LNA for Optimization of Fluorescent Oligonucleotide Probes: Improved

COMMUNICATION pubs.acs.org/bc

LNA for Optimization of Fluorescent Oligonucleotide Probes: Improved Spectral Properties and Target Binding Irina V. Astakhova,† Alexey V. Ustinov,‡ Vladimir A. Korshun,‡ and Jesper Wengel*,† † ‡

Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia

bS Supporting Information ABSTRACT: Mixmer LNA/DNA fluorescent probes containing the 1-(phenylethynyl)pyrene fluorophore attached to 20 arabino-uridine were synthesized and studied. The conjugates displayed significantly higher hybridization affinity to target DNA, increased fluorescence quantum yields of single-stranded oligonucleotides and their duplexes, and improved ability to form an interstrand excimer compared to analogous non-LNA probes.

’ INTRODUCTION DNA- and RNA-derived fluorescent probes of various constructs that result in clear fluorescence signals upon binding to targets constitute a paradigm for modern, relatively inexpensive, and sequence-specific, homogeneous detection of nucleic acids.1,2 For effective nucleic acid targeting, high-affinity and sequence-specific synthetic oligonucleotides (ONs) are required. Locked nucleic acid (LNA, Chart 1) is a synthetic nucleotide analogue3
7 that provides high-affinity and selective recognition of cDNA and RNA.8
13 High-affinity LNA/DNA ONs containing different fluorescent dyes have recently been applied for single-nucleotide polymorphism (SNP) analysis,9
13 real-time PCR,14
16 FISH analysis,17
20 and other applications.21,22 In all these assays, fluorescent LNA/DNA probes were superior in thermal affinity (Tm values increased up to 8
C per one LNA monomer incorporation measured in a medium salt buffer) and in SNP discrimination of target sequences, when compared to the analogues DNA probes. Another important aspect of fluorescent ON probes is suitable fluorescent dyes or dye pairs that are sensitive, photostable, and relatively inexpensive. Most fluorescent LNA/DNA probes reported so far have contained terminally attached fluorescent dyes of the xanthene or cyanine families (e.g., FAM, JOE, HEX, Cy5, etc.), which signal hybridization by fluorescence effect of choice (e.g., by dye-quencher proximity or by light-up effect of a single dye).23
30 However, being exposed into the medium, these dyes did not allow more informative studies of biomolecules and their segments, or of their specific interactions. Fluorescent ONs containing polyaromatic hydrocarbons (PAHs) are alternative probes for nucleic acid studies which due to the PAH electronic π
π* structure sense even minor changes in their microenvironment by fluorescence.31 Among various PAH dyes, pyrene is of increased interest because of the long lifetime of its excited state and its ability in π-stacking, r 2011 American Chemical Society

resulting in exciplex and excimer formation.31 The main disadvantage of the pyrene label is its low quantum yields within biomolecules and relatively short absorption and emission wavelengths crossing the autofluorescence region of cells. Recently, the 1-(phenylethynyl)pyrene fluorophore (1-PEPy, Chart 1) was introduced as a refined alternative to parent pyrene with advantages of increased absorption, bathochromic shift of the absorption and emission maxima, and increased fluorescence quantum yield within biomolecules.32
35 Previously, we attached the 1-PEPy fluorophore to 20 -arabino-uridine via a carbamate linker and showed the utility of this modification for detection of nucleic acid hybridization.36 Even though 1-PEPy attached to DNA exhibited short excited lifetime (0.96
2.40 ns), the dye was able to form inter- and intrastrand excimers.36 On the other hand, we observed that internal 1-PEPy modification, i.e., 1-PEPy attached to an internal 20 -arabino-uridine monomer, significantly reduced thermal stabilities of DNA duplexes (Tm values decreased by 3
8
C per one modification in a medium salt buffer) which can become an obstacle for its utility.36 The negative effect of 1-PEPy on probe affinity can be reduced by incorporation of two or more LNA nucleotides into ONs. However, recent structural investigations of LNA/DNA ONs have showed that, being a RNA mimic, LNA nucleotides significantly change duplex geometry by decreasing the B-type character of doublestranded DNA toward intermediate A/B-type.8
13 Therefore, we propose that the effect of LNA within ONs on spectral properties of any PAH label can be significant and need to be studied in detail. We herein evaluate ONs having the 1-PEPy label attached to 20 -arabino-uridine via a carbamate linker in combination with LNA nucleotides. We report synthesis, thermal denaturation, Received: November 16, 2010 Published: March 14, 2011 533

dx.doi.org/10.1021/bc1005027 | Bioconjugate Chem. 2011, 22, 533–539

Bioconjugate Chemistry

COMMUNICATION

Chart 1. Structures of LNA, 1-PEPy, Modified Phosphoramidites 136 and 236 and the Corresponding Monomers X and Y

Table 1. ONs Used in This Study and Thermal Stabilities of Modified Duplexesa

a

AL, GL, and MeCL denote LNA adenin-9-yl, guanin-9-yl, and 5-methylcytosin-1-yl monomers, respectively. Melting temperatures were measured in a medium salt buffer using 1.0 μM concentrations of ONs. The blue and gray droplets represent monomer X and LNA monomers, respectively. ΔTm* and ΔTm** values were calculated relative to unmodified double-stranded DNA ON12:ON1 (Tm = 58.0
C) and 1-PEPy modified LNA-free doublestranded DNA ON13:ON1 (Tm = 50.0
C), respectively.

improve both binding affinity and spectral properties of fluorescent ON probes.

and spectral studies of these resulting 1-PEPy-labeled LNA/ DNA probes, and demonstrate how incorporation of LNA can 534

dx.doi.org/10.1021/bc1005027 |Bioconjugate Chem. 2011, 22, 533–539

Bioconjugate Chemistry

COMMUNICATION

Table 2. Spectroscopic and Photophysical Properties of Modified ONs and Duplexesa λabsmax, bands I, II (nm)

λflmax (nm)

τ1 (ns)

S1 (%)

τ2 (ns)

S2 (%)

Æτfæ (ns)

Φf

ON2

394, 371

402

0.75

32

2.68

68

2.06

0.25

ON2:ON1

390, 370

402

0.63

38

2.46

62

1.76

0.24

ON3

395, 373

402

0.79

27

2.86

73

2.30

0.39

ON3:ON1

391, 372

404

0.68

40

2.76

60

1.93

0.26

ON4

393, 370

401

1.05

11

2.87

89

2.67

0.63

ON4:ON1

390, 367

403

0.82

7

2.81

93

2.67

0.76

ON5

396, 373

402

0.66

20

2.70

80

2.29

0.34

ON5:ON1 ON13

390, 370 397, 372

402 405

0.67 0.60

26 35

2.68 2.50

74 65

2.15 1.83

0.32 0.16

ON13:ON1

391, 370

405

0.90

44

2.68

56

1.89

0.23

#

Absorption and steady-state fluorescence emission spectra measured in a medium salt buffer using 0.1
1.0 μM concentrations of ONs. λabsmax and λflmax are absorption and fluorescence maxima, respectively; τ1 and τ2 are two fluorescence lifetime components; S1 and S2 are contribution of τ1 and τ2 components in percentages; average fluorescence lifetime Æτfæ is calculated using equation: Æτfæ = ΣSiτi /ΣSi.26 a

’ RESULTS AND DISCUSSION Initially, we prepared four different ONs having the 1-PEPy fluorophore (monomer X)36 and two LNA nucleotides3
7 in four different relative positions (Table 1; ON2
ON5). Synthesis of the modified ONs was performed using phosphoramidite derivative 136 and commercially available LNA phosphoramidites.3
7 Additionally, compounds 1 and 236 were utilized to prepare six complementary sequences to ON2
ON5 having monomers X and Y positioned near the 50 -end (Table 1; ON6
ON11). The latter was done in order to study 1-PEPy interstrand excimer formation within DNA duplexes. Finally, we prepared the LNA-free fluorescent probe ON13, which was used as a reference to verify the effect of LNA on 1-PEPy properties. The sequence of ON13 was equivalent to the one that was previously used to investigate 1-PEPy-20 -arabino-uridine attached to DNA.36 All modified oligonucleotides were prepared using standard protocols except for extended coupling times (10 min) for the phosphoramidites of monomers X and Y, and for LNA phosphoramidites. Moreover, we observed better coupling yields of the phosphoramidite reagents 1 and 2 using 0.6 M pyridine hydrochloride in MeCN as activator in comparison with standard 1H-tetrazole in MeCN (99% vs 89
90%, respectively). All modified ONs were purified by RP-HPLC and their identities and purities were confirmed by MALDI-MS and IC-HPLC, respectively. First, we investigated the binding of ON2
ON5 and ON13 to DNA by UV thermal denaturation experiments using a medium salt buffer (100 mM NaCl, 10 mM Na-phosphate, 0.1 mM EDTA, pH 7.0), and compared the data to the unmodified and X-labeled references ON12:ON1 and ON13: ON1, respectively (Table 1). Insertion of the two LNA monomers completely neutralized the destabilizing effect of 1-PEPy-20 arabino-uridine (Table 1, Tm and ΔTm data for ON2
ON5). Interestingly, the specific positioning of the LNA monomers along the probe sequences had a remarkable effect on Tm values as well. The highest duplex stabilization by LNAs was observed for the duplex ON4:ON1 having LNA monomers separated by two nucleotides from the 1-PEPy (ΔTm = þ12.0
C compared to ON13:ON1). Next, we evaluated spectroscopic and photophysical properties of the 1-PEPy-containing LNA/DNA conjugates (Table 2). Steady-state fluorescence emission spectra were recorded in a medium salt buffer using an excitation wavelength of 360 nm.

Generally, all spectra exhibited a typical 1-PEPy-monomer emission of the 1-PEPy dye in the region 380
480 nm with λflmax ∼ 400 nm. The insertion of LNA monomers into ON2
ON5 did not significantly affect either absorption or fluorescence emission wavelengths of the 1-PEPy dye. Next, fluorescence quantum yields Φf of single-stranded ON2
ON5 and their duplexes Φf = 16
76% were approximately 160
390% higher than those for ON13 and also remarkably higher that typical quantum yields of pyrene label attached to DNA. We propose that the high quantum yields of 1-PEPy within LNA/DNA conjugates might result from combination of two factors: (1) short fluorescence lifetime of 1-PEPy and (2) shielded positioning of the 1-PEPy fluorophore within the polar groove environment preventing interactions of the fluorophore with quenchers of fluorescence such as medium, oxygen, and nucleobases. As it was demonstrated for the fluorescent pyrene label attached directly to 20 -amino-LNA,37,38 this specific positioning can be achieved due to structural alterations within the duplexes induced by the LNA nucleotides. The high fluorescence quantum yields of the 1-PEPy-LNA/DNA significantly decrease probe detection limits, which is of high importance for biological studies and biomedical assays. However, the high Φf values of conjugates ON2
ON5 (0.25
0.63) were only slightly affected by hybridization with complementary unmodified DNA. Additionally, slightly increased average fluorescence lifetimes of the 1-PEPy dye within LNA-containing ONs and duplexes compared to the references ON13 and ON13:ON1 were observed. Moreover, even in presence of the two RNA-mimicking LNA nucleotides still two fluorescent excited states of the 1-PEPy were observed, and both had fluorescence lifetimes and contribution values of the same range as ON13. This implies that insertion of the LNA nucleotides remarkably affects positioning of the fluorophore within the probes and increase their fluorescence quantum yields without affecting basic photophysical characteristics of the dye, such as the number of fluorescent excited states and their lifetimes. Remarkably high Φf values were observed for both singlestranded ON4 and its duplex ON4:ON1 in which 1-PEPy and LNAs were separated by two nucleotides (Table 2). Such high fluorescence quantum yields of the dye within a single-stranded conjugate are quite surprising and may result from LNA-induced formation of a secondary structure in ON4 preventing quenching interaction of the fluorophore. The formation of a preorganized structure by ON4 was also suggested by the more structured CD 535

dx.doi.org/10.1021/bc1005027 |Bioconjugate Chem. 2011, 22, 533–539

Bioconjugate Chemistry

COMMUNICATION

spectra obtained for single-stranded ON4 compared to the CD spectra of LNA/DNA probes ON2, ON3, and ON5, and reference probe ON13 (Figure 1 in Supporting Information). To further investigate how the 1-PEPy can be protected from quenchers of fluorescence within single-stranded LNA/DNA, molecular models were built (Figure 2). They showed that stacked structures from two spatially accessible G:C base pairs allow placing of 1-PEPy in a cave isolating it from quenching by the buffer medium, oxygen, or nucleobases (Figure 2C,D). Next, pyrene excimer fluorescence (λflmax ∼ 480 nm) is proven to be a useful tool for structural studies in biochemistry and molecular biology as “probing of proximity” since the excimer signal is observed when the two pyrenes display some degree of coplanarity and are positioned within a distance less than 4 Å.31,39
41 Previously, pyrene attached to the sugar part of

different nucleosides formed interstrand excimers being located in “þ2 zipper”,42
44 “þ1 zipper,37,43,44 or “
1 zipper”37,45 modes (for description of the “zipper” nomenclature, see caption of Table 3). Recently, we also confirmed weaker inter- and intrastrand excimers formed by 1-PEPy, although it has such a short excited lifetime (