The Zhu Ting research group of the College of Life Sciences realized mirror T7 transcription

Both natural nucleic acids and natural proteins that make up life have single chiral properties: known natural nucleic acids are composed of D-type ribose, and natural proteins are almost all composed of
L-type amino acids.
Zhu Ting's research group is committed to starting from the law of genetic information center, using chemistry, biology and other multidisciplinary means to construct a "mirror biology system"
with the chirality opposite of natural biomolecules 。 So far, the research group has preliminarily realized the mirror nucleic acid replication, transcription, reverse transcription and other processes in the mirror center rule, broken through the technical bottleneck of large mirror protein total chemical synthesis and thousand base length mirror gene synthesis, developed mirror nucleic acid sequencing, mirror DNA information storage, mirror nucleic acid directed evolution and other technologies, and is currently focusing on building a mirror protein translation system to achieve a complete mirror center rule, and trying to expand the practical application of
mirror biology system.

The key to building a mirror protein translation system is the synthesis of mirror ribosomes
.
Ribosome RNA is the structure and catalytic core of ribosomes, and its molecular weight accounts for about 2/3 of the total molecular weight of ribosomes, so the difficulty in synthesizing mirror ribosomes lies in the preparation of mirror 5S, 16S and 23S ribosome RNAs
with lengths of about 120 nt, 1.
5 kb and 2.
9 kb, respectively.
However, limited by existing techniques, the longest mirror RNA that can be obtained before is only 120 nt, which is far from enough to achieve the goal of
preparing long mirror RNA.
Through nearly six years of research and exploration, Zhu Ting's research group fully chemically synthesized high-fidelity mirror T7 RNA polymerase with a molecular weight of 100 kDa, and used the mirror polymerase to transcribe various mirror RNAs, including mirror 5S, 16S and 23S ribosome RNA, mirror ribose switch (riboswitch), mirror ribozyme, etc.
, and carried out stability studies
on a variety of mirror RNAs 。 The research paper, titled "Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs," was published in the journal
Science on October 28, 2022.

Figure 1: Synthesis of mirrored T7 RNA polymerase

In order to break through the limitation of protein size by total chemical synthesis, the researchers used the strategy of splitting protein to synthesize three fragments of T7 RNA polymerase with a total length of 883 amino acids with a length of 363, 238 and 282 amino acids, respectively, and jointly renatured them in vitro to correctly fold them into a 100 kDa high-fidelity mirror T7 RNA polymerase with complete function (Figure 1).
It is the largest fully chemically synthesized mirror protein
ever reported.
At the same time, the researchers also used the mirror polymerase to transcribe and purify all three mirror ribosome RNAs, the longest transcription length was 2.
9 kb (Figure 2), which is the longest mirror RNA
reported so far.
The whole-chemical synthesis strategy of large mirror proteins and the assembly and transcription technology of thousand base length mirror genes used in this experiment solve the problem of preparing large mirror biomolecules that have long restricted the development of mirror biology, and lay a foundation
for the subsequent construction of mirror protein translation system, the realization of a complete mirror center rule and the expansion of the practical application of mirror biology system.

Figure 2: Transcribing and purifying mirror ribosome RNA

In addition, the researchers also attempted to conduct a stability study of mirror RNA (Figure 3), storing purified natural and mirror 16S ribosome RNA with a length of 1.
5 kb in DEPC treated ultrapure water to remove ribonuclease (RNase), and the natural RNA was completely degraded after 4 hours.
Even when RNase inhibitors are added to the sample, they are completely degraded after 48 h; The mirror RNA is completely degraded
after 30 days.
This experiment shows that DEPC-treated ultrapure water still has trace RNase contamination, but the unique biological stability of mirror RNA makes it unaffected by
RNase contamination.
The researchers also stored natural and mirror 16S ribosome RNA in lotus pond water taken from the natural environment, and the results showed that the natural RNA was completely degraded after 4 hours, with or without the addition of RNase inhibitors, while the mirror RNA was completely degraded
after 7 days.
Due to the unique biological stability of mirror RNA, it is expected to become an RNA research model system
that is not affected by RNase contamination in the future.

Figure 3: Studying the stability of mirrored 16S ribosome RNA

In summary, the study reported the preparation of a variety of mirror RNAs, including mirror ribosome RNA and functional RNA, using fully chemically synthesized 100 kDa high-fidelity mirror T7 RNA
polymerase.
The mirrored T7 transcription system is expected to be used in diagnostic therapy, information storage, molecular computing, biological imaging, and RNA-related basic research
.
Xu Yuan, a 2017 doctoral student at the School of Life Sciences of Tsinghua University, is the first author of the paper, and Professor Zhu Ting is the corresponding author
.
The research was supported
by grants from the National Natural Science Foundation of China, the Joint Center for Life Sciences at Tsinghua-Peking University, and the Beijing Center for Frontier Research in Biostructures.

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https://cen.
acs.
org/biological-chemistry/synthetic-biology/Mirror-image-polymerase-makes-key/100/web/2022/10