Development of Recombinant RNA Technology

限制性内切酶通常识别和切割长度为4到6个碱基对（bp）的特定DNA序列。然而，在DNA中存在的4096个可能的bp序列中，只有一小部分被限制酶识别。此外，市售限制性内切酶的质量往往很差；制剂不纯，酶浓度低。本提案旨在开发重组RNA技术，利用核糖酶可以切割和连接RNA分子，以服务于目前由限制性内切酶和连接酶服务的功能。可能产生4096种不同的核酶，每种核酶都具有特定的6个核苷酸（nt）序列。所有的核酶都将通过相同的方法产生和纯化，每个都将在相同的反应缓冲液中起作用。因此，应该有可能生产出一贯高质量的廉价核酶。这项技术将显著提高我们产生重组DNA分子的能力。重组分子将在RNA水平上通过自剪接II族内含子序列催化的体外反式剪接产生。这些重组RNA分子将通过逆转录酶复制到DNA中，并通过聚合酶链反应（PCR）扩增产生重组DNA分子。本研究将确定重组RNA技术作为核酸操作的实用工具的可行性。II类内含子能够在体外将外显子拼接在一起（Peebles et al . 1986）。这个反应是自催化的。如图1所示，内含子催化其自身从前体RNA中移除。两个要连接的序列（外显子1和2）用矩形表示。内含子由六个按顺序编号的保守结构域组成。分子生物学依靠重组DNA技术来操纵和克隆DNA分子。限制性内切酶和连接酶专门用于切割和连接DNA分子。这项技术被广泛使用，我们很少考虑到它的局限性。限制性内切酶通常识别和切割长度为4到6个碱基对（bp）的特定DNA序列。然而，在DNA中存在的4096个可能的6 bp序列中，只有一小部分被限制性内切酶识别。此外，市售限制性内切酶的质量往往很差；制剂不纯，酶浓度低。本提案旨在开发重组RNA技术，利用核糖酶（RNA催化剂）切割和连接RNA分子，以服务于目前由限制性内切酶和连接酶（连接核酸片段的酶）服务的功能。可能产生4096种不同的核酶，每种核酶具有特定的6个核苷酸（nt）序列特异性。所有的核酶都将通过相同的方法产生和纯化，每个都将在相同的反应缓冲液中起作用。因此，应该有可能生产出一贯高质量的廉价核酶。这项技术将显著提高我们产生重组DNA分子的能力。在体外以RNA水平生成重组分子，然后通过逆转录酶将RNA分子复制成DNA，逆转录酶将RNA分子复制成DNA，再通过聚合酶链反应扩增得到重组DNA分子。本研究将确定重组RNA技术作为核酸操作的潜在工具的可行性。II类内含子能够在体外将外显子拼接在一起（Peebles al. 1986）。这个反应是自催化的。如图1所示，内含子催化其自身从前体RNA中移除。两个要连接的序列（外显子1和2）用矩形表示。内含子由六个按顺序编号的保守结构域组成。

Jarrell 9400562 Restriction enzymes typically recognize and cleave specific DNA sequences that are 4 to 6 basepairs (bp) in length. However, only a small subset of the 4096 possible bp sequences present in DNA are recognized by restriction enzymes. In addition, commercially available restriction enzymes are often of poor quality; the preparations are impure and the enzyme concentration is low. This proposal seeks to develop recombinant RNA technology making use of ribozymes that can cut and join RNA molecules to serve the functions currently served by restriction enzymes and ligase. Potentially, 4096 different ribozymes, each with a particular 6 nucleotide (nt) sequence specifically, can be generated. All of the ribozymes will be generated and purified by the same method and each will function in the same reaction buffer. Thus, it should be possible produce inexpensive ribozymes of consistently high quality. This technology will significantly improve our ability to generate recombinant DNA molecules. The recombinant molecules will be generated at the RNA level using in vitro trans splicing catalyzed by self splicing group II intron sequences. These recombinant RNA molecules will be copied into DNA by reverse transcriptase and amplified by the polymerase chain reaction (PCR) to yield the recombinant DNA molecules. This research will determine the feasibility of recombinant RNA technology as a practical tool for nucleic acid manipulation. Group II introns are capable of splicing together exons in vitro (Peebles et al 1986). This reaction is autocatalytic. As shown in Figure 1, the intron catalyzes its own removal from a precursor RNA. The two sequences to be joined (exons 1 and 2) are represented by rectangles. The intron consists of six conserved structural domains that are numbered sequentially. %%% Molecular Biology relies upon recombinant DNA technology to manipulate and clone DNA molecules. Restriction enzymes and ligase are used to specifically cleave and join DNA molecules. This technology is so widely used that we seldom consider its limitations. Restriction enzymes typically recognize and cleave specific DNA sequences that are 4 to 6 basepairs (bp) in length. However, only a small subset of the 4096 possible 6 bp sequences present in DNA are recognized by restriction enzymes. In addition, commercially available restriction enzymes are often of poor quality; the preparations are impure and the enzyme concentration is low. This proposal seeks to develop recombinant RNA technology making use of ribozymes (RNA catalysts) that can cut and join RNA molecules to serve the functions currently served by restriction enzymes and ligase the enzyme that joins nucleic acid fragments. Potentially, 4096 different ribozymes, each with a particular 6 nucleotide (nt) sequence specificity, can be generated. All of the ribozymes will be generated and purified by the same method and each will function in the same reaction buffer. Thus, it should be possible to produce inexpensive ribozymes of consistently high quality. This technology will significantly improve our ability to generate recombinant DNA molecules. The recombinant molecules will be generated at the RNA level in vitro and then copied into DNA by the enzyme reverse transcriptase that can copy RNA molecules into DNA and amplified by the polymerase chain reaction to yield the recombinant DNA molecules. This research will determine the feasibility of recombinant RNA technology as a potential tool for nucleic acid manipulation. Group II introns are capable of splicing together exons in vitro (Peebles al. 1986). This reaction is autocatalytic. As shown in Figure 1, the intron catalyzes its own removal from a precursor RNA. The two sequences to be joined (exons 1 and 2) are represented by rectangles. The intron consists of six conserved structural domains that are numbered sequentially.