"Over the past month the world has been surprised by several terrorist attacks, in Turkey, Lebanon, France and most recently Mali," National Coalition chief Khaled Khoja said.
"The recent attack in Mali was claimed by Al-Qaeda, and in this context, I renew my call to Al-Nusra to break its ties with Al-Qaeda," Khoja said in Turkey.
Khoja has in the past urged Al-Nusra to break with Al-Qaeda, which officially embraced the group as its Syrian affiliate in April 2013.
World powers meeting in Vienna early this month hashed out a plan for the creation of a transitional government, a new constitution and elections.
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The plan also envisions a ceasefire across Syria, except in territory controlled by the Islamic State group, which in theory would apply to regions under the control of Al-Nusra and its allies.
Al-Nusra is part of a powerful alliance known as the Army of Conquest that captured Syria's northwestern province of Idlib earlier this year, and it has a strong presence in other parts of the country.
Al-Nusra is regarded as a partner by many factions in the opposition because it has focused on fighting Syrian troops rather than seeking to set up a "caliphate" like the one established by its rival IS.
Saudi Arabia is to host a meeting next month of Syrian armed and political opposition members to build a common platform for peace talks.
Jordan is drawing up a list of opposition delegations that will take part in the peace talks.