molecule.
As we talked at the very beginning, we synthesized 4 different DNA sequences, which are listed in the form below. W1 and W2 can form a native DNA double strand. W1
with W4 or W2 with W3 can form a DNA double strand with single selenium modified. W3 and W4 can form a double selenium modified DNA double strand.
Table 3.6 DNA sequences we synthesized which can bind with the small molecule DB2429 and DB2457.
DNA Sequences Sequence Name
5’-d(CCAAAGTTGC)-3’ W1
5’-d(GCAACTTTGG)-3’ W2
5’-d(CCAAAGT5-SeTGC)-3’ W3
5’-d(GCAACTT5-SeTGG)-3’ W4
We used the synthesized DNA sequences alone or binding with small molecule trying to create some crystal to do the X-ray diffraction. The figures in the following are some of the crystals we obtained.
Figure 3.19 Experimental DNA crystals obtained.
The crystal quality is scaled by its shape and size. The higher scores the crystal get, the more possibility to get useful diffraction data. Perfect crystal shall get 5 scores while poor crystal can only get 1.
W3-W4 Scale: 2 W2-W3-DB2429 Scale: 2 W 3-W4-DB2429 Scale: 2 W1-W2-DB2457 Scale: 2 W1-W4-DB2429 Scale: 1 W2-W3-DB2457 Scale: 1
From the crystal figure, the crystal is small and numerous, and the shape is irregular. We sent the crystals to do X-ray diffraction. Unfortunately, we didn't get useful X-ray diffraction data with those DNA sequences. Subsequent work will continue to optimize crystallization conditions for better crystals.
3.6.2 Crystallization study for DNA sequences with 3’-position overhangs
Since we cannot get good crystals, we tried to add a GC overhang at 3’-position for each DNA sequence. As the overhangs introduced, the DNA double-strand shall form a GC sticky end. The sticky end could connect two DNA double strands easily which may make it easier to form better crystals. The form below shows the DNA sequences we synthesized.
Table 3.7 DNA sequences we synthesized which has a GC overhang at 3'-position.
DNA Sequences Sequence Name
5’-d(CCAAAGTTGCGC)-3’ Top
5’-d(GCAACTTTGGGC)-3’ N
5’-d(GCAAC5-Se TTTGGGC)-3’ S
5’-d(GCAAC5-Se T5-Se TTGGGC)-3’ D
5’-d(GCAAC5-Se T5-Se T5-SeTGGGC)-3’ T
The Top sequence can form a double strand DNA with S, D or T. The newly formed DNA double-strand should contain single, double or triple selenium modified.
Table 3.8 The DNA double brand could be formed by the synthesized DNA sequences. To be read vividly, the bottom sequence is presented in 3' to 5' direction and the GC overhangs are highlighted.
Name DNA Double Brand Sequences
3‘-d(CGGGTTTCAACG )-5’
S 5-d( CCAA AGTTGCGC)-3’
3‘-d(CGGGTT(5-SeT)CAACG )-5’
D 5’-d( CCA A AGTTGCGC)-3’
3‘-d(CGGGT(5-SeT)(5-SeT)CAACG )-5’
T 5’-d( CC A A AGTTGCGC)-3’
3‘-d(CGGG(5-SeT)(5-SeT)(5-SeT)CAACG )-5’
However, we couldn’t get any good shape crystals by using these DNA sequences. Still, we did a screening test. The screening result is shown in the form below.
Table 3.9 Screening result for the DNA double strands with 3'-overhangs.
Although we didn’t have good crystal which can do the X-ray diffraction to finish the structure study, we can still come to the conclusion that DNAs with selenium modified could be easier to form crystals. As we can see in the table above, the more selenium atoms in the DNA double strand, the faster and the more crystal we observed.
3.6.3 Crystallization study for DNA sequences with 5’-position overhangs
We cannot get better crystal with 3’-position GC overhangs. So, we had another trial with 5’-position overhangs. The sequences we synthesized were listed below.
Table 3.10 DNA sequences we synthesized which has a GC overhang at 5'-position.
DNA Sequences Sequence Name
5’-d(CGCCAAAGTTGC)-3’ Top
5’-d(CGGCAACTTTGG)-3’ N
5’-d(CGGCAAC TT5-Se TGG)-3’ S
5’-d(CGGCAAC T5-Se T5-Se TGG)-3’ D
5’-d(CGGCAAC5-Se T5-Se T5-SeTGG)-3’ T
The Top sequence can form a double strand DNA with S, D or T. The newly formed DNA double-strand should contain single, double, or triple selenium modified.
Table 3.11 The DNA double brand could be formed by the synthesized DNA sequences. To be read vividly, the bottom sequence is presented in 3' to 5' direction and the GC overhangs are highlighted.
Name DNA Double Brand Sequences
N 5’-d(CGCCAAAGTTGC)-3’
3‘-d( GGTTTCAACGGC)-5’
S 5-d(CGCC AAAGTTGC)-3’
3‘-d( GG (5-SeT)TTCAACGGC)-5’
D 5’-d(CGCC A AAGTTGC)-3’
3‘-d( GG(5-SeT)(5-SeT)TCAACGGC)-5’
T 5’-d(CGCC A A AGTTGC)-3’
N Scale: 3 N Scale: 4
N + 2429 Scale: 3 N + 2429 Scale: 3.5
S Scale: 3 S Scale: 3
S + 2457 Scale: 3 S + 2457 Scale: 2.5
D Scale: 3 D Scale: 2.5
D + 2429 Scale: 2.5 D + 2429 Scale: 2.5
We are happy to see that we got some good crystals when we introduced GC overhangs at 5’-position. We mounted the crystals and tried to do the X-ray diffraction experiment. However, we still didn’t get good data even if we use the crystals which have regular shapes.
T Scale: 2.5 T Scale: 3
T + 2429 Scale: 2 T + 2429 Scale: 2
T + 2457 Scale: 2.5 T + 2457 Scale: 2.5
3.6.4 The effect on the crystallization of different MPD concentration.
Table 3.12 The crystallization result for the single selenium modified DNA double- strand with 5'-position GC overhangs. The MPD concentration is distributed from 20% to 45%. The pictures are what the crystals looked like on the third day.
Table 3.13 The crystallization result for the single selenium modified DNA double- strand with 5'-position GC overhangs binding with small molecule DB2429. The MPD concentration is distributed from 20% to 45%. The pictures are what the crystals looked like on the third day.
Table 3.14 The crystallization result for the single selenium modified DNA double- strand with 5'-position GC overhangs binding with small molecule DB2457. The MPD
concentration is distributed from 20% to 45%. The pictures are what the crystals looked like on the third day.
Table 3.15 The crystallization result for the double selenium modified DNA double- strand with 5'-position GC overhangs. The MPD concentration is distributed from 20% to 45%. The pictures are what the crystals looked like on the third day.
Table 3.16 The crystallization result for the double selenium modified DNA double- strand with 5'-position GC overhangs binding with small molecule DB2429. The MPD
concentration is distributed from 20% to 45%. The pictures are what the crystals looked like on the third day.
Table 3.17 The crystallization result for the double selenium modified DNA double- strand with 5'-position GC overhangs binding with small molecule DB2457. The MPD concentration is distributed from 20% to 45%. The pictures are what the crystals looked like on the third day.
Table 3.18 The crystallization result for the triple selenium modified DNA double- strand with 5'-position GC overhangs. The MPD concentration is distributed from 20% to 45%. The pictures are what the crystals looked like on the third day.
Table 3.19 The crystallization result for the triple selenium modified DNA double- strand with 5'-position GC overhangs binding with small molecule DB2429. The MPD concentration is distributed from 20% to 45%. The pictures are what the crystals looked like on the third day.
Table 3.20 The crystallization result for the triple selenium modified DNA double- strand with 5'-position GC overhangs binding with small molecule DB2457. The MPD
concentration is distributed from 20% to 45%. The pictures are what the crystals looked like on the third day.
Through the screening result above, we can conclude that higher MPD concentration can form crystals easier and better. And the sequences with more Se- modification can create crystal more easily.
3.6.5 Summary
Single overhang and double overhangs DNA can form better crystals than triple overhangs DNA. The 5’-overhang DNA can form better crystals than 3’-overhang DNA. Single overhang and double overhangs DNA can form better crystals than triple overhangs DNA. The 5’-overhang DNA can form better crystals than 3’-overhang DNA. Higher MPD concentration can form more crystals. The sequences with more Se-modification can form crystal more easily.
After acquiring the DNA crystal, the structure can be determined by X-ray diffraction. The crystal structure of each DNA molecule can be obtained by analyzing the X-ray diffraction data.
However, although we sent the DNA crystals to do the X-ray diffraction, we didn’t get any useful diffraction data.
4 CONCLUSIONS
The two major challenges in nucleic acid X-ray crystallography, crystallization and phasing determination, can be partially solved by introducing selenium modification into DNA. 5-Se-thymidine was successfully synthesized and incorporated into DNA oligonucleotides via solid-phase synthesis to facilitate the crystallization and phasing.
Small molecule DB2429 and DB2457 were co-crystalized with Se-DNAs and nice crystals were obtained, which are better than the native crystals. 5’-d(CCAAAGTTGC)-3’ and 5’-d(GCAACTTTGG)-3’ duplex can crystallize but doesn’t have useful X-ray diffraction data even that it’s selenium modified. The complex of DNA duplex binding with small molecule doesn’t have good diffraction data, as well.
The melting study reveals that the selenium modification has no signification perturbation to the DNA stability.
Since we couldn’t get good structure data from the regular DNA double-strands (native and Se-modified) after the X-ray diffraction, we tried to introduce overhangs to the duplex through which we could get a sticky end. The 5’-overhang DNA can form better crystals than 3’-overhang DNA. Single overhang and double overhangs DNA can form better crystals than triple overhangs DNA. We also find out that higher MPD concentration can form more crystals quickly and 35% MPD aqueous solution shall be the best reservoir solution for crystallization. The sequences with more Se-modification can create crystal more efficiently which means that selenium atom in the DNA molecule affects crystallization.
After obtaining the DNA crystal, the structure can be determined by X-ray diffraction. The crystal structure of each DNA molecule can be obtained by analyzing the X-ray diffraction data.
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