Synthesis and Structural Characterization of 2′-Deoxy-2′-fluoro-l-uridine Nucleic Acids

ﬂ uoro ‑L ‑uridine Nucleic Acids

Letter

Synthesis and Structural Characterization of 2′-Deoxy-2′-

Yuliya Dantsu, Ying Zhang, and Wen Zhang*

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ABSTRACT: Despite the development of artiﬁcial L-RNA/DNA

as therapeutic molecules, the in-depth investigation on their

chemical modiﬁcations is still limited. Here, we synthesize a

chemically derivatized 2′-deoxy-2′-ﬂuoro-L-uridine building block

and incorporate it into oligonucleotides. Our thermo-denaturiza-

tion and enzymatic digestion experiments reveal their superior

stability. Furthermore, one crystal structure of L-type ﬂuoro-DNA

is determined to characterize its handedness. Our results reveal the

increase of L-helix stability by ﬂuoro-modiﬁcation and provide the

foundation for its future functional application.

ucleic acid ligands are able to fold into three-dimensional

stable signal ampliﬁer, the design of antisense L-/D-

structures with high aﬃnities for a variety of molecular

oligodeoxynucleotide chimeras, and the L-DNA containing

nanotechnology for material science and drug delivery.

targets, from small ions to entire organisms. The development

of Systematic Evolution of Ligands by EXponential enrichment

Despite the enhancement in stability, it is still critical to

further explore the chemical optimization of L-nucleic acids in

order to expand their functionalities. It is evident that chemical

modiﬁcations on D-nucleic acid aptamer can considerably

improve thermal and structural stabilities (in certain regions)

and oﬀer additional physicochemical diversity. However,

compared to the D-aptamer, much less chemical diversity has

been pioneered in L-aptamer SELEX. Indeed, the inadequacy

of chemically modiﬁed L-nucleic acid is a bottleneck that

restricts the success rate of selection. To date, the reported

example includes the utilization of L-5-aminoallyl-uridine,

which has the chemical moiety at the 5-position of uridine

(

SELEX) has enabled the isolation of functional oligonucleo-

tides from combinatorial libraries through in vitro selection

methods. As a result of feasibility with DNA and RNA

libraries, aptamers are becoming attractive advanced tools for

use in disease diagnostic and therapeutic application. Never-

theless, one of the seemingly intractable challenges of aptamer

drugs is their short half-life in vivo. For instance, the ubiquitous

nucleases in human body can cause the rapid degradation of

natural RNA/DNA molecules, which limits their practicability

in pharmaceutical application. To date, tremendous eﬀorts

have been devoted targeting nucleic acid structures to

engender the stable candidates in biological ﬂuids. Some

that plays a critical role for molecular interaction. Besides,

the ﬂuoro-modiﬁcation has also been successfully introduced

nuclease-resistant oligonucleotides have been successfully

isolated by selection from the modiﬁed libraries or post-

modiﬁcation of aptamers.

into the 2′-position of L-deoxyribose of pyrimidine, but there

is a lack of detailed thermodynamic and structural character-

izations. On the other hand, the native D-type 2′-deoxy-2′-

ﬂuoro-ribonucleotides have wide beneﬁcial applications,

because of not only the nuclease resistance in vivo but also

the enthalpy-based stability enhancement as a result of 2′-F

induced strengthening of H-bonding and stacking interac-

One approach has been adapted to potently optimize nucleic

acid therapies by utilizing the DNA/RNA molecules with

radically diﬀerent chirality from their native counterparts. The

principle is bolstered by the observation that the L-type

enantio-nucleic acid ligands display the identical physical,

chemical, and structural properties in terms of solubility and

structural stability as D-nucleic acids. Furthermore, due to the

chiral inversion, the L-nucleic acids are unrecognizable by any

nucleases in plasma, resulting in the greatly extended half-life in

tions. The electron-withdrawing ﬂuoro-derivatization at the

′-position of L-deoxyribose should also restrain the sugar

Received: May 1, 2021

Published: June 18, 2021

vivo. There have been various applications of L-DNA/RNA in

nucleic acid therapeutics and diagnostics, including the L-

aptamers with distinct three-dimensional structures and

excellent binding aﬃnities to molecular targets, the microarray

platform involving design of probes with L-DNA sequence

stems, the design of a catalytic L-DNA hairpin assembly as the

2021 American Chemical Society

007

Org. Lett. 2021, 23, 5007−5011

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Letter

predominantly to L-type C3′-endo conformation, thereby

enhancing the helical structural and thermal stability of L-

duplex. Herein, we employed the chemical synthesis of L-type

(DCI). Furthermore, DCI, as an activator, also promotes a

coupling step in the solid-phase synthesis of oligonucleotides.

Compared to DNA, the ﬂuoro-functionality at the 2′-

position of our synthetic nucleoside phosphoramidite may

cause the steric hindrance eﬀect during the coupling step of

solid-phase synthesis. Consequently, an extended coupling

′-deoxy-2′-F-uridine building block and its oligonucleotides,

and further carried out comprehensive studies to reveal more

insights into structural and functional properties.

The idea of incorporation of a relatively small ﬂuorine atom

into a carbohydrate moiety of nucleosides relies on its ability to

mimic the features of the hydroxyl group (similar van der

Waals radii and polarity) and act as an acceptor by creating

hydrogen bonds. Investigations of ﬂuoro-modiﬁed nucleotides

revealed better binding aﬃnity to targets and nucleases

resistance. For instance, the replacement of 2′- and 3′-hydroxyl

groups by ﬂuorine lead to nucleosides with potential antiviral

eﬀect, while the presence of two geminal ﬂuorine atoms at C-2′

time of 180 s was applied in each L-U coupling. To avoid the

unintended ﬂuoride degradation, we chose the concentrated

ammonium hydroxide solution, instead of AMA solution, for

the nucleobase deprotection. The modiﬁed L-U monomer was

successfully incorporated into the oligonucleotides at quanti-

tative yield.

In the practical application, some chemical modiﬁcations

have been introduced to the aptamers in order to reduce the

conformational ﬂexibility and increase the thermal stability.

position resulted in chemotherapy medication Gemcitabine.

In order to explore the potential stabilizing eﬀect caused by the

2′-F moiety, we performed the thermal denaturation studies to

measure the melting temperatures of various L-type duplexes.

In this work, we attempted to chemically modify the nucleic

acid molecules by both converting the handedness to L-type

and introducing an additional 2′-F-substituent to the back-

bone.

The Sybr green I ﬂuorescent dye was used in T measurement,

which could nonspeciﬁcally bind to double-stranded, not

The organic synthesis of the target phosphoramidite started

from the commercially available L-type 2,2′-anhydrouridine 1,

followed by protecting the 3′ and 5′ positions by a

single-stranded, nucleic acid to induce ﬂuorescent signal.

We ﬁrst decided to examine the eﬀect of L-2′-F

derivatization in L-DNA duplexes. The melting temperature

of L-DNA duplex 5′-ATGGTGCTC-3′/5′-GAGCACCAT-3′

tetrahydropyranyl group (Scheme 1), as described earlier.

(

entry a, Table 1) was measured to be 49.7 °C. It is worth

Scheme 1. Synthesis of L-Type 2′-Deoxy-2′-ﬂuorouridine

Phosphoramidite (6) and Oligonucleotide (7)

Table 1. MS Analysis and T Measurement of Synthetic L-

Oligonucleotides (n.d. = Not Determined)

isotopic mass m/z

duplex T_m

(°C)

entry

sequences

measured (calcd)

L-5′-dATGGTGCTC-3′

[M−2H]²⁻: 1363.3

49.7 ± 0.4

43.2 ± 0.2

41.8 ± 0.3

44.9 ± 0.5

43.7 ± 0.6

48.5 ± 0.5

49.3 ± 0.2

49.9 ± 0.7

52.4 ± 0.3

n.d.

(

1363.2)

L-5′-dATGGdUGCTC-3′

L-5′-dATGGdUGCdUC-3′

[M−2H]²⁻: 1356.7

(

1356.7)

[M−2H]²⁻: 1349.7

(

1349.7)

L-5′-dATGGU GCTC-3′

[M−2H]²⁻:1365.2

(

1365.2)

L-5′-dATGGU GCU C-3′

[M−2H]²⁻: 1367.2

(

1367.2)

L-5′-rGCAAAUUUGC-3′

[M−2H]²⁻: 1570.8

(

1570.7)

L-5′-rGCAAAUUU GC-3′

[M−2H]²⁻: 1571.7

Subsequent hydrolysis reaction and ﬂuorination with DAST

reagent gave rise to the compound 3 in 64% overall yield. The

deprotection of 2′-deoxy-2′-ﬂuoro-3′,5′-di-O-tetrahydropyran-

yl-β-L-uridine could be realized by treatment with either p-

(

1571.7)

L-5′-rGCAAAUU U GC-3′ [M−3H]3−_:₁₀₄₇_.₉

(1048.1)

L-5′-dGCAAATTTGC-3′

L-5′-dGTGTACAC-3′

[M−3H]³⁻: 1007.3

(

1007.5)

TsOH in MeOH at room temperature for 3 h or with

[M−2H]²⁻: 1202.8

Amberlite (H ) in aqueous methanol overnight. However,

we found that the ﬁrst method generated traces of p-

toluenesulfonic acid that might catalyze detritylation reaction

and reduce the yield of the target compound. In this work, we

(

1203.2)

L-5′-dGU GTACAC-3′

[M−2H]²⁻: 1205.1

n.d.

(

1205.1)

chose to deprotect the compound 3 under Amberlite (H )

resin in aqueous methanol in 83% overall yield. The tritylation

reaction was carried out by treating substance 4 with 4,4′-

dimethoxytrityl chloride in dry pyridine. The key phosphor-

amidite 6 could be synthesized by applying two commonly

used phosphitylation reagents, such as 2-cyanoethyl N,N-

diisopropylchlorophosphoramidite (PCl) and 2-cyanoethyl

N,N,N′,N′-tetraisopropylphosphorodiamidite (PN2). How-

ever, by treating substance 5 with PCl, the phosphitylation

reaction could only be successfully accomplished for a small

scale (up to 200 mg). The target compound 6 was ﬁnally

synthesized in high yield in a larger scale in the presence of

PN2 and activating reagent such as 4,5-dicyanoimidazole

noting that our synthetic 2′-F-dU monomer is lacking a methyl

group at the 5-position compared to L-thymidine, and it is

known that the C5 group can help to stabilize the DNA helical

conformation. Therefore, to have the comprehensive

comparison, we synthesized two additional L-DNA duplexes,

which contained L-deoxyuridine residues to replace L-

thymidines at the corresponding positions (entries b and c).

When one L-T was substituted by L-dU, the melting

temperature was dropped to 43.2 °C; moreover, when two L-

dUs were present at the same duplex, the T_mwas further

decreased to 41.8 °C. This result fully revealed the importance

of 5-methyl in the thymidine base when stabilizing the L-DNA

008

Org. Lett. 2021, 23, 5007−5011

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Letter

Figure 1. Crystal structures of ﬂuoro-modiﬁed L-DNA. A: Crystals of ﬂuoro-modiﬁed -DNA octamer. B: Stereoview of ﬂuoro-L-DNA 8-mer (left,

resolution = 1.03 Å) and its mirror-image D-DNA 8-mer (right, PDB 1d79). C: Superimposed structures of ﬂuoro-modiﬁed L-DNA (green) and the

mirror reﬂection of D-DNA structure (purple). D: Modiﬁed L-U :L-A Watson−Crick base pair. Gray mesh indicates the corresponding 2F −F maps

contoured at 1.0 σ. E: 2′-deoxy-2′-F-L-uridine in the 3′-endo-conformation.

double helix. When we replaced one L-deoxyuridine by L-2′-F-

(oligo f: L-5′-rGCAAAUUUGC-3′, oligo g: L-5′-

′-deoxyuridine (entry d), the T was enhanced by 1.7 °C, to

rGCAAAUUU GC-3′, oligo f′: D-5′-rGCAAAUUUGC-3′,

4.9 °C. Besides, when 2 ﬂuoro-modiﬁed residues were

oligo g′: D-5′-rGCAAAUUU GC-3′). The RNA samples

introduced to replace L-deoxyuridines (entry e), the T_m

could be improved by 1.9 °C, to 43.7 °C. These melting

studies lend evidence that bringing in ﬂuoro-modiﬁcation to

the 2′-position could give rise to a more stable L-DNA duplex,

although the 2′-exo-F conformationally disagrees with the 2′-

endo conformation in canonical L-deoxyribose. Even so, the 5-

methyl group of L-thymidine has a more consequential impact

on the stability of L-DNA.

were incubated with 0.5% human serum at 37 °C, followed

by denaturing PAGE analysis (Figures S22 and S23). It was

observed that the native oligo f′ was completely digested

within 1 h without any intact strand detectable. Even after

modiﬁcation with one 2′-F-Uridine residue, the D-RNA g′ only

displayed slightly improved stability and could not survive in

serum longer than 2 h. In contrast, the L-RNA f and g, either

wild-type or modiﬁed by a ﬂuoride group, remained intact after

24 h of incubation with serum, without any fragmental

cleavage product observed. It reveals that both ﬂuoro-modiﬁed

and nonmodiﬁed L- nucleic acids have dramatically enhanced

biological stability in physiological environments, which is

much stronger than the traditional 2′-F modiﬁcation in gene

therapy.

We then performed the circular dichroism studies to

characterize the chiralities of our synthetic oligonucleotides.

Two sets of ﬂuoro-modiﬁed duplexes, including both L- and D-

formed oligonucleotides (DNA d and d′ and RNA g and g ′),

were tested. The corresponding spectra are shown in Figure

S24 . The data revealed that the D-form 2′-F-modiﬁed DNA

molecule adopted a B-form-like conformation, which provided

a conservative CD spectrum with the following amplitude

bands: a high positive band around 278 nm and a short

negative one around 238 nm. In contrast, its mirror-image

counterpart showed the chiral inversion (high negative around

278 nm and short positive around 238 nm, Figure S24A). For

the native 2′-F-modiﬁed RNA, the CD spectrum was

characterized as the A-form-like duplex, by a high positive

band at 266 nm, a negative one at 236 nm, and a short positive

band at 224 nm. Its chiral inversion was displayed when the

modiﬁed L-RNA oligonucleotide was assessed (Figure S24B).

The CD characterization illustrates the synthetic ﬂuoro-

modiﬁed L-DNA and L-RNA adopt the mirror-image helical

conformations of their D-form ﬂuoro-counterparts.

On the other hand, after incorporating the 2′-F into the L-

RNA backbone, we observed slightly enhanced thermal

stability_.The self-complementary L -RNA 5′-

GCAAAUUUGC-3′ (entry f) displayed a T of 48.5 °C,

while the introduction of 2′-F moieties increased the T by 0.8

and 1.4 °C, respectively (entries g and h). Considering that the

modiﬁcations existed pairwise in the self-complementary

duplexes, this ﬁnding indicates that the small ﬂuorine atoms

caused a mild T_mincrease (∼0.4 °C per modiﬁcation),

probably due to the conformational rigidity to the L-type A-

form. Our results are consistent with the possibility that the 2′-

F group could, due to enthalpy, stabilize the native RNA

structures. When the entire oligonucleotide was converted to

L-DNA with the same sequence (entry i), the melting

temperature was measured to be 52.4 °C, much higher than

the duplexes containing the same sequence but all the ribo-

residues. We expect that this striking boost is engendered by

the three consecutive L-thymidines in the oligo, which totally

leads to six 5-methyl groups in the double helix. For

comparison, we also measured the thermostability of a set of

′-F modiﬁed D-RNA (Table S1). The conclusion is similar to

our observation regarding L-oligonucleotides, that one ﬂuoro-

modiﬁcation could enhance the T by ∼0.4 °C. Interestingly,

the overall T values of D-RNAs are lower than the mirror-

image L-counterparts, and the reason might be the asym-

metrical nature of the intercalating SYBR green dye, which

causes the diﬀerent binding patterns between D- and L- forms.

Susceptibility to nuclease degradation is a signiﬁcant

limitation to the design and utilization of aptamer drugs in

vivo. Here we examined the stability of 2′-ﬂuoro-modiﬁed L-

oligonucleotides in human serum solution. We used four

diﬀerent RNA oligonucleotides, which shared the same

sequence, but possessed diﬀerent chirality and modiﬁcation

In order to investigate the structural features of the 2′-ﬂuoro

moiety on the L-ribose backbone, we then attempted to

crystallize a self-complementary L-DNA 8-mer, 5′-

GU GTACAC-3′, by screening 384 commercial conditions

at 18 °C. Subsequently, we performed optimization of

promising crystal growth, which included amplifying the

crystallization drop and adjusting the concentrations of

009

Org. Lett. 2021, 23, 5007−5011

Organic Letters

The Supporting Information is available free of charge at

Letter

precipitant and DNA. The best crystal obtained was a rod-

shaped specimen (Figure 1A) grown under 0.08 M NaCl, 0.02

regular Watson−Crick base pairs, including the modiﬁed L-

type U :A pair (Figure 1D). These data indicate that the

M MgCl , 0.04 M sodium cacodylate trihydrate pH 5.5, 35%

ﬂuoro-modiﬁcation does not generate local and overall

structural perturbation to the left-handed duplex. Moreover,

the high-resolution reveals that the 2′-F-ribose displays the L-

type C3′-endo pucker conformation (Figure 1E), consistent

with the native D-type 2′-F nucleic acid structure.

(

v/v) MPD, 0.002 M hexammine cobalt(III) chloride.

Notably, the crystal showed a strong X-ray diﬀraction to 1.03

Å during data collection. Due to the limited number of

published L-nucleic acid crystal structures as a model, we ﬁrst

intended to solve this novel ﬂuoro-modiﬁed L-DNA structure

by Single-wavelength Anomalous Diﬀraction (SAD). After

soaking the optimal crystals in the iridium(III) hexamine

chloride solution, although the strong iridium anomalous

signal was observed during data collection and data processing,

our attempts to search for Ir³heavy atoms with high

occupancy and build helical model failed.

In summary, we have synthesized the L-type uridine

monomer containing 2′-F-modiﬁcation, as the analogue of L-

uridine and mirror reﬂection of native 2′-F-uridine. Sub-

sequently, we successfully incorporated the residue into L-DNA

and L-RNA in quantitative yield. Our systematic thermal study

and enzymatic assessment reveal that the ﬂuoro-L-oligonucleo-

tides have notable stability. The CD experiment results validate

that the ﬂuoro-modiﬁed L-type DNA and RNA adopt the left-

handed helical conformations, which present the mirror-image

characteristics of their D-type counterparts. Furthermore, our

We then turned to the data collected from the unsoaked

crystal and performed molecular replacement. We utilized a

published D-DNA structure containing the same sequence

(

PDB 1d79 ) and transformed the coordinates into its mirror-

X-ray crystal structure of oligo L-5′-GU GTACAC-3′ is the

image reﬂection across the x axis. Using the created L-DNA as

the search model, we successfully obtained the solution and

determined the structure (PDB 7KW4) using Phaser in the

ﬁrst structure of chemically modiﬁed L-nucleic acid. The

atomic level resolution allows us to examine the structural

insights into the modiﬁed A-form L-DNA. Indeed, the modiﬁed

L-DNA presents great structural stability, as evidenced by the

overall and local parameters in the L-helix. Although the L-type

2′-F-uridine monomer and its oligonucleotide have been

CCP4 suite. The space group was assigned as hexagonal

P6 22, with one single strand in an asymmetric unit. After

establishing the structure, we re-examined the data from the

crystal soaked with Ir . We successfully solved the structure by

molecular replacement, but could not assign any obvious

pioneered before, our study here for the ﬁrst time

comprehensively demonstrates its superiority and provides

the structural foundation. Therefore, these ﬁndings will

provide the theoretical framework for the L-nucleic acid

therapy design where thermostability is important, including

the L-aptamer for disease treatment, L-nanoparticle for drug

delivery, and L-molecular beacon for diagnosis.

electron density belonging to Ir . It seemed that the observed

intense anomalous signal was either from the disordered heavy

atoms, which weakly and nonspeciﬁcally bound to L-DNA, or

from the Ir only adhering to the surface of the crystal. The

possible reasons for our failure likely include the compact

molecular packing inside crystal to inhibit molecular diﬀusion

or the hexamine iridium(III) chloride only binding to weak

G:U wobble pair.

ASSOCIATED CONTENT

Supporting Information

■

sı

As a reﬂection of the native D-type DNA octamer, our L-

DNA duplex adopts a left-handed A-form conformation. The

structural features of the L-helical geometry resemble those in

the native A-form duplex (Table 2). The base pairs in our L-

https://pubs.acs.org/doi/10.1021/acs.orglett.1c01498.

Experimental procedures, NMR characterization data

and LC-MS characterizations for all new compounds,

LC-Q-ToF characterizations, melting temperature meas-

urement, serum digestion analysis, circular dichroism

measurement and crystallographic statistics for all

synthetic oligonucleotides (PDF )

Table 2. Average Helical Parameters for L- and D-Nucleic

Acids 8-mer

L-5′-GU GTACAC-3′

D-5′-GTGTACAC-3′ (1d79)

Rise (Å)

3.10

3.17

Accession Codes

Roll (deg)

Slide (Å)

Tilt (deg)

Shift (Å)

Twist (deg)

7.12

−1.13

5.22

0.39

−33.02

6.91

−1.15

5.46

0.43

31.98

Atomic coordinates and structure factors for the reported

crystal structures have been deposited with the Protein Data

bank under accession code 7KW4.

AUTHOR INFORMATION

Corresponding Author

■

helical duplex has an average stepwise rise of 3.10 Å, roll angel

of 7.12°, slide of −1.13 Å, tilt of 5.22°, and shift of 0.39 Å. The

average base pair twist is −33.02°. All the helical parameters

are close to those of its native counterpart D-DNA, except for

the average twist of base pairs in the opposite direction. All

eight L-ribose rings present the C3′-endo or C3′-endo-2′-exo

conformations. The entire structure represents the mirror

reﬂection of native D-DNA crystal structure (Figure 1B). We

transformed the published structure of D-5′-GTGTACAC-3′

Wen Zhang − Department of Biochemistry and Molecular

Biology, Indiana University School of Medicine, Indianapolis,

Indiana 46202, United States; orcid.org/0000-0003-

4811-4384 ; Email: wz15@iu.edu

Authors

Yuliya Dantsu − Department of Biochemistry and Molecular

Biology, Indiana University School of Medicine, Indianapolis,

Indiana 46202, United States

(

PDB 1d79) into its mirror reﬂection and superimposed it with

our determined L-8-mer structure. The two structures are

highly superimposable, and the r.m.s. deviations between them

are 0.088 Å (Figure 1C). The L-DNA duplex contains eight

Ying Zhang − Department of Biochemistry and Molecular

Biology, Indiana University School of Medicine, Indianapolis,

Indiana 46202, United States

010

Org. Lett. 2021, 23, 5007−5011

Organic Letters

Complete contact information is available at:

Letter

https://pubs.acs.org/10.1021/acs.orglett.1c01498

(15) Pallan, P. S.; Greene, E. M.; Jicman, P. A.; Pandey, R. K.;

Manoharan, M.; Rozners, E.; Egli, M. Unexpected origins of the

enhanced pairing affinity of 2 ′-fluoro-modified RNA. Nucleic Acids

Res. 2011, 39, 3482−3495.

Notes

(16) Gesto, D. S.; Cerqueira, N. M. F.S. A.; Fernandes, P. A.; Ramos,

The authors declare no competing ﬁnancial interest.

M. J. Gemcitabine: a critical nucleoside for cancer therapy. Curr. Med.

Chem. 2012, 19, 1076−1087.

(17) Shi, J.; Du, J.; Ma, T.; Pankiewicz, K.; Patterson, S. E.; Hassan,

ACKNOWLEDGMENTS

■

A. E.; Tharnish, P. M.; McBrayer, T. R.; Lostia, S.; Stuyver, L. J.

Synthesis and in vitro Anti-HCV Activity of β -d-and l-2 ′-Deoxy-2 ′-

Fluororibonucleosides. Nucleosides, Nucleotides Nucleic Acids 2005, 24,

We thank the Zhang lab for helpful discussions, insightful

commentary, and careful revision of the manuscript. We thank

Dr. L. Zeng and the Chemical Genomics Core at IUSM for

LC-MS and NMR analysis. We thank Dr. Zdzislaw Wawrzak

from the Life Sciences Collaborative Access Team beamline

75−879.

(18) Lewis, M.; Meza-Avina, M. E.; Wei, L.; Crandall, I. E.; Bello, A.

M.; Poduch, E.; Liu, Y.; Paige, C. J.; Kain, K. C.; Pai, E. F. Novel

interactions of fluorinated nucleotide derivatives targeting orotidine

1-ID-D at the Advanced Photon Source, Argonne National

19) Ni, S.; Yao, H.; Wang, L.; Lu, J.; Jiang, F.; Lu, A.; Zhang, G.

′-monophosphate decarboxylase. J. Med. Chem. 2011, 54, 2891−

Laboratory (USA). This research used resources of the

Advanced Photon Source, a U.S. Department of Energy

901.

(

DOE) Oﬃce of Science User Facility operated for the DOE

Chemical modifications of nucleic acid aptamers for therapeutic

purposes. Int. J. Mol. Sci. 2017, 18, 1683.

Oﬃce of Science by Argonne National Laboratory under

Contract No. DE-AC02-06CH11357. Use of the LS-CAT

Sector 21 was supported by the Michigan Economic

Development Corporation and the Michigan Technology

Tri-Corridor (Grant 085P1000817).

(20) Hernández, M.; Rodríguez-Lázaro, D.; Esteve, T.; Prat, S.; Pla,

M. Development of melting temperature-based SYBR Green I

polymerase chain reaction methods for multiplex genetically modified

organism detection. Anal. Biochem. 2003, 323, 164−170.

(21) Wärmländer, S.; Sponer, J. E.; Sponer, J.; Leijon, M. The

influence of the thymine C5 methyl group on spontaneous base pair

REFERENCES

■

breathing in DNA. J. Biol. Chem. 2002, 277, 28491−28497.

E.; Egli, M. 2 ′-Fluoro RNA Shows Increased Watson −Crick H-

1) Wilson, D. S.; Szostak, J. W. In vitro selection of functional

nucleic acids. Annu. Rev. Biochem. 1999 , 68 , 611 −647.

(22) Patra, A.; Paolillo, M.; Charisse, K.; Manoharan, M.; Rozners,

Bonding Strength and Stacking Relative to RNA: Evidence from

NMR and Thermodynamic Data. Angew. Chem. 2012, 124, 12033−

2) Ellington, A. D.; Szostak, J. W. In vitro selection of RNA

molecules that bind specific ligands. Nature 1990 , 346 , 818 −822.

3) Zhou, J.; Rossi, J. Aptamers as targeted therapeutics: current

potential and challenges. Nat. Rev. Drug Discovery 2017 , 16 , 181 −202.

4) Wang, R. E.; Wu, H.; Niu, Y.; Cai, J. Improving the stability of

aptamers by chemical modification. Curr. Med. Chem. 2011, 18,

126−4138.

5) Urata, H.; Shinohara, K.; Ogura, E.; Ueda, Y.; Akagi, M. Mirror-

image DNA. J. Am. Chem. Soc. 1991, 113, 8174−8175.

6) Williams, K. P.; Liu, X.-H.; Schumacher, T. N.; Lin, H. Y.;

2036.

23) Thota, N.; Li, X.; Bingman, C.; Sundaralingam, M. High-

resolution refinement of the hexagonal A-DNA octamer d

GTGTACAC) at 1.4 Å. Acta Crystallogr., Sect. D: Biol. Crystallogr.

993, 49, 282−291.

24) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.

Ausiello, D. A.; Kim, P. S.; Bartel, D. P. Bioactive and nuclease-

D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl.

(

Crystallogr. 2007, 40, 658−674.

resistant L -DNA ligand of vasopressin. Proc. Natl. Acad. Sci. U. S. A.

997, 94, 11285−11290.

7) Dey, S.; Sczepanski, J. T. In vitro selection of l-DNA aptamers

that bind a structured d-RNA molecule. Nucleic Acids Res. 2020, 48,

669−1680.

8) Hauser, N. C.; Martinez, R.; Jacob, A.; Rupp, S.; Hoheisel, J. D.;

(25) Bundgaard Jensen, T.; Pasternak, A.; Stahl Madsen, A.;

Petersen, M.; Wengel, J. Synthesis and Structural Characterization of

Matysiak, S. Utilising the left-helical conformation of L -DNA for

′-Fluoro-α -L -RNA-Modified Oligonucleotides. ChemBioChem 2011,

12, 1904−1911.

(

analysing different marker types on a single universal microarray

platform. Nucleic Acids Res. 2006, 34 , 5101 −5111.

(9) Kabza, A. M.; Sczepanski, J. T. l-DNA-Based Catalytic Hairpin

Assembly Circuit. Molecules 2020, 25, 947.

10) Damha, M. J.; Giannaris, P. A.; Marfey, P. Antisense L /D -

oligodeoxynucleotide chimeras: nuclease stability, base-pairing prop-

erties, and activity at directing ribonuclease H. Biochemistry 1994, 33,

11) Lin, C.; Ke, Y.; Li, Z.; Wang, J. H.; Liu, Y.; Yan, H. Mirror

877−7885.

image DNA nanostructures for chiral supramolecular assemblies.

Nano Lett. 2009, 9, 433−436.

(12) Gawande, B. N.; Rohloff, J. C.; Carter, J. D.; von Carlowitz, I.;

Zhang, C.; Schneider, D. J.; Janjic, N. Selection of DNA aptamers

with two modified bases. Proc. Natl. Acad. Sci. U. S. A. 2017, 114,

13) Kabza, A. M.; Sczepanski, J. T. An l-RNA Aptamer with

898−2903.

Expanded Chemical Functionality that Inhibits MicroRNA Bio-

genesis. ChemBioChem 2017, 18, 1824−1827.

(14) Shi, J.; Du, J.; Ma, T.; Pankiewicz, K. W.; Patterson, S. E.;

Tharnish, P. M.; McBrayer, T. R.; Stuyver, L. J.; Otto, M. J.; Chu, C.

K. Synthesis and anti-viral activity of a series of d-and l-2 ′-deoxy-2 ′-

fluororibonucleosides in the subgenomic HCV replicon system.

Bioorg. Med. Chem. 2005, 13, 1641−1652.

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