried out the experiment under the same

condition using dU ON, and ON with the same sequence as

4SdU ON except for altering the 4SdU with dU. As expected,

no detectable oxidative amination product was observed for

dU ON as characterized by MS (Figure S3 ), suggesting the

TFEA derivatization indeed occur selectively on 4SdU. To

further illustrate the reaction mechanism, we conducted

oxidative amination on the small molecule 4-thiouracil with

1−43

tans.

This chemical reactivity of 4-thiouridine (s U), the

ribonucleosides of 4-thiouracil, has been utilized for the

researches of RNA metabolism through s U labeling and

4,45

recording.

We speculate this reaction can also be adopted

for modiﬁcation of ONs with diverse chemical groups using

the corresponding amines. The deoxyribonucleoside version of

-thiouracil, 4-thio-2′-deoxyuridine (4SdU) can be incorpo-

rated into ONs through a well-developed solid-phase synthesis

method, and 4SdU-containing ONs are commercially available

from vendors.

6,47

Thus, we consider 4SdU may serve as a

reactive handle for postsynthetic modiﬁcation of ONs, and a

series of functional amines can be derived to ONs via oxidative

amination of 4SdU (Scheme 1).

NaIO and TFEA and found the generation of TFE-derived

Modiﬁcation of 4SdU ON with Alkyl Amines through

Oxidative Amination. We initially studied the performance

of oxidative amination of a 4SdU-containing ON (4SdU ON,

cytosine according to nuclear magnetic resonance (NMR)

analysis (Figure S4 ).

TFEA is a relatively weak base and its protonated form has a

low pK value (pK = 5.7), which ensures suﬃcient TFEA

Scheme 1) with 10 mM NaIO and 600 mM triﬂuoroethyl-

amine (TFEA) in 100 mM sodium acetate buﬀer (pH 5.2),

molecules presented as the nonprotonated form in the acetate

Bioconjugate Chem. 2021, 32, 721−728

Bioconjugate Chemistry

dansylamide (Dan) for ﬂuorescent signal generation. A series

Article

Table 1. Yields of Isolated Products Obtained by the

Oxidative Amination Reaction of 4SdU ON with NaIO and

the Amines

of alkyl amine-modiﬁed ONs were obtained as Alk-dC ON,

Az-dC ON, Bio-dC ON, and Dan-dC ON respectively, and

their production were evidenced via MALDI-TOF MS and

HPLC-UV analysis (Figures S7 and S8 and Table 1).

Next, we examined the functions of these alkyl amine-

derived ONs. We ﬁrst performed the copper-catalyzed azide−

alkyne cycloaddition (CuAAC) reaction on 4SdU ON, Alk-dC

ON, and Az-dC ON, each with ﬂuorescent alkyne and azide,

ALKYNE-FLUOR 488 (Alk488) and AZIDE-FLUOR 545

sample name

yields of isolated products

TFE-dC ON

Et-dC ON

Bz-dC ON

Alk-dC ON

Az-dC ON

Bio-dC ON

Dan-dC ON

Ph-dC ON

Fl-dC ON

93%

84%

92%

91%

82%

76%

74%

95%

66%

63%

(

Az545). Polyacrylamide gel electrophoresis (PAGE) analysis

demonstrated click reactions selectively occurred between Alk-

dC ON and Az545 as well as Az-dC ON and Alk488 (Figure

b). MALDI-TOF MS analysis of the CuAAC products,

Az545-Alk-dC ON and Alk488-Az-dC ON, showed no

unreacted Alk-dC ON and Az-dC ON left after reaction with

Az545 and Alk488, suggesting Alk-dC ON and Az-dC ON

were highly active for biorthogonal chemistry (Figure S9). We

next investigated the interaction between Bio-dC ON and

streptavidin magnetic beads (SA-MBs) by UV spectral and

PAGE analysis. Following the user guide, 4SdU ON and Bio-

dC ON were treated with SA-MBs adsorption. Based on the

quantiﬁcation of original and leftover ONs by UV absorbance

at 260 nm, we found approximately 47% of molecules were

adsorbed by SA-MBs from the samples of Bio-dC ON, while

Pyr-dC ON

buﬀer (pH 5.2). Considering the majority of alkyl amines tend

to be protonated in acidic solution, we adjusted the pH

condition to neutral for keeping the alkyl amines in our work

reactive to oxidative amination. First, 100 mM ethylamine (Et-

NH ) and benzylamine (Bz-NH ) were tested to react with

SdU ON and 10 mM NaIO in 50 mM sodium phosphate

buﬀer (pH 7.0) containing 25% volume of dimethylformamide

DMF). The amination products, Et-dC ON and Bz-dC ON,

(

4SdU ON stayed almost untouched (Figures 1c and S10). The

appeared predominantly in MALDI-TOF MS and HPLC-UV

analysis (Figures S5 and S6 and Table 1), indicating oxidative

amination of 4SdU ON could still proceed with common alkyl

amines. Then, we further evaluated oxidative amination of

speciﬁc binding of Bio-dC ON to SA-MBs was further veriﬁed

by disappearance of upper biotinylated ON band in the PAGE

analysis (Figure S11). Collectively, Bio-dC ON prepared by

our method preserved the capacity of biotin for streptavidin-

binding. Then we measured the ﬂuorescence of Dan-dC ON

SdU with alkyl amines carrying useful functional groups,

including alkyne (Alk) and azide (Az) for biorthogonal

chemistry, biotin (Bio) for streptavidin adsorption, and

and compared it with that of Dan-NH . The ﬂuorescence

intensity of 10 μ M Dan-dC ON was at the same order of

Figure 2. Modiﬁcation of 4SdU ON with aromatic amines through oxidative amination. (a) Fluorescence intensity at 520 nm of 1 μM Fl-NH , 10

μM Fl-NH , 1 μM dU ON, and 1 μM 4SdU ON treated with NaIO and Fl-NH . Condition: 100 mM sodium phosphate buﬀer (pH 8.0), λ =

90 nm. (b) Fluorescence intensity at 436 nm of 0.01 μM Pyr-NH , 0.1 μM Pyr-NH , 1 μM Pyr-NH , 10 μM dU ON, and 10 μM 4SdU ON

2 2 2

treated with NaIO and Pyr-NH . Condition: 90% DMF + 10% 10 mM sodium phosphate buﬀer (pH 7.0), λ = 372 nm. (c) The images of amino-

containing ﬂuorophores as well as ONs treated with NaIO and the corresponding ﬂuorophores under white light (top) and UV light (365 nm,

bottom).

Bioconjugate Chem. 2021, 32, 721−728

Bioconjugate Chemistry

Article

magnitude with that of 10 μM Dan-NH , though there was

ﬂuorescence intensity change, we could further conﬁrm the

formation of ﬂuorophore-arylated cytosine in ONs. Therefore,

our method using oxidative amination of 4SdU ON could be a

promising approach for facile creation of arylated nucleobase

in ONs.

about 40% reduction of ﬂuorescence mainly due to

uncomplete ﬂuorescence labeling of ON (Figure 1d). The 10

μM dU ON treated with NaIO and Dan-NH displayed weak

ﬂuorescence, which was not arising from covalent bond

formation between Dan-NH₂and dU ON because there

were no visible Dan-derived ON bands emerged in PAGE

analysis (Figure S12 ). Accordingly, the chemically modiﬁed

ONs prepared by our method reserved the functions of

modiﬁcations, revealing that oxidative amination did not cause

undesired chemical changes to functional groups.

Modiﬁcation of 4SdU ON with Aromatic Amines

through Oxidative Amination. After the achievement in

modifying 4SdU ON with various alkyl amines as shown

above, we set to explore whether the oxidative amination of

CONCLUSIONS

■

In summary, we developed a general method for postsynthetic

modiﬁcation of ONs with a wide range of functional groups

based on oxidative amination of 4SdU. Both alkyl amines and

aromatic amines could be used for modifying 4SdU ON

through our method. The as-prepared ONs, including Alk-dC

ON, Az-dC ON, Bio-dC ON, and Dan-dC ON, reserved the

activities of their conjugated functional moieties for bio-

rthogonal chemistry, streptavidin adsorption and ﬂuorescing

respectively after the oxidative amination. Our postsynthetic

modiﬁcation approach, in particular, could produce the

arylated cytosine in ONs. The obtained ﬂuorophore-arylated

ONs, Fl-dC ON, and Pyr-dC ON exhibited altered

ﬂuorescence properties, compared to the aromatic amine

ﬂuorophores before derivatization. Despite the requirement of

high concentration amines for eﬃcient oxidative amination, we

still expect this method will be a promising strategy to

construct various functionalized ONs for nucleic acid−based

biosensing and therapy.

SdU ON was also compatible with aromatic amines. In

principle, when oxidative amination occurs between aromatic

amine and 4-thiouracil, arylated cytosine may be formed. As an

extended pi-system, arylated cytosine potentially exhibit

unparalleled properties and valuable functions. First, to testify

the tolerance of oxidative amination to aromatic amines, we

applied 250 mM aniline (Ph-NH ) to react with 4SdU ON and

0 mM NaIO in 100 mM sodium acetate buﬀer (pH 5.2)

containing 50% volume of DMF. The eﬃcient production of

Ph-dC ON was validated by MALDI-TOF MS and HPLC-UV

analysis (Figures S13 and S14 and Table 1). Subsequently, we

moved on to select two aromatic amine-containing ﬂuo-

rophores, aminoﬂuorescein (Fl-NH ) and aminopyrene (Pyr-

EXPERIMENTAL SECTION

■

NH ) for 4SdU amination on the ON, and the obtained ON

Oxidative Amination Reaction of Nucleic Acids with

TFEA. The oxidative amination reaction was conducted in an

aqueous solution according to the literature: NaIO (100

mM, 4 μL) was added to a solution of oligonucleotides (200

μM, 10 μL), EDTA (10 mM, 4 μL), TFEA (1.88 μL, 24

μmol), and NaAc-HAc buﬀer (pH 5.2, 200 mM, 20 μL). The

reaction was incubated at 45 °C for 1 h. Then, the

oligonucleotides were puriﬁed using Amicon-3k ultraﬁlter

with water to remove small molecules.

^H^N^M^R^Aⁿ^a^l^y^sⁱ^s^o^f⁴^-^T^hⁱ^o^u^r^a^cⁱ^l^T^r^e^a^t^e^d^wⁱ^t^h^N^a^I^O4

and TFEA. 4-Thiouracil (3.2 mg, 25 μmol) and NaIO (9.1

^m^g^,⁴²^.⁵^μ^m^o^l⁾^w^e^r^e^dⁱ^s^s^o^l^v^e^dⁱⁿ²⁵⁰^μ^L^o^f^D^M^S^O^-^d6

respectively. TFEA (2.6 μL, 33 μmol) was added into 4-

conjugates, Fl-dC ON and Pyr-dC ON were characterized by

MALDI-TOF MS and HPLC-UV analysis (Figures S15 and

S16 and Table 1). The selectivity of oxidative amination for Fl-

NH and Pyr-NH conjugated to 4SdU ON was analyzed by

PAGE, where 4SdU ON derived by Fl-NH and Pyr-NH

migrated slightly slower and faster than 4SdU ON respectively,

while dU ON could be derived by neither Fl-NH nor Pyr-

NH (Figure S12).

Afterward, we investigated the ﬂuorescence properties of Fl-

dC ON and Pyr-dC ON. The solution of 1 μM Fl-dC ON

exhibited 24-fold ﬂuorescence enhancement than 1 μM Fl-

NH in the same buﬀer, while the ﬂuorescence of dU ON after

treatment with NaIO and Fl-NH was negligible (Figure 2a).

thiouracil. Subsequently NaIO was added into 4-thiouracil

On the contrary, 10 μM Pyr-dC ON reduced the ﬂuorescence

and TFEA mixture. The reaction was incubated at 45 °C for 4

to less than 1% of even 1 μM Pyr-NH in the same solvent, and

h, and H NMR spectrum was measured immediately.

dU ON treated with NaIO₄and Pyr-NH₂was almost

nonﬂuorescent (Figure 2b). This phenomenon is attributed

to the diﬀerent mechanisms of derivation-caused ﬂuorescence

intensity change for Fl-NH and Pyr-NH . Fl-NH has a low

Synthesis of Dansyl Ethylenediamine. Dansyl ethyl-

enediamine (Dan-NH ) was prepared according to a published

procedure: Dansyl chloride (Dan-Cl, 487.39 mg, 1.8 mmol)

was dissolved in 20 mL of DCM. The Dan-Cl was added

dropwise to a 50 mL of DCM solution of ethylenediamine (6

mL, 90 mmol) in ice bath conditions. The mixture was stirred

at room temperature for 1 h. The reaction was monitored by

TLC with ethyl acetate vs acetone = 1:1 (V/V). The solution

was acidiﬁed by 150 mL of 1 M HCl. The aqueous phase was

collected and basiﬁed by 30 mL of 5 M NaOH. The product

was extracted by 100 mL of DCM. The organic phase was

quantum yield due to intramolecular excited-state electron

transfer from the amino-substituted phenyl to xanthene ring.

Fl-NH recovers the strong luminous emission of ﬂuorescein

when its primary amine is made unavailable by covalent

labeling or electrostatic interaction. On the other hand, Pyr-

NH itself is a high quantum yield ﬂuorophore in organic

solvents. When the amine of Pyr-NH is attached with an

electron-drawing group, its ﬂuorescence will be quenched

resulting from photoinduced electron transfer (PET) eﬀect.

dried over Na

obtained as a gray-white solid (461.3 mg, yield 87.1%). H

NMR (400 MHz, CDCl ) δ 8.51 (d, 1H), 8.29 (d, 1H), 8.22

(d, 1H), 7.53 (dd, 1H), 7.49 (dd, 1H), 7.15 (d, 1H), 2.88 (dd,

SO and evaporated to dryness. Dan-NH was

In comparison to the ON modiﬁed with alkyl amine-

containing ﬂuorophore like Dan-NH retaining its intrinsic

emission characteristic, conjugation of aromatic amine-

containing ﬂuorophores directly to the heterocycle of ON

nucleobase could signiﬁcantly aﬀect their luminescent proper-

ties (Figure 2c). Based on these derivatization-caused

2H), 2.86 (s, 6H), 2.67 (dd, 2H). C NMR (101 MHz,

DMSO-d ) δ 151.89, 136.61, 129.88, 129.62, 129.59, 128.82,

128.34, 124.12, 119.61, 115.63, 46.65, 45.58, 42.01. ESI: calc.

Bioconjugate Chem. 2021, 32, 721−728

Bioconjugate Chemistry

The Supporting Information is available free of charge at

Article

for C H O N S, [M + H] 294.12762, found 294.12695; [M-

was 1.0 mL/min. The gradient elution procedure was as

follows: 0 min 100% eluent A, 35 min 70% eluent A + 30%

eluent B, 40 min 100% acetonitrile, 45 min 100% acetonitrile,

50 min 100% eluent A, and 60 min 100% eluent A. The yields

of reactions were quantiﬁed according to the peak area ratio of

the isolated product to the reactant, and these peak areas were

normalized by that of inert impurity dC ON as an internal

standard. The isolated products were collected and dried in

vacuo and subsequently identiﬁed by MALDI-TOF MS

analysis.

−

H] 292.11197, found 292.11230.

Oxidative Amination Reaction of Nucleic Acids with

Alkyl Amines. NaIO (100 mM, 4 μL) was added to a

solution of oligonucleotides (250 μM, 2 μL), EDTA (10 mM,

μL), alkyl amines dissolved in DMF (400 mM, 10 μL),

sodium phosphate buﬀer (pH 7.0, 200 mM, 10 μL), and 10 μL

of H O. For the reaction with Dan-NH , the proportion of

DMF was adjusted to 50%. The reaction was incubated at

room temperature for 3 h. Then, the oligonucleotides were

puriﬁed using Amicon-3k ultraﬁlter with water.

Click Chemistry of Nucleic Acids Modiﬁed with

Alkyne or Azide. The CuAAC reaction was carried out

following the reported protocol: Sodium ascorbate (150

ASSOCIATED CONTENT

■

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acs.bioconjchem.1c00016.

mM, 10 μL) was added to a solution of oligonucleotides (25

μM, 20 μL), CuSO (20 mM, 5 μL), THPTA (50 mM, 10

μL), Alk488 or Az545 (1 mM, 5 μL), and sodium phosphate

buﬀer (pH 7.0, 200 mM, 50 μL). The reaction was incubated

at room temperature for 2 h and stopped by adding 10 μL of

Material and instrumentation, MALDI-TOF MS anal-

ysis, NMR analysis, absorption measurements, PAGE

analysis (PDF)

00 mM EDTA. Then, the oligonucleotides were puriﬁed

using Amicon-3k ultraﬁlter with water.

PAGE Analysis of Nucleic Acids. The samples for

denaturing PAGE analysis were prepared by mixing 10 pmol

of nucleic acids, with equal volume of 8 M urea and 1/10

volume of DNA loading buﬀer. The samples were loaded on

AUTHOR INFORMATION

■

Corresponding Author

Aijun Tong − Department of Chemistry, Beijing Key

Laboratory for Microanalytical Methods and

Instrumentation, Key Laboratory of Bioorganic Phosphorus

Chemistry and Chemical Biology (Ministry of Education),

Tsinghua University, Beijing 100084, China; orcid.org/

0% denaturing PAGE and run for 2 h at 3 W. Green

ﬂuorescence of nucleic acids was observed by imaging using

SYBR Green mode. Afterward, the gels were stained by SYBR

Gold in 1 × TBE for 8 min and imaged using SYBR Gold

mode.

0000-0003-0633-4098 ; Email: tongaj@

mail.tsinghua.edu.cn

Binding of Biotin-Derived Nucleic Acids by SA-MBs.

SA-MBs (4 mg/mL, 300 μL) was added into a 1.5 mL of

microcentrifuge tube. Apply magnet to side of tube for 30 s.

The supernatant was removed and discarded. The beads were

washed with 600 μL of binding buﬀer (containing 20 mM Tris-

HCl at pH 7.5, 1 mM EDTA and 0.5 M NaCl) for three times.

Then 100 μL of 2 μM biotin-derived nucleic acids was added

into beads. The beads were vortexed and incubated at room

temperature for 30 min. A magnet was applied and then the

supernatant was transferred to a clean microcentrifuge tube.

Oxidative Amination Reaction of Nucleic Acids with

Authors

Jingyi Wang − Department of Chemistry, Beijing Key

Laboratory for Microanalytical Methods and

Instrumentation, Key Laboratory of Bioorganic Phosphorus

Chemistry and Chemical Biology (Ministry of Education),

Tsinghua University, Beijing 100084, China

Jiachen Shang − Department of Chemistry, Beijing Key

Laboratory for Microanalytical Methods and

Instrumentation, Key Laboratory of Bioorganic Phosphorus

Chemistry and Chemical Biology (Ministry of Education),

Tsinghua University, Beijing 100084, China

Yu Xiang − Department of Chemistry, Beijing Key Laboratory

for Microanalytical Methods and Instrumentation, Key

Laboratory of Bioorganic Phosphorus Chemistry and

Chemical Biology (Ministry of Education), Tsinghua

University, Beijing 100084, China

Aromatic Amines. NaIO (100 mM, 4 μL) was added to a

solution of oligonucleotides (250 μM, 2 μL), EDTA (10 mM,

μL), aromatic amines dissolved in DMF (500 mM, 20 μL),

and NaAc-HAc buﬀer (pH 5.2, 200 mM, 10 μL). The reaction

was incubated at 45 °C for 3 h. Then, the oligonucleotides

were puriﬁed by ethanol precipitation or n-butanol extraction

and subsequently washed using Amicon-3k ultraﬁlter with

water.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.bioconjchem.1c00016

Fluorescence Measurement of Fluorophore-Derived

Nucleic Acids. The concentration of nucleic acids was

determined by its absorbance at 260 nm. The 200 μL of a

solution of nucleic acids was used for measurement of

ﬂuorescence spectra. The bandwidth of excitation and

emission was set at 5 nm.

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

HPLC-UV Analysis of Nucleic Acids for Quantiﬁcation

of Oxidative Amination Reaction. The reactant or product

nucleic acids were analyzed on a HPLC system equipped with

a UV detector. A C18 column (5 μM particle size, 120 Å pore

size, 250 mm length, 4.6 mm inner diameter) was used at 35

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

C with 50 mM triethylammonium acetate (TEAA) buﬀer

We are very grateful for the ﬁnancial support from the National

Natural Science Foundation of China (Nos. 21775084,

21974076, and 21621003).

(

pH 7.0)-acetonitrile (93:7) as eluent A and 50 mM TEAA

buﬀer (pH 7.0)-acetonitrile (50:50) as eluent B. The ﬂow rate

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Bioconjugate Chemistry

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