芘基对在dsDNA中的构建:基于8-氮-7-去氮-2'-脱氧腺苷的7位取代及连接臂对荧光性质的影响

doi:10.7503/cjcu20180182

摘要/Abstract

摘要：

通过7-芘乙炔基-8-氮-7-去氮-2'-脱氧腺苷(2)和7-芘乙基-8-氮-7-去氮-2'-脱氧腺苷(3)2个脱氧腺苷类似物在双螺旋DNA(dsDNA)中构建芘基对, 同时保持2'-脱氧腺苷的碱基配对专一性, 探讨了芘基对的多种组合在dsDNA中的荧光光谱变化及其与dsDNA形成和解离的关系. DNA双螺旋的热稳定性和圆二色光谱表征结果反映了连接臂与芘基在dsDNA大沟区的相互作用. 芘基荧光光谱表明, 化合物2在dsDNA中的荧光光谱反映了双螺旋的变化, PY02+D02在形成双螺旋前后发生了荧光猝灭, 而PY03+PY05在形成双螺旋前后呈现荧光, 这2个芘基对组合在含双螺旋结构域的功能核酸和生物传感器中具有潜在应用价值.

关键词: 芘基, 脱氧腺苷类似物, 双螺旋DNA, 荧光光谱, 热稳定性

Abstract:

Pyrenyl excimer is well characterized to be a fluorescent tag suitable for the construction of nucleic acids-based biosensors. dsDNA was used as one of the major motifs for the construction of the pyrenyl pairs in the biosensors. Here, 7-pyrenylethynyl-8-aza-7-deaza-2'-deoxyadenosine(2) and 7-pyrenylethyl-8-aza-7-deaza-2'-deoxyadenosine(3) were used to introduce pyrenyl group to duplex DNA, by which the specific base pairing of 2'-deoxyadenosine was kept, and the pyrenyl group was located at the major groove of the duplex. In the B-duplex DNA formed by 5'-d(TAGGTCAATACT)-3' and 3'-d(ATCCAGTTATGA)-5', the canonical residue 2'-deoxyadenosine(dA) was replaced by residue 2 or 3 at several positions, respectively. All the DNA sequences were synthesized on an ABI 392 DNA/RNA synthesizer and characterized by mass spectrum. The thermal stability of the modified duplexes was significantly affected. It was observed that T_m was dramaticly decreased by residue 2 and dramaticly increased by residue 3. It was supposed that the large hydrophobic pyrenyl group was protruded to the hydrophilic major groove by the rigid ethynyl linkage in residue 2, while the flexible linkage in residue 3 permitted the pyrenyl group to stack into the neighboring basepair. The diffe-rent location of the pyrenyl group from residues 2 and 3 was also confirmed by the circular dichroism(CD), the spectra from 300 nm to 450 nm indicated that more pyrenyl stacking formed in the duplexes with residue 2. Especially, the fluorescence switch-on was observed in the formation of the duplex PY03+PY05 and switch-off in the formation of the duplex PY02+D02, respectively. These results demonstrated that the residue 2 was more sensitive to the conformational changes of duplex DNA, and these two pyrenyl group combinations were worthwhile to be further studied in the context of functional nucleic acids for the construction of the pyrenyl-based sensors.

Key words: Pyrenyl, Deoxyadenosine analogue, Duplex DNA, Fluorescence spectrum, Thermal stability

中图分类号:

O629

TrendMD:

李阳, 李志文, 朱俊飞, 刘世会, 何军林. 芘基对在dsDNA中的构建:基于8-氮-7-去氮-2'-脱氧腺苷的7位取代及连接臂对荧光性质的影响. 高等学校化学学报, 2018, 39(10): 2206.

LI Yang, LI Zhiwen, ZHU Junfei, LIU Shihui, HE Junlin. Construction of Pyrenyl Pairs in dsDNA: Fluorescent Properties of Multiple Pyrenyl-attached dsDNAs Through 7-Substituted 8-Aza-7-deaza-2'-deoxyadenosine Analogues^†. Chem. J. Chinese Universities, 2018, 39(10): 2206.

图/表 5

参考文献 52

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Name	Sequence	MS(calcd.), m/z	Name	Sequence	MS(calcd.), m/z
PY01	5'-d(TAGGTC22TACT)-3'	4092.2(4090.8)	PY06	5'-d(TAGGTC33TACT)-3'	4100.4(4101.0)
PY02	5'-d(TAGGTC2AT2CT)-3'	4092.7(4090.8)	PY07	5'-d(TAGGTC3AT3CT)-3'	4100.1(41010)
PY03	5'-d(TAGGTC2ATACT)-3'	3867.9(3868.7)	PY08	5'-d(TAGGTC3ATACT)-3'	3871.7(3872.7)
PY04	5'-d(TAGGTCA2T2CT)-3'	4092.0(4090.8)	PY09	5'-d(TAGGTCA3T3CT)-3'	4100.2(41010)
PY05	5'-d(AGT2TTGACCTA)-3'	3868.1(3868.7)	PY10	5'-d(AGT3TTGACCTA)-3'	3872.1(3872.7)

Name	Sequence	MS(calcd.), m/z	Name	Sequence	MS(calcd.), m/z
PY01	5'-d(TAGGTC22TACT)-3'	4092.2(4090.8)	PY06	5'-d(TAGGTC33TACT)-3'	4100.4(4101.0)
PY02	5'-d(TAGGTC2AT2CT)-3'	4092.7(4090.8)	PY07	5'-d(TAGGTC3AT3CT)-3'	4100.1(41010)
PY03	5'-d(TAGGTC2ATACT)-3'	3867.9(3868.7)	PY08	5'-d(TAGGTC3ATACT)-3'	3871.7(3872.7)
PY04	5'-d(TAGGTCA2T2CT)-3'	4092.0(4090.8)	PY09	5'-d(TAGGTCA3T3CT)-3'	4100.2(41010)
PY05	5'-d(AGT2TTGACCTA)-3'	3868.1(3868.7)	PY10	5'-d(AGT3TTGACCTA)-3'	3872.1(3872.7)

Name	Sequence	T_m/℃	ΔT_m/℃	Name	Sequence	T_m/℃	ΔT_m/℃
D01+D02	5'-d(TAGGTCAATACT)-3'	49.8		PY06+D02	5'-d(TAGGTC33TACT)-3'	45.9	-3.9
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTTATGA)-5'
PY03+D02	5'-d(TAGGTC2ATACT)-3'	39.4	-10.4	PY07+D02	5'-d(TAGGTC3AT3CT)-3'	61.7	11.9
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTTATGA)-5'
PY08+D02	5'-d(TAGGTC3ATACT)-3'	54.0	4.2	PY09+D02	5'-d(TAGGTCA3T3CT)-3'	59.5	9.7
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTTATGA)-5'
D01+PY05	5'-d(TAGGTCAATACT)-3'	41.0	-8.8	PY01+PY05	5'-d(TAGGTC22TACT)-3'	48.8	-1.0
	3'-d(ATCCAGTT2TGA)-5'				3'-d(ATCCAGTT2TGA)-5'
D01+PY10	5'-d(TAGGTCAATACT)-3'	57.0	7.2	PY02+PY05	5'-d(TAGGTC2AT2CT)-3'	54.0	4.2
	3'-d(ATCCAGTT3TGA)-5'				3'-d(ATCCAGTT2TGA)-5'
PY03+PY05	5'-d(TAGGTC2ATACT)-3'	49.8	0	PY04+PY05	5'-d(TAGGTCA2T2CT)-3'	60.0	10.2
	3'-d(ATCCAGTT2TGA)-5'				3'-d(ATCCAGTT2TGA)-5'
PY01+D02	5'-d(TAGGTC22TACT)-3'	44.3	-4.5	PY06+PY10	5'-d(TAGGTC33TACT)-3'	55.2	5.4
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTT3TGA)-5'
PY02+D02	5'-d(TAGGTC2AT2CT)-3'	36.5	-13.3	PY09+PY10	5'-d(TAGGTCA3T3CT)-3'	59.4	9.6
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTT3TGA)-5'
PY04+D02	5'-d(TAGGTCA2T2CT)-3'	52.1	2.3	PY07+PY10	5'-d(TAGGTC3AT3CT)-3'	58.6	8.8
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTT3TGA)-5'
PY08+PY10	5'-d(TAGGTC3ATACT)-3'	64.2	14.4
	3'-d(ATCCAGTT3TGA)-5'

Name	Sequence	T_m/℃	ΔT_m/℃	Name	Sequence	T_m/℃	ΔT_m/℃
D01+D02	5'-d(TAGGTCAATACT)-3'	49.8		PY06+D02	5'-d(TAGGTC33TACT)-3'	45.9	-3.9
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTTATGA)-5'
PY03+D02	5'-d(TAGGTC2ATACT)-3'	39.4	-10.4	PY07+D02	5'-d(TAGGTC3AT3CT)-3'	61.7	11.9
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTTATGA)-5'
PY08+D02	5'-d(TAGGTC3ATACT)-3'	54.0	4.2	PY09+D02	5'-d(TAGGTCA3T3CT)-3'	59.5	9.7
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTTATGA)-5'
D01+PY05	5'-d(TAGGTCAATACT)-3'	41.0	-8.8	PY01+PY05	5'-d(TAGGTC22TACT)-3'	48.8	-1.0
	3'-d(ATCCAGTT2TGA)-5'				3'-d(ATCCAGTT2TGA)-5'
D01+PY10	5'-d(TAGGTCAATACT)-3'	57.0	7.2	PY02+PY05	5'-d(TAGGTC2AT2CT)-3'	54.0	4.2
	3'-d(ATCCAGTT3TGA)-5'				3'-d(ATCCAGTT2TGA)-5'
PY03+PY05	5'-d(TAGGTC2ATACT)-3'	49.8	0	PY04+PY05	5'-d(TAGGTCA2T2CT)-3'	60.0	10.2
	3'-d(ATCCAGTT2TGA)-5'				3'-d(ATCCAGTT2TGA)-5'
PY01+D02	5'-d(TAGGTC22TACT)-3'	44.3	-4.5	PY06+PY10	5'-d(TAGGTC33TACT)-3'	55.2	5.4
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTT3TGA)-5'
PY02+D02	5'-d(TAGGTC2AT2CT)-3'	36.5	-13.3	PY09+PY10	5'-d(TAGGTCA3T3CT)-3'	59.4	9.6
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTT3TGA)-5'
PY04+D02	5'-d(TAGGTCA2T2CT)-3'	52.1	2.3	PY07+PY10	5'-d(TAGGTC3AT3CT)-3'	58.6	8.8
	3'-d(ATCCAGTTATGA)-5'				3'-d(ATCCAGTT3TGA)-5'
PY08+PY10	5'-d(TAGGTC3ATACT)-3'	64.2	14.4
	3'-d(ATCCAGTT3TGA)-5'