Enhanced Binding Affinity for an i-Motif DNA Substrate Exhibited by a Protein Containing Nucleobase Amino Acids

Article abstract of DOI:10.1021/jacs.6b11825

Several variants of a nucleic acid binding motif (RRM1) of putative transcription factor hnRNP LL containing nucleobase amino acids at specific positions have been prepared and used to study binding affinity for the BCL2 i-motif DNA. Molecular modeling suggested a number of amino acids in RRM1 likely to be involved in interaction with the i-motif DNA, and His24 and Arg26 were chosen for modification based on their potential ability to interact with G14 of the i-motif DNA. Four nucleobase amino acids were introduced into RRM1 at one or both of positions 24 and 26. The introduction of cytosine nucleobase 2 into position 24 of RRM1 increased the affinity of the modified protein for the i-motif DNA, consistent with the possible Watson-Crick interaction of 2 and G14. In comparison, the introduction of uracil nucleobase 3 had a minimal effect on DNA affinity. Two structurally simplified nucleobase analogues (1 and 4) lacking both the N-1 and the 2-oxo substituents were also introduced in lieu of His24. Again, the RRM1 analogue containing 1 exhibited enhanced affinity for the i-motif DNA, while the protein analogue containing 4 bound less tightly to the DNA substrate. Finally, the modified protein containing 1 in lieu of Arg26 also bound to the i-motif DNA more strongly than the wild-type protein, but a protein containing 1 both at positions 24 and 26 bound to the DNA less strongly than wild type. The results support the idea of using nucleobase amino acids as protein constituents for controlling and enhancing DNA-protein interaction. Finally, modification of the i-motif DNA at G14 diminished RRM1-DNA interaction, as well as the ability of nucleobase amino acid 1 to stabilize RRM1-DNA interaction.

Full text of DOI:10.1021/jacs.6b11825

Subscriber access provided by Fudan University
Communication
Enhanced Binding Affinity for an i-Motif DNA Substrate
Exhibited by a Protein Containing Nucleobase Amino Acids
Xiaoguang Bai, Poulami Talukder, Sasha M Daskalova, Basab Roy,
Shengxi Chen, Zhongxian Li, Larisa M Dedkova, and Sidney M. Hecht
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b11825 • Publication Date (Web): 06 Mar 2017
Downloaded from http://pubs.acs.org on March 7, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 6
Journal of the American Chemical Society
1
2
3
4
TOC Graphic
5
6
7
8
9
NH2
NH2
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
N
OH
O
OH
O
O
NH2
NH2
O
NO2
HN
N
O
N
OH
OH
O
O
NH2
NH2
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 6
1
2
3
4
5
6
7
8
9
Enhanced Binding Affinity for an i‐Motif DNA Substrate Exhibited by
a Protein Containing Nucleobase Amino Acids
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Xiaoguang Bai,† Poulami Talukder,† Sasha M. Daskalova, Basab Roy, Shengxi Chen, Zhongxian Li,‡
Larisa M. Dedkova,* and Sidney M. Hecht*
Biodesign Center for BioEnergetics, and School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287,
United States
Supporting Information Placeholder
O
N
NO2
NH2
NH2
N
ABSTRACT: Several variants of a nucleic acid binding motif
(RRM1) of putative transcription factor hnRNP LL containing nu-
cleobase amino acids at specific positions have been prepared and
used to study binding affinity for the BCL2 i-motif DNA. Molecu-
lar modeling suggested a number of amino acids in RRM1 likely to
be involved in interaction with the i-motif DNA, and His24 and
Arg26 were chosen for modification based on their potential ability
to interact with G14 of the i-motif DNA. Four nucleobase amino
acids were introduced into RRM1 at one or both of positions 24 and
26. The introduction of cytosine nucleobase 2 into position 24 of
RRM1 increased the affinity of the modified protein for the i-motif
DNA, consistent with the possible Watson-Crick interaction of 2
and G14. In comparison, the introduction of uracil nucleobase 3
had a minimal effect on DNA affinity. Two structurally simplified
nucleobase analogues (1 and 4) lacking both the N-1 and the 2-oxo
substituents were also introduced in lieu of His24. Again, the
RRM1 analogue containing 1 exhibited enhanced affinity for the i-
motif DNA, while the protein analogue containing 4 bound less
tightly to the DNA substrate. Finally, the modified protein contain-
ing 1 in lieu of Arg26 also bound to the i-motif DNA more strongly
than the wild-type protein, but a protein containing 1 both at posi-
tions 24 and 26 bound to the DNA less strongly than wild type. The
results support the idea of using nucleobase amino acids as protein
constituents for controlling and enhancing DNA−protein interac-
tion. Finally, modification of the i-motif DNA at G14 diminished
RRM1−DNA interaction, as well as the ability of nucleobase amino
acid 1 to stabilize RRM1−DNA interaction.
HN
N
N
N
O
OH
OH
OH
O
OH
O
O
O
O
NH2
NH2
NH2
NH2
3
4
1
2
OH
NH2
OH
OH
OH
O
O
O
NH2
NH2
NH2
7
6
5
Figure 1. Nucleobase amino acids (1-4), and amino acids (5-7), prepared
and used to activate a suppressor tRNACUA and incorporated into RRM1 via
in vitro ribosomal synthesis.
While a number of nucleic acid–protein binding motifs have been
documented, including the helix-turn-helix1 and leucine zipper2
DNA binding motifs, the de novo design of new/modified protein
motifs to enable selective targeting of predetermined DNA and
RNA structures is still quite challenging. As all nucleic acid struc-
tures are assembled using only a small number of nucleobases, and
only a limited number of base pairing/association schemes are op-
erative, a novel strategy for nucleic acid recognition might involve
the use of nucleobases as amino acid side chains, enabling nucleic
acid recognition by familiar and well understood interactions such
as base pairing and stacking.
There are numerous proteins which play important roles in cellu-
lar functions by selective interaction with DNA or RNA. These pro-
teins include those involved in transcriptional regulation, DNA rep-
lication and repair of DNA damage. DNA interactive proteins in-
clude topoisomerases, gyrases, recombinases, transcription factors
and DNA polymerases. Proteins which recognize RNA selectively
include aminoacyl-tRNA synthetases, the initiation, elongation and
termination factors which participate in protein translation, riboso-
mal proteins, RNA polymerases and proteins involved in RNA
splicing complexes. Many of these proteins take part in essential
cellular functions, such that modifying their selective targeting
could in principle modulate (aberrant) cellular function.
Although there is as yet no report of any protein which recognizes
a nucleic acid target selectively by employing a nucleobase incor-
porated within the protein, the basic concept is implicit within a
number of related studies involving peptides. Of particular note are
the efforts of the Mihara laboratory in preparing a series of peptides
containing L-amino--nucleobase butyric acids for the purpose of
RNA binding.3 For the synthesis of larger protein mimetics, pep-
tides containing a nucleobase amino acid were synthesized chemi-
cally and condensed by means of native chemical ligation.3b
A
number of interesting results were obtained in this study. For ex-
ample, when three nucleobase acid residues were included within
arginine-rich α-helical peptides targeted to a hairpin RNA, the af-
ACS Paragon Plus Environment

Page 3 of 6
Journal of the American Chemical Society
finities varied with the nature of the nucleobases present.3a The in-
new pyrimidine-like nucleobases (1 and 4, Figure 1) which lack
1
2
3
4
5
6
7
8
troduction of nucleobase acids within the dimerization domain of
HIV-1 protease enhanced affinity, but lowered the catalytic activity
to some extent.3b
both the 2-oxo and N-1 substituents of pyrimidines. In spite of its
structural differences from 2, nucleobase 1 seemed to function
analogously to 2 in its DNA binding properties when introduced
into position 24 of RRM1. As anticipated, nucleobase 4 diminished
RRM1−i-motif DNA interaction when introduced into position 24.
Also included to facilitate interpretation of the results obtained with
the nucleobase amino acids were amino acids p-NH2Phe (5), tyro
sine (6) and phenylalanine (7), all of which have been incorporated
into proteins previously by nonsense codon suppression.7
The nucleobase amino acids employed for this study are of a type
first described by the Diederichsen laboratory4 for the preparation
of nucleobase functionalized peptide nucleic acids4a and -pep-
tides.4b These contain a single methylene group between the nucle-
obase and -carbon atom of the derived amino acid, i.e. the same
spacing as in the four proteinogenic aromatic amino acids (histi-
dine, phenylalanine, tyrosine and tryptophan). We have recently
described5 the synthesis of the amino acids corresponding to the
five canonical nucleobases in RNA and DNA (uracil, thymine, cy-
tosine, adenine and guanine), two of which (2 and 3, Figure 1) are
employed in the present study. While these amino acids were pre-
viously incorporated into two proteins, the potentially altered func-
tions of the derived proteins were not explored.5
9
The chemical synthesis of derivatives of 1 began from commer-
cially available 5-methyl-2-aminopyridine (8) (Scheme 1).8a The
amino group was oxidized to afford the respective 2-nitropyridine
(9) in 60% yield, the latter of which was treated with N-bromosuc-
cinimide8b in CCl4 to provide benzylic bromide 10. Regioselective
lithiation of the Schöllkopf chiral auxiliary with n-BuLi,8c followed
by admixture with 10, gave 11 which was isolated as a single dia-
stereomer.8d Mild hydrolysis using 2 N HCl then afforded the α-
substituted amino acid methyl ester 12 in 84% yield. Following hy-
drogenation of the nitro group over Pd/C, and NVOC protection of
amino acid 13 as the activated cyanomethyl ester 14, treatment with
the dinucleotide pdCpA9 provided NVOC-1 as its pdCpA ester (15)
in 54% yield. The aminoacylated dinucleotide was then ligated to
an abbreviated suppressor tRNA transcript (tRNACUA-COH) using
T4 RNA ligase, and the NVOC protecting group was removed by
photolysis prior to its use for the synthesis of analogues of RRM1.
Closely analogous transformations employing synthetic intermedi-
ate 12 were used to prepare a suppressor tRNA activated with 4 (SI,
Scheme S1).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
B
A
Scheme 1. Synthesis of the pdCpA and tRNACUA Esters of Nucleobase
Amino Acid 1
Figure 2. (A) Structure of the BCL2 i-motif DNA.6 The cytidine rich i-motif
stem is shown in yellow with two of the lateral loops and the central loop.
(B) Human hnRNP LL RRM1 domain, including His24, generated by I-
Tasser software using the structure of Mus musculus RRM domain of
BAB28521 protein (pdb 1WEX) as a template.15
NO2
N
NH2
NO2
NO2
O
Schollkopf auxiliary,
2.5 M n-BuLi,
2 N HCl, THF
conc. H2SO4/H2O2
0 oC to rt, overnight
60%
NBS, AIBN
CCl4, reflux, 4 h
54%
N
N
N
N
oC to rt, 1.5 h
84%
anh. THF, -78 oC to rt, 16 h
32%
0
N
CH3
CH3
CH2Br
O
9
8
10
11
Presently, we prepared several constructs of RRM1, one of the
nucleic acid recognition motifs of transcription factor hnRNP LL
which has recently been shown to bind to the lateral loops of the
BCL2 i-motif DNA and to unfold the DNA.6 We identified His24
of RRM1 as a potential binding partner for G14 of the i-motif DNA
and report that the modified RRM1 containing cytosine nucleobase
2 in lieu of His24 binds to the i-motif DNA more strongly than
wild-type RRM1, while the introduction of uracil nucleobase 3 pro-
duces no increase in binding. We also report the syntheses of two
NH2
NO2
NH2
1) 1 N LiOH,
1:3:1 H2O-THF-MeOH,
rt, 2 h
N
N
N
,
1) 10% Pd/C, 1 atm H2
MeOH, rt, overnight
TBA-pdCpA
O
CN
O
O
anh. DMF/Et3N,
sonication, 8 h
2) NVOC-Cl, K2CO3
1:1 dioxane-H2O,
rt, overnight
,
2) chloroacetonitrile,
O
O
O
NaHCO3, anh. DMF,
NHNVOC
NH2
NHNVOC
54%
rt, 15 h
14
13
23% (two steps)
32% (two steps)
12
NH2
N
N
N
tRNA-CC
NH2
O
N
O
P
O
N
O
T4 RNA ligase,
tRNA-COH
O
O
H2
N
O
O
OH
OpdCpA
NHNVOC
N
NHR
15
16, R = NVOC
17
hv
, R = H
As a model system for studying the interaction of nucleobase-con-
taining proteins with DNA, we chose the RRM1 from hnRNP LL,
which has been expressed in a cell free system and shown to bind
to the lateral loops of the i-motif DNA formed in the promoter re-
gion of BCL2.6 Based on an analysis of the likely molecular mode
of hnRNP LL−DNA interaction using iTASSER,10 DP-bind,11 and
NCBI CDD12 as well as a knowledge of the important RNA binding
roles of the invariant residues His105 and Arg107 in the closely
related protein hnRNP L,13,14 we chose the analogous residues
(His24 and Arg26) in RRM1 for modification. The structures of the
i-motif DNA and RRM1 are shown in Figure 2.
Three RRM1 DNA constructs were created, enabling the intro-
duction of different nucleobase amino acids in lieu of His24 or
Arg26, or at both of these positions. The yields of modified RRM1s
obtained using a suppressor tRNA activated with 1, 2 or 3 were
studied (Figure 3A). As shown in the Figure, the suppression yield
of modified RRM1 containing 1 at position 24 was ~3-fold greater
than for 2 or 3 (71% vs 23 and 24%).16 Also studied comparatively
Figure 3. SDS-Polyacrylamide gel showing (A) incorporation of nucleo-
base amino acids 1, 2 and 3 into position 24 of RRM1, (B) incorporation of
amino acid 1 into position 24, position 26 or both positions of RRM1. Sup-
pression yields are shown. No, absence of activated suppressor tRNA.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 6
were the efficiencies of incorporation of 1 into positions 24 (55%)
bind to G14 via a wobble-type interaction (SI, Figure S8), explain-
ing its more modest affinity.19 Less surprising was the finding that
an RRM1 having nucleobase amino acid 4 at position 24 exhibited
greatly diminished DNA affinity (BC50 110 ± 10 nM) (Table 2).
This seems consistent with what might have been expected from
the introduction of a nitro substituent with its dipolar covalent bond
in lieu of a H-bond donor/acceptor.
1
2
3
4
5
6
7
8
and 26 (54%), and into both positions (6%) (Figure 3B). While the
yield of the modified RRM1 containing two amino acid 1 residues
was lower as anticipated, it was still sufficient to provide material
enabling biochemical studies, a critical finding since the implemen-
tation of a strategy to use nucleobase side chains in proteins to
achieve selective nucleic acid recognition will clearly require the
use of two or more closely spaced nucleobase amino acids. Also
prepared to aid in interpretation of i-motif DNA binding studies
were RRM1s containing nucleobase amino acid 4, p-NH2Phe (5),
Tyr (6) or Phe (7) at position 24.
Also studied were RRM1 analogues having p-NH2Phe (5), Tyr (6)
or Phe (7) at position 24 (Table 2 and SI, Figure 7C). RRM1 con-
taining 5 had a BC50 value of 92
± 9 nM. This loss of affinity rela-
9
tive to the RRM1 containing 1 at position 24 (BC50 53 ± 12 nM),
argues that the ring N atom must function as a H-bond acceptor,
and suggests that enhanced binding in this system is not due to en-
hanced base stacking. The lesser affinity of RRM1 6-24 Arg (BC50
141 ± 5 nM) reinforces the role of the exocyclic N atom in 1 as a
source of enhanced affinity, logically through H-bonding. Introduc-
tion of Phe into RRM1 7-24 Arg further reduced DNA binding.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Quantification of the Binding of Modified RRM1s Containing
1 to BCL2 i-Motif DNA
Protein
BC50 value (nM)
96 ± 13
RRM1 His24 Arg26 (wt)
RRM1 1-24 Arg26
RRM1 His24 1-26
RRM1 1-24 1-26
53 ± 12
63 ± 11
130 ± 28
Table 3. Quantification of the Binding of Modified RRM1s Containing
1 to wild-type (G14) and Modified (A14) BCL2 i-Motif DNAs
Table 2. Quantification of the Binding of Modified RRM1s Containing
2 - 7 to BCL2 i-Motif DNA
Protein
BC50 value (nM)
G14 i-motif
A14 i-motif
99 ± 8
81 ± 9
Protein
BC50 value (nM)
80 ± 10
RRM1 His24 Arg26 (wt)
RRM1 1-24 Arg26
84 ± 3
47 ± 5
55 ± 10
RRM1 His24 Arg26 (wt)
RRM1 2-24 Arg26
RRM1 3-24 Arg26
RRM1 4-24 Arg26
RRM1 5-24 Arg26
RRM1 6-24 Arg26
RRM1 7-24 Arg26
54 ± 11
82 ± 16
110 ± 10
92 ± 9
141 ± 5
RRM1 His24 1-26
78 ± 11
We also studied the effect of altering the i-motif DNA binding
partner for RRM1. The binding interaction involves the lateral
loops of the i-motif, where G14 is located.6 Replacement of G14
with A14 diminished RRM1 affinity for the i-motif (Table 3).
Interestingly, while the introduction of 1 at position 24 or 26 re-
sulted in stronger binding of RRM1 to the modified i-motif, the
improvement in affinity was much less pronounced than for the na-
tive RRM1 sequence (Table 3, SI, Figure S9).
165 ± 4
Four RRM1 samples prepared by in vitro protein synthesis (wild-
type and three modified proteins containing 1) were purified by
Strep-Tactin chromatography, and analyzed by denaturing SDS-
polyacrylamide gel electrophoresis (Figure S3A), verifying the
electrophoretic homogeneity of the samples. RRM1 containing 1 at
position 24 was analyzed by MALDI-MS/MS (Figure S4), verify-
ing the presence and position of this nucleobase amino acid.
Binding of the BCL2 i-motif DNA to each of the RRM1s was
studied using an electrophoretic mobility shift assay (EMSA).17,18
The results are shown in Table 1 and SI, Figure S7A. The RRM1s
containing amino acid 1 at position 24 (BC50 53 ± 12 nM) or 26
(BC50 63 ± 11 nM) exhibited increased affinity for the BCL2 i-motif
DNA relative to wild-type RRM1 (BC50 96 ± 13 nM). Thus replace-
ment of either His24 or Arg26 by 1 resulted in energetically im-
proved contacts with the target DNA, relative to the normal amino
acids. The selective nature of the interactions with i-motif DNA
mediated by 1 at position 24 or 26 can be appreciated from the ob-
servation that the simultaneous introduction of 1 into both positions
actually substantially diminished the affinity of the resulting RRM1
for the DNA (BC50 130 ± 28 nM).
Analysis of two other mutant proteins RRM1-24-2 and RRM1-
24-3, having cytosine and uracil-based amino acids in position 24,
prepared analogously (Figure S3B), provides mechanistic insights
(Table 2, SI, Figure S7B). The presence of cytosine nucleobase 2
at RRM1 position 24 also increased DNA affinity relative to wild
type (BC50 54 ± 11 nM), providing affinity similar to that of RRM1
containing 1 at position 24, while RRM1 having uracil nucleobase
3 at that position had an affinity comparable to that of wild-type
RRM1 (BC50 82 ± 16 vs 80 ± 10 nM). While not definitive, the
foregoing observations are consistent with the thesis that H-bond-
ing interactions between the nucleobase moieties of amino acids 1
– 3 and the nucleobase of G14 play a key role in enhanced binding
of the modified RRM1s to the i-motif DNA. While the enhanced
binding observed for RRM1 containing 1 would be consistent with
the formation of Watson-Crick like H-bonds between 1 and G14,
one might have expected a diminution of RRM1−DNA binding for
the RRM1 containing 3. Plausibly, the RRM1 containing 3 may
Figure 4. Computationally docked models showing the binding of RRM1
to the i-motif DNA via His24 and Arg26 interaction. The modeling was
done using SWISS-MODEL20 homology modeling (A) and HADDOCK21
web server was used for data-driven docking (B). A scoring algorithm used
factors such as backbone flexibility, surface resides and specific amino ac-
ids to rank order the docked structures. The structure shown is a high scor-
ing model illustrating the potential distance between His24 and G14.
ACS Paragon Plus Environment

Page 5 of 6
Journal of the American Chemical Society
We have used computational docking to attempt to understand the
Notes
1
2
3
4
5
6
7
8
molecular basis for the observation that introduction of nucleobase
1 into position 24 or 26 of RRM1 enhanced DNA binding but the
presence of 1 in both positions significantly diminished binding.
Figure 4A shows one of the most energetically favorable docked
structures. The Arg26 guanidine moiety is within H-bonding dis-
tance to O6 and N2 of G14 of the i-motif DNA (as well as O2 of C-
13), while the His24 imidazole N is more than 7 Å from all DNA
bases (and ~8 Å from N2 of G14). Another very favorable docked
structure is shown in Figure 4B. The His24 imidazole N is ~4 Å
from O6 of G14, while Arg26 has been displaced close to the DNA
backbone. Thus, in these energetically favorable models, either
His24 or Arg26 (but not necessarily both) can be H-bonded to G14.
This is consistent with the experimental observations noted in Ta-
ble 1 when 1 was introduced in lieu of one or both of these amino
acids.
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This study was supported by Grant GM 103861 from the National
Institute of General Medical Sciences, National Institutes of
Health. Z. L. thanks the NFSC-joint fund for talent cultivation in
Henan Province for a fellowship (U1404206).
REFERENCES
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(1) Brennan, R. G.; Matthews, B. W. J. Biol. Chem. 1989, 264, 1903.
(2) (a) Landschulz,W. H.; Johnson, P. F.; McKnight, S. L. Science 1988,
240, 1759. (b) Vinson, C. R.; Sigler, P. B.; McKnight, S. L. Science 1989,
246, 911. (c) Pavletich, N. P.; Pabo, C. O. Science 1991, 252, 809.
(3) (a) Miyanishi, H.; Takahashi T.; Mihara, H. Bioconjugate Chem. 2004,
15, 694. (b) Takahashi, T., Yana, D., Mihara, H. ChemBioChem 2006, 7,
729. (c) Watanabe, S.; Tomizaki, K.; Takahashi, T.; Usui, K.; Kajikawa K.;
Mihara, H. Peptide Sci. 2007, 88, 131.
(4) (a) Diederichsen, U. Angew. Chem. Int. Ed. Engl. 1996, 35, 445. (b)
Chakraborty, P.; Diederichsen, U. Chem. Eur J. 2005, 11, 3207.
(5) Talukder, P.; Dedkova, L. M.; Ellington, A. D.; Yakovchuk, P.; Lim,
J.; Anslyn, E. V. Hecht, S. M. Bioorg. Med. Chem. 2016, 24, 4177. For the
purposes of this study, we define nucleobase amino acids as species having
at least one N atom within the aromatic substituent.
(6) (a) Kang, H.-J.; Kendrick, S.; Hecht S. M.; Hurley, L. H. J. Am. Chem.
Soc. 2014, 136, 4172. (b) Talukder, P.; Chen, S.; Roy, B.; Yakovchuk, P.;
Spiering, M. M.; Alam, M. P.; Madathil, M. M.; Bhattacharya, C.; Benkovic
S. J.; Hecht, S. M. Biochemistry, 2015, 54, 7457. (c) Roy, B.; Talukder, P.;
Kang, H.-J.; Tsuen, S. S.; Alam, M. P.; Hurley, L. H.; Hecht, S. M. J. Am.
Chem. Soc. 2016, 138, 10950.
It seems logical to anticipate that optimizing selective protein−nu-
cleic acid interactions will require the use of multiple nucleobase
amino acids in a single protein. In this regard a few observations
seem worthy of note. First, because modified nucleobase amino ac-
ids such as 1 and 416 appear to be incorporated with greater effi-
ciency than “canonical” nucleobase amino acids such as 2 and 3,
once fully optimized they should facilitate the introduction of mul-
tiple nucleobase amino acids into a protein. Second, our recent de-
velopment of modified ribosomes capable of incorporating dipep-
tides into proteins22 may enable the introduction of two contiguous
nucleobase amino acids in a single ribosomal event. The finding
(Table 1) that incorporation of amino acid 1 both into positions 24
and 26 of RRM1 substantially reduced the affinity of this protein
for the i-motif (BC50 130 ± 28 vs 96 ± 13 nM), as compared with
the increases in affinity observed following either of the same sub-
stitutions made singly, indicates that developing nucleic acid bind-
ing proteins that function with favorable affinity and selectivity will
demand creative new strategies, and that the full repertoire of inter-
actions noted for nucleic acid interactions, including stacking, van
der Waals and electrostatic interactions, may find utility in addition
to the apparent H-bonding putatively involved in the current study.
Finally, while the increases in protein affinity observed in the pre-
sent study were not large, they may arguably be sufficient in many
cases to alter the specificity for their nucleic acid target. The intro-
duction of multiple nucleobase amino acids into a single protein
should also conduce to that outcome. Presumably, increasing affin-
ity would at some point negatively impact biological function.
(7) Gao, R.; Zhang, Y.; Dedkova, L.; Choudhury, A. K.; Rahier, N. J.;
Hecht, S. M. Biochemistry 2006, 45, 8402.
(8) (a) Zhang, F.; Zhang, S.; Duan, X. F. Org Lett. 2012, 14, 5618. (b)
Jones, P. A.; Wilson, I.; Morisson-IveSon, V.; Jones, C.; Woodcraft, J.;
Jackson, A.; Wynn, D. International Patent WO 106564 A2, 2009. (c)
Schöllkopf, U.; Groth, U.; Westphalen, K. O.; Deng, C. Z. Synthesis 1981,
969. (d) Talukder, P.; Chen, S. X.; Arce, P. M.; Hecht, S. M. Org Lett. 2014,
16, 556.
(9) Robertson, S. A.; Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.;
Schultz, P. G. Nucleic Acids Res. 1989, 17, 9649.
(10) Roy, A.; Kucukural, A.; Zhang, Y. Nat. Prot. 2010, 5, 725.
(11) Hwang, S.; Gou, Z.; Kuznetsov, I. B. Bioinformatics 2007, 23, 634.
(12) Marchler-Bauer, A.; Derbyshire, M. K.; Gonzales, N. R.; Lu, S.,
Chitsaz, F.; Geer, L. Y.; Geer, R. C.; He, J.; Gwadz, M.; Hurwitz, D.
I.; Lanczycki, C. J.; Lu, F.; Marchler, G. H.; Song, J. S.; Thanki, N.; Wang,
Z.; Yamashita, R. A.; Zhang, D.; Zheng, C.; Bryant, S. H. Nucleic Acids
Res. 2015, 43, (database issue), D222.
(13) Zhang, W.; Zeng, F.; Liu, Y.; Zhao, Y.; Lv, H.; Niu, L.; Teng, M.; Li,
X. J. Biol. Chem. 2013, 288, 22636.
(14) These two residues correspond to His 74 and Arg 76 in hnRNP LL.
A summary of potential DNA interactive residues in RRM1 is summarized
in the Supporting Information (SI) Figure S1.
ASSOCIATED CONTENT
Supporting Information
Experimental procedures for the synthesis and characterization of
the pdCpA derivatives of 1 and 4, for the synthesis and biochemical
evaluation of modified RRM1s, and the EMSA data for the pro-
tein−DNA binding experiments.
(15) Zhang, Y. BMC Bioinformatics 2008, 9, 40.
(16) The incorporation of nucleobase amino acid 4 into positions 24 and
26 was carried out in a separate experiment; the suppression yields were 61
and 48%, respectively (SI, Figure S2).
(17) Kendrick, S.; Akiyama, Y.; Hecht, S.M.; Hurley, L.H. J. Am. Chem.
Soc. 2009, 131, 17667.
(18) The assay was optimized using wild-type RRM1 elaborated in E. coli
(SI, Figures S5 and S6). Wild-type RRM1 prepared in vitro had an affinity
for the i-motif DNA quite similar to the sample isolated from E. coli (SI,
Figure S6).
(19) For another possible reason for the behavior of RRM1 1-24 Arg26
see: Hildbrand, S.; Blaser, A.; Parel, S. P.; Leumann, C. J. J. Am. Chem.
Soc. 1997, 119, 5499.
(20) Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. Bioinformatics 2006,
22, 195.
(21) Dominguez, C.; Boelens, R.; Bonvin, A. M. J. Am. Chem. Soc. 2003,
125, 1731.
(22) Maini, R.; Dedkova, L. M.; Paul, R.; Madathil, M. M.; Roy Chow-
dhury, S.; Chen, S.; Hecht, S. M. J. Am. Chem. Soc. 2015, 137, 11206.
AUTHOR INFORMATION
Corresponding Authors
*Email: sidney.hecht@asu.edu. Phone: (480) 965-6625. Fax:
(480) 965-0038.
*Email: larisa dedkova@asu.edu. Phone: (480) 965-8248. Fax:
(480) 965-0038
Present Addresses
‡Z.L.: High & New Technology Research Center, Henan Academy
of Sciences, Zhengzhou, 450002, P. R. China.
Author Contributions
†X.B. and P.T. contributed equally.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 6
1
2
3
4
5
6
7
8
9
NH2
NH2
N
N
N
OH
O
OH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
NH2
NH2
O
NO2
HN
N
O
N
OH
OH
O
O
NH2
NH2
5
ACS Paragon Plus Environment