Development of Recombinant RNA Technology

重组RNA技术的发展

• 批准号: 9400562
• 负责人: Kevin Jarrell
• 金额: $ 5万
• 依托单位: Trustees of Boston University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9400562&HistoricalAwards=false
• 关键词: Development; Recombinant; RNA; Technology

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.

Jarrell 9400562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，存在于DNA中的4096个可能的BP序列中，只有一小部分被限制性内切酶识别。此外，市售的限制性内切酶往往质量不佳，制剂不纯，酶浓度低。这项提议寻求开发重组RNA技术，利用核酶可以切割和连接RNA分子，以服务于目前由限制性内切酶和连接酶起作用的功能。潜在地，可以产生4096种不同的核酶，每个核酶具有特定的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，并且每个核酶都将在相同的反应缓冲液中发挥作用。因此，应该有可能生产出持续高质量的廉价核酶。这项技术将显著提高我们产生重组DNA分子的能力。重组分子将通过自剪接II组内含子序列催化的体外反式剪接在RNA水平上产生。这些重组的RNA分子将通过逆转录酶复制到DNA中，并通过聚合酶链式反应(PCR)扩增得到重组DNA分子。这项研究将确定重组RNA技术作为一种实用工具进行核酸操作的可行性。第二组内含子能够在体外将外显子拼接在一起(Peeble等人，1986)。这个反应是自动催化的。如图1所示，内含子催化自己从前体RNA中移除。待连接的两个序列(外显子1和外显子2)用矩形表示。内含子由六个保守的结构域组成，这些结构域是按顺序编号的。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被用来专门切割和连接DNA分子。这项技术应用如此广泛，以至于我们很少考虑它的局限性。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，存在于DNA中的4096个可能的6bp序列中，只有一小部分被限制性内切酶识别。此外，市售的限制性内切酶往往质量不佳，制剂不纯，酶浓度低。这项提议寻求开发重组RNA技术，利用核酶(RNA催化剂)可以切割和连接RNA分子，以服务于目前由限制酶起作用的功能，并连接连接核酸片段的酶。可能产生4096种不同的核酶，每种核酶都具有特定的6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，并且每个核酶都将在相同的反应缓冲液中发挥作用。因此，生产出价格低廉、质量稳定的核酶应该是可能的。这项技术将显著提高我们产生重组DNA分子的能力。重组分子将在体外的RNA水平上产生，然后通过能将RNA分子复制到DNA中的酶逆转录酶复制到DNA中，并通过聚合酶链式反应进行扩增，得到重组DNA分子。这项研究将确定重组RNA技术作为一种潜在的核酸操纵工具的可行性。第二组内含子能够在体外将外显子拼接在一起(Peeble al.1986)。这个反应是自动催化的。如图1所示，内含子催化自己从前体RNA中移除。待连接的两个序列(外显子1和外显子2)用矩形表示。内含子由六个保守的结构域组成，这些结构域是按顺序编号的。