Investigation of DNA-protein sequence-specific interactions with a ds-DNA array.

The sequence specific recognitions between DNAs and proteins play important roles in many biological functions. The use of double-stranded DNA arrays (ds-DNA arrays) for studying sequence specific recognition between DNAs and proteins is a promising method. Here we report the use of a ds-DNA probe with multi operation sites of restriction proteins in the middle sequence to investigate DNA-protein sequence-specific interactions including methylation. We arranged EcoR I site and Rsa I site on the same duplex DNA probe to fabricate ds-DNA arrays. We used the ds-DNA arrays to study DNA-restriction enzyme reactions before and after duplex DNA methylation under different probe concentration and reaction time conditions. Our results indicated that the ds-DNA arrays can be further biochemically modified and made accessible for interactions between DNAs and proteins in complex multi-step gene-regulation processes.

Introduction

A major pursuit in biological research is to understand how cells maintain normal cellular homeostasis and how they behave in an intricately controlled manner in response to their surroundings. DNA-proteins sequence-specific interactions play important roles in regulating cellular processes such as transcription [1], recombination [2], restriction [3], replication [4] and DNA-drugs interaction [5,6]. Several methods have been developed to analyze the DNA-protein sequence-specific interactions, which are mainly gel-shift assays [7,8] and DNase I foot-printing assays [9]. However, they are not only laborious, time-consuming and incapable of providing highly parallel analysis, but also they are not suitable for analyzing the complex multi-step protein-DNA interactions.

Oligonucleotide arrays or DNA arrays have provided a platform for high-throughput detection of biomolecules and for analyzing the interaction between biological molecules [10,11,12,13], and now are beginning to be used in detecting DNA-binding proteins [14,15,16,17]. Most DNA arrays are made of ssDNA, and recognize the target DNA through hybridization. Unlike arrays used for gene resequencing, a new type of DNA arrays, double-stranded DNA (ds-DNA) arrays should be fabricated for detecting sequence-specific regulatory DNA-binding proteins. Preliminary studies of double-stranded DNA arrays suggested that these ds-DNA arrays are well suited for the analysis of DNA–protein interactions, particularly for the discovery of the sequences recognized by transcription factors and the quantitative assessment of those important interactions. Bulyk was the first to convert single-stranded to a double-stranded array to perform highly parallel investigations of DNA–protein interactions by synthesizing a constant sequence at every position on an array and then annealing and enzymatically extending a complementary primer and he first used the ds-DNA arrays to study the DNA-protein sequence-specific interaction [14].

As we know, a number of DNA–protein interactions require that the DNA be biochemical modified in some way. For example, restriction-modification systems occur in many bacterial species, and constitute a defense mechanism against the introduction of foreign DNA into the cell [18,23]. Our group had also published two methods for fabrication of ds-DNA arrays and here we also use restriction enzymes as proteins to investigate DNA-proteins interactions, for restriction enzymes have very stringent sequence requirements, they have been used as model systems to study the sequence specificity of DNA–protein interactions [19,20,21,22]. We arranged EcoR I site and Rsa I recognition site on the same duplex DNA probe to fabricate ds-DNA arrays and labeled the ds-DNA probes during the ds-DNA arrays creation previously and then used the ds-DNA arrays to study DNA-restriction enzyme reactions before and after duplex DNA methylation under different probe concentration and reaction time conditions. Our results indicated that the ds-DNA arrays can be further biochemically modified and that the DNA is accessible for interaction with DNA-binding proteins in complex multi-step gene-regulation processes.

Results and Discussion

Restriction enzyme digestion

To verify and study the DNA/protein interactions on the fabricated ds-DNA arrays, ds-DNA arrays which had been inserted with Cy3-dUTP were used for digestion with EcoR I and Rsa I, respectively. The relative fluorescence intensity variations before and after endonuclease digestions are shown in Figure 1 and Figure 2, and summarized in Table 1 and Table 2.

Figure 1

The images of EcoR I and Rsa I digestion on ds-DNA array. (A): the image of the ds-DNA array which created by insertion with Cy3-dUTP (a) digested by EcoR I under 1h(b) and 12h(c); (B): the image of ds-DNA array (a) digested by Rsa I under 1 h(b) and 12 h(c) digestion. The four different concentrations of Oligo II (C, 1, 2, 3 and 4 below the two images indicate control, 80 µM, 40 µM, 20 µM and 10 µM Oligo II, respectively.) were spotted onto the array.

Figure 2

The relative fluorescence intensity variation before and after restriction enzyme digestion. Oligo I, which didn’t contain the two restriction enzyme recognition sites, was spotted as a control.

Table 1

The ratios between experiment spots and controls of EcoR I digestion

	Control	80µM	40µM	20µM	10µM
Pre-digestion	1	0.65	0.40	0.35	0.22
1h-digestion	1	0.25	0.17	0.15	0.14
12h-digestion	1	0.07	0.06	0.05	0.05

Table 2

The ratios between experiment spots and controls of Rsa I digestion

	Control	80µM	40µM	20µM	10µM
Pre-digestion	1	0.85	0.53	0.39	0.26
1h-digestion	1	0.26	0.16	0.11	0.07
12h-digestion	1	0.15	0.11	0.07	0.05

To determine whether the digestion efficiency was affected by the concentrations of the ds-DNA probes and reaction time, four different concentrations (80 µM, 40 µM, 20 µM, 10 µM) and two different reaction times (1 h, 12 h) were tested in this experiment. To avoid the signal intensity loss caused by background decrease, we calculated the intensity ratios between experimental spots and controls to compare the signal change before and after incubation. As the data shown in Table 1 and Table 2 indicate, it is suggested that the fluorescence intensities decreased as the digestion time increased. On the other hand, the probe concentrations were not the main factors influencing the digestion efficiency. It indicated that the probes density below 80 µM was not congested enough to encumber the enzyme molecules’ movement.

Restriction enzyme digestion after the treatment of EcoR I methylase

The ds-DNA arrays labeled with Cy3-dUTP were treated with EcoR I methylation enzyme before the two enzymes digestion. The results are shown in Figure 3 and Figure 4 and Table 3. The data showed that there were no significant signal losses after the EcoR I digestion of the ds-DNA array which had been previously treated with EcoR I methylation enzyme. However, the ds-DNA array after Rsa I incubation presented significant signal decrease when previously treated with EcoR I methylase.

Figure 3

The images of array one digested by EcoR I and Rsa I after methylation. (A): Images of array one which created by inserted Cy3-dUTP (a) was treated by EcoR I methylation enzyme (b) and then by EcoR I (c); (B): Images of array one which created by inserted Cy3-dUTP (a) was treated by EcoR I methylation enzyme (b) and then by Rsa I (c). (C and E below the images indicate Control probe and Oligo I, respectively)

Figure 4

The fluorescence intensity variation of EcoR I and Rsa I digestion on the same array after methylation. EM indicated methylation enzyme and ER indicated EcoR I.

Table 3

The ratios between experiment spots and controls of Restriction enzymes digestion after the treatment of EcoR I methylase

	Control	Un-treated	EM*-treated	RE*-treated
EcoR I digestion	1	0.85	0.84	0.82
Rsa I digestion	1	0.65	0.62	0.19

*EM and RE refer to EcoR I methylase and Restriction enzyme respectively.

Conclusions

There are Restriction Modification (RM) systems which are species/strain specific, and they only allow the survival of DNA received from a like species or strain having the same RM system [18,23]. This combination of a specific methylase and endonuclease functioned as a type of immune system for individual bacterial strains, protecting them from infection by foreign DNA. In any cell with a RM system, both the restriction and modification enzymes have the same sequence specificity. The EcoR I endonuclease within the same bacteria will not cleave the methylated DNA. Foreign viral DNA, which is not methylated at the sequence "GAATTC" will therefore be recognized as "foreign" DNA and will be cleaved by the EcoR1 endonuclease [23]. Our ds-DNA array platform would be useful in studying the efficiency and influence factors in this kind of complex RM systems.

In summary, we anticipate that double-stranded DNA arrays designed in various formats will have broad applications in studying protein-DNA interactions, including inhibitors/activators of sequence specific transcription factors, or synergy among transcription regulators. We also anticipate that ds-DNA arrays will be very effective substitutes for many tedious protein-DNA interaction assays currently used in the field, such as gel mobility shift assays, filter binding assays, etc.

Table 5

Probe and primer sequences

Name	Sequence	Length (nt)
Oligo I	5’…NH2-TTTTTTGTTGCATTTCCGGGTTTGGCAAGCTTTTAAGCTT… 3’	40
Oligo II	3’…NH2-TTTTTCTCCCCTGAAAGGGTATAGCTTTTATTTATTAT…5’	44
Oligo III	5’…GAG GGGACTTT CCC ATATCG…3’	26

The blocked area indicates the endonuclease recognition site.